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1 INTRAMOLECULAR INTERACTIONS OF MARCKS AND ITS RELATIONSHIP TO TUMOR NECROSIS FACTOR-ALPHA AND RHEUMATOID ARTHRITIS By IMAN MOHAMMAD ASHRAF AL-NAGGAR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008
2 2008 Iman Mohammad Ashraf Al-Naggar
3 I dedicate this work to Allah (God Almighty) in partial fulfillment of His mandate to seek Knowledge; to my husband, mother, father, grandm other, grandfather and siblings, whose love and support kept me going; to the Bubb, Edison a nd Long Labs who provided a great scientific atmosphere for me to flourish; and finally, to all my children, born (Salma) and yet to come, whom I love greatly.
4 ACKNOWLEDGMENTS I would like to begin by thanking God for giving me this great opportunity to learn and for putting in my path wonderful people who have he lped me and shown me the way. I want to thank all those people, and will try my best to remember them all here. I want to start with my loving husband, Mr. Ahmad M. Mahmoud. To put it simply, this would not have been possible without his love and support. Support is such a small word to describe everything Ahmad has done for me over the last 5 years. Ahmad spent hours walking around the ARB and the College of Medicine looki ng for information about a graduate program I could attend at UF. He would drop me off each morning and pick me up every evening, take me to eat when I was hungry, take me back to th e lab at 2 and 3 and 4 am when I had crazy experiments running late. Ahmad didnt care whether or not I cooked, cleaned or did laundry because I had so much studying and lab work to do and would go grocery shopping because I hated to. He traveled with me to conferen ces and took care of our daughter and me. Ahmad always sincerely asked me whether there was anyt hing he could do for me. He also came with me to lab parties where he didnt know anyone and felt out of place because it made me happy. He endured lots of stubbornness and hard headed ness and still forgave me and made me feel loved. He was wonderful to my family when they came to visit because I needed them, regardless of how busy he was and he understood th at every year I wanted to spend the holidays visiting my family rather than taking that road tr ip of his dreams. He al ways made me feel like the more important graduate student even when he was in an equally challenging program, and, when he was drowning in failing experiments, he still found a way to convince me not to give up and that mine would work someday. Ahmad forced me to socialize when I felt the least social, forcing me on a trip when I didnt want to be cause he knew I needed it Support was all those dinners of pasta, Ragu and frozen meatballs he put together for us while I wrote this thing.
5 Support was everything nice, every kind word and gesture he did for me during my most trying years yet. All that said, I thank my husband Ahmad for his support. I would not have made it this far without him. I would like to thank my parents, Dr. Ashraf Al-Naggar and Ms. Wafaa Badawy for pushing me towards graduate school. They have always known whats best for me and have always insisted that I do it, regardless of what I wanted. I have finally understood that they were right all along, and that they knew better. They realized my potent ial long before I did and I am very glad they did not allow me to waste it. Th ey have gone to great measures to ensure that I had everything I needed to help me survive gr aduate school and not to have to worry about anything else along the way. They bought me a beau tiful house and ensured my comfort. They visited me whenever they could. I am lucky to have them as parents: very hardworking, smart, selfless and dedicated, they set a wonderful example for my siblings and me. I really appreciate everything they have done for me and pray that I will always be the daughter they want me to be. I would like to thank my grandparents Dr. Wadie Badawy and Ms. Haneya Shokri for valuing education beyond any limits and pushing me and ev eryone they know to pursu e higher levels of education. I also thank them fo r their unconditional love and endle ss prayers that have kept me on track. I would like to thank Dr. Michae l R. Bubb and Dr. Arthur S. Edison for agreeing to mentor me and doing a fantastic job at it! They are both great scientists a nd I am in awe of their critical thinking. I really could not hope for better teachers: kind, smart, knowledgeable, patient, supportive, understanding, loving and caring. I am inspired by their hard work and dedication to science and education. I am humbled by their modesty and humanity. I am honored by their
6 friendship. Not only am I a far better scientist th an I thought I could become in 5 years, I am a better human being for having known them and worked with them. I thank them for everything! I would like to thank my committee members for their guidance and great ideas: Dr. Sally Litherland, Dr. Eric Sobel and Dr. Shannon Holliday. I have been fortunate to have a diverse, knowledgeable committee that had answers to all my questions, solutions to my problems, and great advice and encouragement wh en I needed them most. I would like to thank Dr. Minor u Satoh for all his help and problem solving in my ELISA experiments, and for always being there when I needed him. Dr. Satoh is a very smart and knowledgeable person from whom I was lucky to get help. His kindness and giving know no boundaries. Although he was not officially a member of my committee, he gave me the full support one can expect from a committee me mber. I thank him for all his help. I would like to thank Dr. Joanna Long for be ing a great friend and inspiration. Dr. Long has shown me that it is possible to be both a stro ng, smart, successful scientist and a great mother of four! She is a genuinely wonderful person and I truly am in awe of her. I would like to thank all the people in the Bubb, Edison a nd Long Labs that have helped me throughout my years in graduate school, as we ll as make my long hours in the lab enjoyable: Dr. Elena Yarmola, Dr. Hazel Tapp, Dr. Cheria n Zachariah, Dr. Jiahong Shao, Vijay Antharam Aaron Dossey, Omjoy Ganesh, Ramadan Ajredini, Terry Green, Mini Samuel-Landtiser, Fatma Kaplan, Heather Cornell, Seth McNeil, and too many undergraduates to list. I especially thank Minh Q. Vo, Reuben Judd and Alejandro Sanchez fo r their help and friendship and wish them great luck in their future. I would like to tha nk Zoe Fisher, Firas Kobeissy and Nichole Giordani for great study groups and treasured friendships.
7 I would like to thank Mary Handlogten from Dr Weiners Lab for teaching me how to use the RT-PCR and being there when I had questions and being a great friend. I would like to thank Alfred Chung for making all the challenging peptides I needed for my assays. I would like to thank Scott McMillen Scott McClung and Stan Stevens for their help with mass spectrometry. I would like to thank everyone else who he lped me in any way with my experiments; I hope they can forgive me for not mentioning them by name. I would also like to thank all the staff that ha s assisted me greatly at UF (Department of Biochemistry and Molecular Biology and the Inte rdisciplinary Program in Biomedical Sciences) and at the Malcom Randall Veterans Affairs Medical Center: Joyce Conners, Susan Gardner, Valerie Cloud-Driver, Elise Feagle, Pat Jones, Bradley Moore, Denise Mesa, Terry Ricki, Shelley Jenkins, Shyra Bailey and many more. I would like to thank my sister, Sarah Al-Na ggar, who always made time for me in the midst of her busy Harvard Law School graduate e ducation. She was always there for me when I needed her and she is the best friend I have ever had and I hope she always will be. I would like to thank Dr. Wayne McCormack, Dr Richard Condit, Dr. Susan Frost and Dr. Linda Bloom for all their help, guidance and advice. I would like to thank my friends who have ma de my years in Gainesville less lonely by providing me with a family away from my fa mily. The El-Mahdawi Family (Uncle Ahmed, Auntie Fouz, Lamia, Ahmed, Wafaa, Omar and Fouz), the Enani Family (Auntie Mona, Hanan, Mahmoud and Eman) and the Dietrich Family (Rick, Chris, Todd, Larrett, Ricky, William and Michael) have taken me and my husband and eventually my baby Salma in and made us feel loved and cared for.
8 Finally, I would like to thank th e University of Florida and the College of Medicine for providing such a rich and truly interdisciplinary graduate program in which I am very lucky to have been a part.
9 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF TABLES................................................................................................................. ........11LIST OF FIGURES.......................................................................................................................13LIST OF ABBREVIATIONS........................................................................................................ 15ABSTRACT...................................................................................................................................19CHAPTER 1 INTRODUCTION..................................................................................................................21MARCKS...............................................................................................................................21MARCKS Functions.......................................................................................................23Major Post-Translational Modifications.......................................................................... 25MARCKS Phosphorylation......................................................................................25MARCKS Proteolysis..............................................................................................28Tumor Necrosis Factor.......................................................................................................... .30Rheumatoid Arthritis..............................................................................................................32Apoptosis................................................................................................................................372 INVESTIGATING INTRAMOLECULAR INTERACTIONS OF MARCKS..................... 42Introduction................................................................................................................... ..........42Neutralization of Acidic Residues on MARCKS...................................................................43Methods...........................................................................................................................45Preparation of EDC-modified MARCKS................................................................ 45Actin-binding assays................................................................................................45MIANS-calmodulin binding assay...........................................................................46Results........................................................................................................................ .....47MARCKS Truncations...........................................................................................................48Methods...........................................................................................................................48Generating truncations of MARCKS....................................................................... 48Testing activity of MARCKS truncations................................................................50Results........................................................................................................................ .....51Conclusions.............................................................................................................................523 SEARCHING FOR AN INTERNA L BINDING SITE ON MA RCKS................................. 67Introduction................................................................................................................... ..........67Mass Spectrometric Determination of Internal Binding Site of MARCKS........................... 67
10 Mass Spectrometry.......................................................................................................... 67Methods...........................................................................................................................69AspN pre-column digest...........................................................................................69Glu-C on column digest........................................................................................... 70Results........................................................................................................................ .....71Use of MARCKS Truncations to Determine PSD-Interacting Terminus.............................. 72Fluorescence Anisotropy Assays.....................................................................................72Direct binding anisotropy assay...............................................................................74Competition anisotropy assay.................................................................................. 74Native Gel Electrophoresis with Fluorescently Labeled PSD......................................... 74Native PAGE of rhodamine-labeled PSD and MARCKS truncations..................... 75Native agarose gel electrophoresis of rhodamine-labeled PSD and MARCKS truncations............................................................................................................. 75Results........................................................................................................................ .....76Conclusions.............................................................................................................................764 MARCKS AND TNF............................................................................................................. 94Introduction................................................................................................................... ..........94Methods..................................................................................................................................95Culture conditions and nucleofection.............................................................................. 95TNF treatment and qRT-PCR..........................................................................................96Results.....................................................................................................................................96Conclusion............................................................................................................................1005 MARCKS IN RHEUMATOID ARTHRITIS...................................................................... 119Introduction................................................................................................................... ........119Study Subjects......................................................................................................................121Cell Isolation from Whole Blood......................................................................................... 122Quantitative Real Time RT-PCR for Dete rmination of mRNA Concentration................... 123Detailed qRT-PCR Protocol..........................................................................................123Results........................................................................................................................ ...125Quantitative Sandwich ELISA of MARCKS and TNF....................................................126Quantification of Plasma TNFUsing a Commercial Sandwich ELISA.................... 127Quantification of Cellular MARCKS Using Optimized Sandwich ELISA.................. 128Results........................................................................................................................ ...130Conclusions...........................................................................................................................1316 CONCLUSIONS AND FUTURE DIRECTIONS............................................................... 158LIST OF REFERENCES.............................................................................................................160BIOGRAPHICAL SKETCH.......................................................................................................180
11 LIST OF TABLES Table page 1-1 MARCKS does not appear to be upregulated by TNFin mi croarray data.................... 39 3-1 Mass spectrometry results of GluC MARCKS fragments bound to biotin-PSD. ..............78 4-1 TNF Superarray array layout........................................................................................... 102 4-2 TNF SuperArray gene table............................................................................................. 103 4-3 Cell viability 48 hours after nucleofection....................................................................... 109 4-4 Results of qRT-PCR to measure MARCKS knockdown by RNA interference............ 110 4-5 TNF SuperArray results following RNA inte rference in response to TNF treatment. .... 111 4-6 Differential expression of apoptosis genes in TNF-treated HL-60 cells following MARCKS and non-targeting RNAi................................................................................. 114 4-7 Genes in TNF SuperArray whose expr ession was similar in T NF-treated nontargeting siRNA transfected cells and non-TNF-treated MARCKS siRNA transfected cells..................................................................................................................................115 4-8 Genes in TNF SuperArray whose expr ession was unchanged in RNAi and TNF treated HL-60 cells...........................................................................................................117 4-9 Genes in TNF SuperArray whose expression was unchanged following TN F treatment of both MARCKS and non-targe ting siRNA transfected HL-60 cells............118 5-1 Ct values obtained by quantitative re al time RT-PCR for mononuclear cells from patient samples................................................................................................................ .133 5-2 Ct values obtained by quantitative real time RT-PCR for granulocytes from patient samples........................................................................................................................ .....135 5-3 Ct and fold change calculations for monocytic cells qRT-PCR from patient samples.............................................................................................................................136 5-4 Ct and fold change calculations for monocytic cells qRT-PCR from patient samples.............................................................................................................................139 5-5 Ct and fold change calculations for gra nulocyte qRT-PCR from patient samples..... 142 5-6 Ct and fold change calculations for gra nulocyte qRT-PCR from patient samples..... 144 5-7 Linear regression for corr elations between RA patient RT-PCR data for various parame ters and TNF.........................................................................................................146
12 5-8 R, R-square, adj. R-square and standa rd deviation for corre lations between RA patient RT-PCR data for various parame ters and TNF.................................................... 147 5-9 ANOVA table for correlations between RA patient RT-PCR data for various parame ters and TNF.........................................................................................................148 5-10 MARCKS and TNF protein le vels in patient samples as determ ined by ELISA............ 149
13 LIST OF FIGURES Figure page 1-1 MARCKS cycles between the plasma membrane and the cytosol.................................... 40 2-1 Amino acid sequence of muri ne MARCKS (Accession # NP_032564). .......................... 53 2-2 The basic PSD of MARCKS may be masked by interacting w ith the acidic termini of MARCKS...........................................................................................................................54 2-3 EDC coupling reaction for negative charge neutralization on MARCKS......................... 55 2-4 Effect of EDC neutralization of negative charges on MARCKS on actin polymerization. ................................................................................................................ ..56 2-5 Effect of EDC neutralization of negative charges on MARCKS on actin depolymerization............................................................................................................... .57 2-6 Effect of EDC neutralization of nega tive charges on MARCK S on F-actin binding........ 58 2-7 Effect of EDC neutralization of ne gative charges on MARCKS on calmodulin binding ...............................................................................................................................59 2-8 Amino acid sequence of MARCKS truncation proteins.................................................... 60 2-9 Schematic diagram of MARCKS truncations.................................................................... 61 2-10 Novagens pET-9a bacterial expression vector................................................................. 62 2-11 Effect of MARCKS truncations and PSD on F-actin bundling......................................... 63 2-12 Coomassie stained native gel of MARCK S proteins with actin and rhodami nelabeled PSD........................................................................................................................64 2-13 Actin high speed pelleting a ssay with MARCKS truncations........................................... 65 2-14 Effect of MARCKS truncations on the time course of actin polymerization.................... 66 3-1 Peptide coverage for salt fractions of Asp-N digested MARCKS eluted from biotinPSD colum n.......................................................................................................................83 3-2 Peptide coverage for salt fractions of GluC digested MARCKS eluted from biotinPSD column .......................................................................................................................84 3-3 Direct binding fluorescence anisotro py of MARCKS N-term inus to Rh-PSD.................86 3-4 Direct binding fluorescence anisotropy of MARCKS C-term inus to Rh-PSD..................87
14 3-5 Fluorescence anisotropy competition with MARCKS N-terminus, Rh-PSD and unlabeled PSD....................................................................................................................88 3-6 Fluorescence anisotropy competition with MARCKS C-terminus, Rh-PSD and unlabeled PSD.................................................................................................................. ..89 3-7 Overlay of N-and C-terminus anisotropy and competition d ata........................................ 90 3-8 UV photo of 7% native Tris -tricine polyacrylamide gel of MARCKS proteins with actin and rhodami ne-labeled PSD...................................................................................... 91 3-9 UV photo of 1% native Tris -tricine agarose gel of MARC KS proteins with actin and rhodam ine-labeled PSD.....................................................................................................92 3-10 MARCKS N-terminus forms oligomers in a native gel..................................................... 93 5-1 Histopaque double-gradient for leuko cyte isolation from whol e blood.......................... 151 5-2 Correlation between mRNA levels of TNFand other proteins in mononuclear cells .. 152 5-3 Correlation between mRNA levels of TNFand other proteins in granulocytes.......... 154 5-4 TNF serum protein levels of different patient groups as determined by quantitative sandwich ELISA ..............................................................................................................156 5-5 MARCKS cellular protein levels of di fferent patient groups as determined by quantitative sandwich ELISA .......................................................................................... 157
15 LIST OF ABBREVIATIONS Ab Antibody ANOVA Analysis Of Variance AP Alkaline Phosphatase AspN Endoproteinase Asp-N ATP Adenosine 5' -Triphosphate BSA Bovine Serum Albumin Ca2+ Calcium CaCl2 Calcium Chloride CaM Calmodulin CCL-2 Chemokine (C-C Motif) Ligand 2 Cdk5 Cyclin-Dependent Kinase-5 cDNA Complimentary Deoxyribonucleic Acid Ct Threshold cycle C-term. MARCKS C-Terminus Protein Da Dalton DEAE Diethylaminoethyl DFP Diisopropylfluorophosphate DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid DNase I Deoxyribonuclease I DTT Dithiothreitol ED Effector Domain EDC 1-Ethyl-3-(3-Dimethylaminopropyl ) Carbodiimide Hydrochloride EDTA Ethylenediaminetetraacetic Acid
16 EGTA Ethylene Glycol Tetraacetic Acid ELISA Enzyme-Linked Immunosorbent Assay ERK Externally Regulated Kinases ESI Electrospray Ionization GluC Endoproteinase Glu-C HAP Hydroxy Apatite HCl Hydrogen Chloride HPLC High Pressure/Perform ance Liquid Chromatography IgG Immunoglobulin G IL-17 Interleukin-17 IL-1 Interleukin-1 Beta IPTG Isopropyl -D-1-Thiogalactopyranoside IRB Institutional Review Board JRA Juvenile Rheumatoid Arthritis KCl Potassium Chloride Kd Dissociation Constant kDA kiloDalton Kl Dissociation Constant of Labeled PSD Ku Dissociation Constant of Unlabeled PSD LC Liquid Chromatography LPA Lysophosphatidic Acid LPS Lipopolysaccharide mAb Monoclonal Antibody MALDI Matrix-Assisted Laser Desorption/Ionization MAP Mitogen-Activated Protein Kinase
17 MARCKS Myristoylated Alanine Rich C Kinase Substrate MCP-1 Monocyte Chemotactic Protein-1 MCTD Multiple Connective Tissue Disorder MES 2-( N -morpholino) Ethanesulfonic Acid MgCl2 Magnesium Chloride MH2 MARCKS Homology 2 Domain MIANS 2-(4-Maleimidoanilino) Naphthalene-6-Sulfonic Acid MRI Magnetic Resonance Imaging mRNA Messenger Ribonucleic Acid MS Mass Spectrometry MTX Methotrexate MW Molecular Weight NaN3 Sodium Azide NF B Nuclear Factor Kappa B N-term. MARCKS N-Terminus Protein OD Optical Density PAGE Polyacrylamide Gel Electrophoresis PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction pI Isoelectric Point PIP2 Phosphotidylinositol 4,5-Bisphosphate PMA Phorbol 12-Myristate 13-Acetate PMSF Phenylmethanesulphonylfluoride or Phenylmethylsulphonyl Fluoride Pred Prednisone PRK1 PKC-Related Kinase 1
18 PSD Phosphorylation Site Domain R Correlation RA Rheumatoid Arthritis RASF Rheumatoid Arthri tis Synovial Fibroblasts Rh-PSD Tetramethylrhodamine-5-Maleimide-Labeled PSD RIPA Radio-Immunoprecipitation Assay RNA Ribonucleic Acid RNase Ribonuclease RT Room Temperature RT-PCR Reverse Transcriptase-Polymerase Chain Reaction Sav-HRP Streptavidin-Horseradish Peroxidase SDS Sodium Dodecyl Sulphate SDS-PAGE Sodium Dodecyl Sulphate -Polyacrylamide Gel Electrophoresis SLE Systemic Lupus Erythematosus SOC Super Optimal Broth, C=catabolite repression TBS Tris-Buffered Saline TNFTumor Necrosis Factor-Alpha TNFR1/2 Tumor Necrsosis Factor Receptor 1 or 2 TPKII Tau Protein Kinase II Tris Tris Hydroxymethylaminoethane Und Undetermined UV Ultraviolet
19 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INTRAMOLECULAR INTERACTIONS OF MARCKS AND ITS RELATIONSHIP TO TUMOR NECROSIS FACTOR-ALPHA AND RHEUMATOID ARTHRITIS By Iman Mohammad Ashraf Al-Naggar May 2008 Chair: Michael R. Bubb Cochair: Arthur S. Edison Major: Medical Sciences--Biochemistry and Molecular Biology The myristoylated alanine-rich C kinase substrate, MARCKS, is a natively unfolded protein that plays important roles in multiple cellular processes, e.g., regulation of brain development, regulation of the actin cytoskel eton, control of lipid second messengers (e.g., phosphatidylinositol bisphosphate, PIP2), cellular migration and adhesion, endo-, exoand phago-cytosis, neurosecretion, maintenance of de ndritic spine morphology, regulation of growth cone adhesion and pathfinding, regulation of ai rway mucin secretion, neurite initiation and myoblast migration. MARCKS contains a central effector domai n called the Phosphorylation Site Domain (PSD), which is the site of interaction with its binding partners such as F-actin and Ca2+calmodulin, as well as its site of phosphorylation by protein kinase C (PKC). In our experiments using a non-myristoylated recombinant MARCKS pr otein, we found that th e protein repeatedly failed to bind to and bundle F-actin filaments and binds Ca2+-calmodulin with lower affinity than reported in the literature. We have shown that intramolecular interactions occurring between the basic PSD of MARCKS and its acidic termini ar e masking the PSD, thus making it unavailable to interact with its binding part ners. Charge neutralization assays suggest that these interactions
20 may be ionic in nature, which is further s upported by the unique charge distribution of the protein. We hypothesize that post-translationa l modifications known to occur at the termini flanking the PSD may disrupt these intramolecular interactions, activating the protein in vivo. A series of fluorescence anisotropy and native PA GE experiments using constructs of MARCKS missing either end of the protein have shown th at such intramolecular interactions may occur with either end of the molecule. Mass spectro metric analysis has narro wed these possible sites of interaction on the protein. In addition, we wished to elucidate the uti lity of MARCKS as a marker for diagnosis and prognosis in rheumatoid arthritis (RA). Th e striking upregulation in MARCKS expression previously reported following TNFstimulation of immune cells and the important role this cytokine plays in RA, where the mo st successful therapy today is TNFblockade, prompted us to wonder whether MARCKS has a cau sal role in this disease. Finally, we wanted to look at the role MARCKS plays in the TNFsignal transduction pathway. Microarray evidence suggests that MA RCKS may play an essential role in TNFinduced apoptosis.
21 CHAPTER 1 INTRODUCTION MARCKS The my ristoylated, alanine-rich C kinase substrate, MARCKS, was first characterized in rat cerebral cortex synaptosomes and in quies cent 3T3 cells as a major substrate for Protein Kinase C, PKC [1, 2]. In humans, it is composed of 332 amino acids (MW 31.6 kDa) and in mice it has only 309 amino acids (MW 29.6 kDa). MARCKS is also found in Xenopus laevis chicken, rats, cows, monkeys and in Pacific elect ric rays. MARCKS is a ubiquitously expressed protein, highest in brain, splee n, and lung, moderate in testis, pancreas, adrenal, kidney, and liver, and lowest in heart and skeletal muscle . It is a very aci dic protein (pI 4.2-4.7), composed mainly of alanines, glutamic ac ids, glycines, prolines and serines. MARCKS contains three highly conserved regions: an N-terminal myristoylation consensus sequence, an MH2 domain (MARCKS homology 2 domain) close to the N-terminus which resembles the cytoplasmic tail of the cation-independent mannose-6-phosphate receptor, whose function is unknown and is the only site of intron-splicing, and a central, highly basic phosphorylation site domain. This latter domain, composed of 25 amino acids of which 13 are positively charged (12 Lysines and 1 Arginine), contains two actin-binding sites , a Ca2+calmodulin binding domain , as well as 4 serine residues that are substrates for phosphorylation by PKC , Protein kinase C-Rela ted Kinase (PRK1, ) and Rho-associated kinase  and plays a major role in phospholipid binding . The PSD was also found to be ADP-ribosylated [10, 11]. The structure of MARCKS is best described as natively unfolded . Uversky  has defined natively unfolded proteins as bei ng extremely flexible, essentially noncompact (extended), and [having] little or no ordered s econdary structure under ph ysiological conditions.
22 The extent to which MARCKS is flexible is unk nown. These proteins are described as having large net charges at neutral pH, a low conten t of hydrophobic amino acid residues [14, 15], and are enriched in E, K, R, G, Q, S, P and A resi dues . The intrinsic di sorder of proteins is believed to allow greater plasticity, which allows them to interact efficiently with several targets and thus mediate crosstalk between different cellular processes and signal transduction pathways. Their increased sensitivity to prot eases allows their fast turnover and thus increased regulation. MARCKS is an acidic protein, with only 6 phenylalanines, 5 of wh ich are in the PSD and anchor MARCKS to the plasma membrane, no tyrosine or tryptophan residues, and is made of almost 30% alanine residues and 20% acidic residues (51 glutamic acids and 8 aspartic acids in murine MARCKS, which give MARCKS its aci dic nature) that are equally di stributed in the protein, but completely absent from its effector domain, 34 prolines, 32 glycines, 30 serines and 23 lysines (12 of which are in the PSD, which has a charge of +13). It was s hown by electron microscopy that it is a rod-shaped, elongated molecule with dimensions of about 4.5nm x 36nm  and this was confirmed by CD studies, which showed little or no helix although sequence analysis showed -helical propensity . The protein was found to barely form any secondary and absolutely no tertiary structur e in solution, or when bound to Ca2+-calmodulin [18, 19] or membrane-bound [20, 21]. Following phosphorylation by PKC in the PSD, both full-length MARCKS [17, 20] and its PSD  have been f ound to become a little more compact, but still unstructured. The lack of structure in MARCK S has been the rationale for studies employing a peptide that corresponds to the effector domain of MARCKS. MARCKS exists both in a membrane-bound and cytosolic form. MARCKS binds reversibly to the inner leaflet of the plasma membrane. This inte raction involves both the myristoyl group (a co-translational lipid m odification which adds myristic acid, a C14 saturated
23 fatty acid to the N-terminal gl ycine of MARCKS via an amid e bond to the amino group of the glycine), which inserts itself into the hydrophobic co re of the lipid bilaye r and the electrostatic interaction of its basic domain, the PS D, with acidic phospholipids, namely PIP2 [23-25]. Finally, penetration of the side chains of the five phenylalanine residues found in the PSD into the hydrophobic core of the bilaye r also aids in anchoring MARC KS to the plasma membrane . None of the above -mentioned interactions on its own, how ever, is sufficient to successfully anchor MARCKS to the plasma membrane. This has been named the myristoyl/electrostatic switch model. Phosphorylation of the PSD (as by PKC; ) and actin or Ca2+-calmodulin binding all cause MARCKS to localize to the cytosol. MARCKS Functions MARCKS has unusual biochemi cal properties, whic h make it able to interact with various molecules, and so many functions have been pr oposed for MARCKS. It was found to bind and crosslink F-actin filaments  and so is thought to be an actin crosslinking protein. Accordingly, roles in remodeling of the actin cytoskeleton and affecting cytoskeletal processes such as endocytosis [28, 29], exocytosis [30, 31], phagocytosis and m acropinocytosis [32, 33] control of cell morphology, motility, adhesion, protrusive activity and cortical actin formation in embryonic cells  have been suggested. MARCKS phosphorylation leads to cortical F-actin disassembly and potentiation of secretion in chro maffin cells . MARCKS has also been implicated in inhibition of adherence to extrace llular matrix proteins affecting cell migration , regulation of integrin-med iated muscle cell spreading [3 7, 38], and inbition of myoblast migration [39-41]. Actin binding is preven ted by both phosphorylation of the PSD and Ca2+calmodulin binding. It binds Ca2+-calmodulin and this inte raction is abolished by phosphorylation of MARCKS by PKC , and is thus thought to pl ay a PKC-dependent role in regulating the avai lability of Ca2+-calmodulin for other processes . MARCKS also binds and
24 sequesters PIP2 with high affinity, reversibly inhibiting phospholipase C [25, 43-48] and is hypothesized to play a role in the regulation of PIP2 availability and regu lation and activation of phospholipase D [49-51]. It was also shown to play a role in tumor suppr ession [52-56], learning and synaptic plasticity , and neurosecretion . MARCKS also regulates growth cone adhesion and pathfinding , plays a role in mu cin secretion in human airway epithelial cells [60-63], is involved in neurite initiation and main taining of dendritic sp ine morphology [64-67], Knockout studies of MARCKS in mice show that MARCKS plays a vital role in the normal developmental processes of neurulation, hemi sphere fusion, forebrain commissure formation, and formation of cortical and retinal laminations regulation of brain development and postnatal survival , as well as the pathoph ysiology of mood disorders . MARCKS is also involved in inflammation and ne uropathic pain where it is a substrate of Rho-kinase . It was repor ted that MARCKS is the major protein produced in response to treatment of neutrophils and m acrophages with the cytokine TNF, i.e. 90% of protein synthesized by these cells in respon se to this inflammatory cytoki ne is MARCKS . It also stimulates MARCKS synthesis in the human pr omyelocytic leukemia cell line HL-60 and the human myeloid leukemia cell line U 937 . Harlan et al. identifie d promoter regions within the MARCKS gene which contained multiple transcri ption initiation sites, including a potential NF B transcription start site, which is shown to be activated by TNF[73, 74]. The specific role MARCKS plays as a TNFeffector has not been elucidated. However, changes in the actin cytoskeleton and cell morphology following cell treatment with TNF[75, 76], are very similar to effects of MARCKS on the actin cytoskeleton and hence it is probable that MARCKS plays a role in mediating these effects. This becomes important, as TNFseems to serve as a mediator in various pathologies such as septic shock, cancer, AIDS, transplantation rejection, multiple
25 sclerosis, diabetes, rheumatoid arthritis, trauma, malaria, meningitis, ischemia-reperfusion injury, and adult respiratory distress syndrome. In rheumatoid arthritis, TNFblockade has recently emerged as one of the most potent therapies. Similar effects on MARCKS protein levels are observed with lipopolysaccharide (LPS) treatment of BV-2 microglial cells, and this effect is inhibited by treating cells with inhibitors of NF B, a transcription fact or downstream of both TNFand LPS . This suggests that MARCKS s ynthesis is tightly regulated. However, to our surprise, prior published results show that this up-regulation of MARCKS mRNA synthesis is not apparent in microarray data of cells following TNFtreatment (Table 1-1), suggesting that TNFmight employ a mechanism other than increasing transcription to achieve high protein levels in the cell or that the pathway may be important only in sele cted cell types such as monocytes and macrophages. These could range from stabilizing MARCKS mRNA, to stabilizing the protein itself through either binding by other molecules or inhibition/downregulation of degradation pathways. The MA RCKS mRNA has a half life of 14 hours in quiescent fibroblasts and this is reduced to 2 ho urs upon stimulation of cells with phorbol esters that activate PKC or with growth factors [78, 79]. In fact, it has been shown that the 3`untranslated region of the MA RCKS mRNA contains CU-rich cis -elements that destabilize the MARCKS transcript . This instability is reversed by binding by the mRNA binding proteins of the Hu family, HuD and HuR. In contrast to fibroblasts, in neuron cells that have high levels of HuD and HuR, the MARCKS transcript is well stabilized and its half life does not change upon PKC activation. Major Post-Translational Modifications MARCKS Phosphorylation Initially, MARCKS was believed to be subs trate to phosphorylation by protein kinase C only [42, 81]. Phosphorylation by PKC in the PSD of MARCKS regulates many aspects of
26 MARCKS interactions: it abrogate s its binding to the plasma membrane [82, 83] and PIP2 , making it dislocate to the cytosol, it prev ents it from binding to F-actin  and Ca2+-calmodulin  and it protects it from cleavage by an unidentif ied cysteine protease [ 84] and by the cysteine protease Cathepsin B . It is clear that phosphorylation of MARCKS by PKC in the PSD is one of the most important post-translational modi fication for regulation of the functions of this protein. Palmer et al.  showed that the PKC -related kinase (PRK1) also phosphorylates MARCKS at serines 152, 156 and 163, which corr espond to the PKC-phosphorylated sites of MARCKS, giving it an extra form of regulat ion in different cell signaling pathways. Later it was shown that MARCKS can be a subs trate for other kinases. Taniguchi et al.  showed it to be an in vivo substrate of prolinedirected protein kinases, such as mitogenactivated protein (MAP) kinase or Cyclin-dependent kinase-5 (Cdk5) (which are abundant in brain and phosphorylate similar sequences) using a reverse-ph ase capillary high performance liquid chromatography separation of lysyl endo protease digested calf brain MARCKS followed by electrospray mass spectroscopy (LC/MS) determination of fragment sizes and phosphorylation and Edman degradation to dete rmine sites of phosphorylation. Of seven phosphorylated serine residues they iden tified (bovine MARCKS Ser 27, 46, 81, 100, 117, 134 and 158), only one was in the PSD of MARCKS, wh ile the other six were N-terminal to the PSD and all were followed by a proline residue. Using the more powerful electrospray ionization/ion trap mass spectrometry, they later identified two more phosphorylation sites on the C-terminus of MARCKS in rat brain (Ser291 and Ser299) that are also thought to be substrates for prolinedirected kinases because they are also immediately followed by prolines [ 87] and speculated that, although the C-terminus has no known function, it is regulated by phosphoryltion. Yamamoto et
27 al.  found MARCKS to be phosphorylated by cdc2 kinase and tau protein kinase II (TPKII) in cytosolic fractions of rat br ains. Phosphorylation sites were found to be completely different from PKC phosphorylation site s and occurred on both serine and threonine residues on MARCKS, and phosphorylation increas ed binding to CaM. Finally, Manenti et al.  found that MARCKS was phosphorylated in vitro by Cdk2 and Cdk4, and to a lesser degree, Cdk1. They determined the phosphorylation sites using el ectrospray MS after di gestion of the protein, and found Ser27 and Thr150 to be phosphorylated by cyclin E-Cdk2. When MARCKS was first phosphorylated by PKC, the initial rate of phosphorylation by Cdk2 was improved without modifying the number of sites concerned. Schnwa er et al.  showed that although MA RCKS is phosphorylated at Ser-113 (in mouse, equivalent to Ser-116 in human and cow MARCKS) in vitro by p42 MAP kinase, it is not phosphorylated by MAP kinase in vivo in permeabilized Swiss 3T3 cells after stimulation with platelet-derived growth factor or PMA even though p42 MAP kinase was shown to be active in these cells, and that PKC activation induced MARC KS phosphorylation at the PKC sites. Ohmitsu et al.  showed that MARCKS is phosphorylated by MAP kinase on Ser-113 in rat hippocampal neurons following stimulation of the glutamate receptor, in addition to phosphorylation by PKC on the PKC sites (Ser 152, 156, 163), which was transient. They also showed that phosphorylation of MARCKS at Se r113 by MAP Kinase reduced its CaM binding ability to 75% of control, while binding of F-actin was abolis hed by this phosphorylation, to an extent comparable to that of PKC phosphorylation of the PSD, suggesting that MAP kinase can functionally regulate the propertie s of MARCKS, especially in its interaction with F-actin. Hasegawa et al.  showed that MARCKS is phosphorylated in rat microglia by ERKs (ERK-1
28 and ERK-2, members of the MAPK family of kinases) following stimulation by the amyloid protein. In addition to phosphorylation with PKC, MAP kinases and Cdks, MARCKS was found to be a substrate of Rho-kina se in human neuronal cells in response to stimulation with lysophosphatidic acid (LPA) . Serine 159, wh ich also corresponds to a PKC phosphorylation site, was found to be a substrate for Rho-associated kinase both in vitro and in vivo in these human neuronal teratoma cells, NT-2. The sa me residue, Ser159 was also found to be a substrate for protein kinase A (PKA) in vitro in these cells. These phosphorylation sites were recognized using a phosphoryla tion site-specific antibody ag ainst Ser159-phospho-MARCKS (pS159-Mar-Ab). This provides additional regulation of MARCKS by a different signal transduction pathway, which doesnt involve PKC activation, the Rho/Rho-kinase pathway. MARCKS Proteolysis Because of its importan t cellular functions the regulation of MARCKS concentrations in cells is important. The MARCKS gene is unde r multiple modes of tran scriptional control, including cytokine-and transfor mation-dependent, cell-specific, and developmental regulation. In general, MARCKS protein conc entrations are reported to clos ely parallel its mRNA levels, which in turn can be regulated at the level of gene transcription by several factors. For example, as described previously, it was reported that TNFand LPS can cause dramatic increases in the levels of MARCKS mRNA and protein in neutrophils, macrophages, and related cells [71, 72]. MARCKS expression is also severe ly decreased in cells transfor med with a variety of oncogenes [55, 56, 93, 94]. For example, in vsrc -transformed murine fibroblasts, MARCKS transcription is down-regulated by 68% compared with untransformed cells, and inhibition of v-src -tyrosine kinase activity restores MARCK S mRNA levels to normal, suggesting that the reduced MARCKS mRNA levels are a direct effect of vsrc activity . MARCKS is also tightly
29 transcriptionally regulated during mouse develo pment  and this was shown to be under control of CBF/NF-Y/CP-1-like and Sp1-like transcription factors in Xenopus laevis . In addition to transcriptional regulation of MARCKS levels, specific proteolytic cleavage by multiple kinases may play a role in this regulation. Spizz et al.  first described cleavage of MARCKS by an unknown cysteine protease in to two major fragments between asaparagine 147 and glutamate 148 (3 amino acids N-terminal to the PSD) in human foreskin fibroblasts. They found, however, that only unphosphorylated MARCKS was a substrate for this protease and that PKC-phosphorylation of the PSD protected MARCKS from this cleavage. This could be a mechanism for strict regula tion of MARCKS protein levels in these cells. A year later, Spizz et al.  described another cysteine protease that cl eaved MARCKS in a pH-dependent manner and fragments associated with lysoso mal fractions, suggesting it was a lysosomal cathepsin, and was identified as Cathepsin B in human fibroblasts. Manen ti et al.  showed that the same cleavage of MA RCKS between Asn147 and Glu148 al so occurs in bovine brain, and might be a general mechanism for dow n-regulation of MARCKS activity. More interestingly, Braun et al.  have f ound that macrophages contain a protease which specifically cleaves human MARCKS expr essed in a cell-free system or in E. coli between Lys-6 and Thr-7, and that this cleavage is myristoylation dependent, i.e. unmyristoylated MARCKS is not recognized or cleaved by these proteases. This cleavage of the Nterminus of MARCKS represents an important mechanism of demyristoy lation of MARCKS, which in turn regulates it association with the plasma memb rane and binding to other protei ns, as Matsubara et al.  have demonstrated that the myristoyl group play s a role in calmodulin binding to MARCKS. Finally, it was shown by Dulong et al.  both in vitro and in vivo in myoblasts that the Ca2+dependent cysteine protease calpain cleaves MARCKS and this cleavage is dependent on the
30 phosphorylation of MARCKS by PKC. Interestingly, they showed that calpain cleavage of MARCKS before myoblast fusion was required for th e process to occur, inferring that cleavage of MARCKS may lead to physiolo gically active fragments rather than just down-regulate the protein. Tumor Necrosis Factor Tumor necrosis factor (TNF, TNF, cachectin ) is a pleiotropic pro-inflammatory cytokine that exerts multiple biologic effects. It is synthesized as a 26-kDa type II transmembrane precursor that is displayed on the plasma membra ne, with the N-terminus in the cytoplasm and the C-terminus exposed to the extracellular space. The TNF precursor is proteolytically cleaved by TNF alpha converting enzyme (TACE) to yiel d a biologically active 17-kDa mature TNF . Mature TNF monomers self associate to form a homotrimer . TNF mediates its effect through two different but structura lly homologous TNF receptors: TNFR1 and TNFR2 (both are type I transmembrane glycoproteins). These TNF receptors are present on all types of cells except red blood cells . TNFR1 is considered to be responsible for most biologic actions of TNF [104, 105]. TNF is produced by numerous immune cells (m acrophages/monocytes, na tural killer cells, Kupffer cells, B cells, T cells, basophils, eosinop hils, glial cells, mast cells) as well as nonimmune cells (astrocytes, granulose cells, osteoblasts, cardiac myocytes, fibroblasts, keratinocytes, neurons, neutrophils, T cells, retinal pigment epithelial cells, smooth muscle cells, spermatogenic cells, tumor cells). It is produced in response to an a ssortment of activating stimuli including LPS, antibodies to LFA-3, calcium ionophores, C5a, CD44, CD45, enterotoxin, GM-CSF, hypoxia, IL-1, leukotrienes, mellitin, MIP-1 nitric oxide, oxygen radicals, parasites, phorbol esters, synthetic lipid A, TNF, toxic shock toxin-1, viruses and irradiation .
31 TNF has many functions, including promo ting synthesis of: adhesion molecules, proinflammatory cytokines, chemokines, MMP s, RANK ligand expression, promotion of angiogenesis, activation of cells (T-cells, B-cells, macrophages) and antiviral and antitumor effects . At the signaling level, TNF indu ces several responses including the activation of phospholipases (A2 and C) and acidic sphingomyelinases which in turn generate second messengers such as diacylglycerol and ceramide . TNF also activat es three MAP Kinase cascades, JUN N-terminal Kinase (JNK), caspase cascades, and several transc ription factors such as NF B. TNF is a potent modulator of the actin cytosk eleton in various cell t ypes. It induces Factin depolymerization and actin s ynthesis in epithelial cells and results in vascular leakiness [109, 110]. In human umbilical vein endothelial cells (HUVECs), TNF induces the transient increase in F-actin, the reorganization of the ac tin cytoskeleton, formation of membrane ruffles, filopodia and actin stress fibers leading to cell retraction and forma tion of intercellular gaps. These effects were found to be medi ated by members of the Rho family of small GTPases, Rho, Rac and Cdc42 in endothelial ce lls. In macrophages, where TNF stimulates many responses including migration, TNF was shown to decrease levels of F-actin and inhibit Cdc42-mediated filopodium extension. In mouse fibroblasts, TNF triggers Cdc42 act ivation leading to filopodia and lamellipodia formation, which then disappear, leaving onl y stress fibers behind . TNF affects chemotaxis and motility in macrophages , fibroblasts , Langerhans cells , epidermal keratinocytes , and neutrophils [7 6, 117]. In glomerular epithelial cells, TNF induces actin polymerization a nd redistribution and focal adhesions, through phosphorylation of vinculin, paxillin and focal adhesion kinase, a ffecting how these cells contact the glomerular
32 capillary basement membrane . In pulm onary endothelial cells, TNF-induced apoptosis was shown to require rearrangement of the ac tin cytoskeleton, which was mediated by Rhokinase . In contrast, TNF was found to elic it antiapoptotic effects in opossum kidney cells via redistribution of the actin cy toskeleton through inhi bition of caspase-3; th is was governed by the phosphatidylinositol-3 kinase Cdc42/Rac1, and phospholipase1 . TNF induces signaling events in lung endothelial cells that result in cytoskelet al changes and increases in EC permeability; these changes are mediated through phosphorylation of ERM proteins (ezrin, radixin, and moesin) by protein ki nase C (PKC) . In mouse embryonic fibroblasts, the WDrepeat protein factor associated with nSMase activity (FAN), a member of the family of TNF receptor adaptor proteins that are coupled to specific signaling cascades, was found to be essential for TNF-induced actin cytoskeletal changes, reorganization and filopodium formation, and that this required the presence of the PH domain which localizes FAN to the plasma membrane by binding specifically to phos phatidylinositol-4,5-bisphosphate (PIP2) . Rheumatoid Arthritis RA is an immunologically me diated chroni c inflammatory diseas e of unknown etiology. It is characterized by synovial cell proliferati on and inflammation with destruction of adjacent articular tissue . RA has a prevalence of 1% worldwide; it is believed to be the most common, potentially treatable cause of disability in the Western world . The consequences of RA can vary from hardly any impairment to severe disease with continuing high disease activity and progressive joint destruction resulting in severe loss of function and increased mortality [125-127]. It can be rapidly destructive, with 60% of patients havi ng erosions of joints seen on radiographs within 2 years of disease onset [128, 129]. RA patients suffer from a loss of functional ability leading to an inability to work, causing an economic burden to society .
33 As early as 1953, patients with RA were shown to have an approximate 10-year premature mortality . Despite extensive research, th e cause of RA is unknown. It is believed that it is multifactorial, with genetic and environmental factors playing important roles. The prevailing view is that RA is mediated by antigen-activated T cells that infiltrate the synovial membrane [131, 132], which leads to a series of inflammatory processes, resulting in vascular and synovial cell proliferation with resorp tion of cartilage and bone. Non-immunologic pathways also probably contribute to tissue inju ry and destruction in established RA . Degradation of articular extracellular matrix comp onents is a hallmark of RA and is largely mediated by matrix metalloproteinases (MMPs). RA is strongly associated with MHC class II allele HLA-DR4, and to a lesser extent to HLA-DR-a and DR14. Ac tivated T cells, macrophages, and fibroblasts produce proinflammatory cytokines that play a key ro le in synovitis and tissue destruction in RA. TNF and IL-1 are two of the main cytokines that enhance synovial pro liferation and stimulate secretion of MMPs, other in flammatory cytokines, and adhesion molecules . There is no good early definition of RA. RA is usually defined by the ACR criteria , but it is well known that these criteria are not optimal to disti nguish early RA from undifferentiated (and sometimes self-limited) poly-a rthritis and early mani festations of other auto-immune diseases such as post-viral arth ropathies, early spondyloa rthropathy, and other, self-limiting arthrides that may satisfy the 1987 ACR RA criteria . Current classification criteria, which rely on the presence of clin ical signs and symptoms and laboratory and radiographic findings, lack the ability to differe ntiate RA from similar rheumatic conditions, especially in elderly persons [ 136]. Patients in the early stages of disease and those who have mild RA may not be correctly id entified using these criteria.
34 Immunologic events in RA occu r many years before the onset of clinical disease. Rheumatoid Factor has been shown in patients years before the onset of symptoms . Similarly, anti-cyclic citrullinated peptide antib odies precede disease by 14 years and precede the detection of RF by an average 2.8 years . Increases in highly sensitive C-reactive protein (CRP) have been shown before onset of clinical disease in patients with or without serologic abnormalities . When serologi c events occur in the presen ce of the appropriate genetic environment, disease activation would seem even more likely. In addition, environmental factors are important, particularly heavy smoking, which is associat ed with an increased risk of development of seropositive RA [140, 141]. Th ese data support the hypothesis that the activation of RA is multifactorial with autoimmu ne and genetic factors important, most likely in conjunction with appropriate environmental stimuli. Complementary to the serologic changes detected before clinically manifest disease, im aging and arthroscopy detect synovitis in clinically normal joints. Ultrasound and MRI are able to show the presence of synovitis in patients with early RA in joints with a norma l clinical examination [142-144]. As described, disease may predate symptoms by many years. There is considerable evidence that radiographic damage, loss of func tion , and loss of bone mineral density [146, 147], occur early in the disease pr ocess. In early RA (<6 months of symptoms), 40% of patients have erosive disease at presentation . Even in an early synovitis clinic that attracts patients at the earliest stages of disease, 25% of patients have radiographic erosions at presentation . New imaging techniques show bone changes occur even earlier than was first thought. Bone edema, the MRI precursor to erosions, can be s een in patients after only 4 weeks of symptoms . Ultrasound can show erosions before they are evid ent on plain radiography .
35 Not only does damage occur early, but also revers ibility of functional loss may be lost with time. Patients treated less than 2 years from disease onset showed a significant improvement in function, using the Health Assessment Questionnai re, after intervention co mpared with patients treated beyond this time point in a study of 440 patients . In a review of 11 different studies, Anderson et al  showed that dis ease duration was paramount in predicting response to DMARD therapy. Of patients, 53% presentin g with less than 1 years disease duration showed a response, whereas later groups showed di minished responses with time. Similarly, in a study of 448 RA patients, the patients who presen ted with less than 5 years disease duration maintained a lower mortality ratio over 21.5 years of follow up compared with late presenters . A commonsense approach to the management of a persistent, progressive, damaging condition such as RA would seem to be intervention before the onset of damage, at a stage when disease still may be reversible. Such a phase of disease has been described as a window of opportunity for intervention, a peri od in early-stage RA during wh ich the progression rate of joint damage is set, and th erapeutic interventions can ex ert maximum effects . Unfortunately, due to the lack of a sensitive and specific marker for early RA, this window is often missed. With better understanding of the pathogenesi s of autoimmune diseases and advancing developments in biopharmaceutical technology, biologic therapeutic agents have been introduced. These agents target specific compon ents of the immune response considered central to the etiology of RA. Alt hough traditional DMARDs generally slow joint damage progression, the prevention of joint damage has become a re volutionary possibility, particularly with the biologics inhibiting TNF. These TNF inhibitors suppress disease activity directly and
36 powerfully and lower the disease burden significantly from the moment that treatment is started [155-157]. Due to its pleiotropic effects, TNF has been an attractive therapeuti c candidate and is felt to play a central role in the pathogenesis of RA. A number of lines of evidence that support this hypothesis are: The biologic activity of TNF can account for th e pathologic processe s contributing to RA (e.g., cell recruitment and ac tivation, synovial lining cell proliferation, increased prostaglandin, and matrix-degrading metall oproteinase activity as well as bone and cartilage destruction) ; TNF and TNF receptors are upregulated in rheumatoid synovium [159, 160]; Anti-TNF downregulates IL-1 and other proinflammatory cytokines in vitro ; When TNF is overexpressed in a transgenic m ouse model, it results in the development of a form of erosive arthritis similar to RA . Animal models of arthritis are ameliorated by anti-TNF . TNF blockade results in great clin ical benefit in RA [164-170]. It has been shown that a brief intervention early in the course of RA can reset radiologic progression rates during subsequent years independent of conseque nt therapy . In addition, delayed treatment trials have shown that a de lay of only 3 to 9 mont hs in starting DMARD therapy has a significant negative impact on radi ographic outcome 2 years later [172, 173]. Both these observations support the window of opportunity hypothesis. Becaus e the TNF inhibitors have been proved to stop joint damage progressi on in severe progressive RA, the achievements of these agents in early RA are currently of gr eat interest. Currently, there are three anti-TNF agents available for clinical use: infliximab, a chimeric anti TNF mAb; etanercept, a soluble TNF-receptor construct; and adalimumab, a human anti-TNF mAb. However, as expected for blocking an important immune cytokine, side effects are a c oncern and include a risk of increased infection (in particular TB and opportunistic infections), drug-induced SLE,
37 lymphomas autoantibody formation, demyelinating disease, injection site reactions, cytopenias and congestive heart failure [107, 174, 175]. Apoptosis Apoptosis is the norma l physiologic mechan ism by which cells commit suicide, also known as programmed cell death. It has evolved in multicellular organisms to allow for the elimination of cells in normal and pathologic settings. Apoptosis is crucial for normal development and tissue homeostasis and is subj ect to genetic control [176, 177]. Abnormalities in this important mechanism are associated with various disease states such as cancer [178, 179], autoimmunity [180, 181], and degenerative disord ers [182, 183]. Apoptosis can be triggered by intracellular and extracel lular events, and signaling occurs through different independent pathways, which converge on the activation of a fa mily of cysteine proteases (caspases), which subsequently cleave myriad cellular substrates Caspases can be activated directly by engagement of cell surface receptors (death re ceptors) such as Fas/CD95 or TNFR1, or by signals in response to intracellular damage or stre ss that are transmitted to the Bcl-2 family. In addition to caspases, there are three major func tional groups of molecule s involved in triggering and affecting the apoptotic process: adaptor prot eins (physically link ce ll death effectors and regulators by forming bridges between caspa ses and upstream regulators of apoptosis, controlling the activation of initiator caspases), members of the tumor necrosis factor receptor superfamily, and members of the Bcl-2 family of proteins. As described previously, TNF is a potent proinflammatory cytokine that can induce apoptosis . TNF also elicits antiapoptotic cell signals, leading to suppression of apoptosis, which is mostly dependent on nuclear factorB (NFB), and to inflammatory response [185-190]. It has also been shown that TNF activates cell survival signaling cascades th at result in the inhibition of apoptosis independently of NFB [191, 192]. Whether members of the TNFR supe rfamily will trigger proliferation, survival,
38 differentiation, or death depends on the cell type a nd the other signals that the cell receives . TNF can induce apoptosis by ligating the TNFR1. Once this receptor is ligated, its death domain (DD) motif recruits several adaptor pr oteins via homotypic domain interactions. These interactions allow caspase a ggregation and activation [196-198] which are also mediated by another domain found in adaptor molecules, the Death Effector Domain (DED). Initiator caspases (e.g., caspases 8 and 10) ar e first activated and start an avalanche of increasing caspase activity by processing and activati ng effector caspases (e.g., caspases 3 and 6) . Caspases then carry out cell killing by cleaving and inactiva ting certain vital cellular proteins such as DNA repair enzymes, lamin, gelsolin, MDM2 (inhibitor of p53), and protein kinase C [199, 200], or by directly or indirectly activati ng enzymes that go on to play furthe r roles in apoptosis .
39 Table 1-1. MARCKS does not appe ar to be upregulated by TNFin microarray data. # Cell type Effect of TNFon MARCKS mRNA Transcription Reference 1 Primary RA synovial fibroblasts (RASFs) Insignificant Change  2 Cultured primary human synoviocytes Insignificant Change  3 HeLa cells Showed results for genes upregulated 2.5 foldNo MARCKS data  4 Coronary artery endothelial cells and smooth muscle Showed results for differentially expressed genesNo MARCKS data  5 Hearts of TNFoverexpressing transgenic mice Showed results for top 50 upregulated genesNo MARCKS data  6 Human umbilical vein endothelial cells (HUVECs) Significant Increase in MARCKS mRNA expression  7 Human keratinocytes Showed resu lts for differentially expressed genesNo MARCKS data 
40 De-myristoylation, PSD Phosphorylation, F-actin or Ca2+-calmodulin binding -------------------------------C-terminus ------------N-terminus ----------++++PSD++++ Plasma Membrane Cytosol C-terminus ------------N-terminus ----------++PSD++ F-actin C-terminus ------------N-terminus ----------++PSD++ Ca2+-calmodulin C-terminus ------------N-terminus ----------++PSD++ P P P P De-myristoylation, PSD Phosphorylation, F-actin or Ca2+-calmodulin binding -------------------------------C-terminus ------------N-terminus ----------++++PSD++++ Plasma Membrane Cytosol -------------------------------------------------------------C-terminus ------------N-terminus ----------++++PSD++++ C-terminus ------------C-terminus ------------N-terminus ----------++++PSD++++ ++++PSD++++ Plasma Membrane Cytosol C-terminus ------------N-terminus ----------++PSD++ F-actin C-terminus ------------C-terminus ------------N-terminus ----------++PSD++ F-actin C-terminus ------------N-terminus ----------++PSD++ Ca2+-calmodulin C-terminus ------------C-terminus ------------N-terminus ----------++PSD++ ++PSD++ Ca2+-calmodulin C-terminus ------------N-terminus ----------++PSD++ P P P P C-terminus ------------N-terminus ----------++PSD++ P P P P P P P P Figure 1-1. MARCKS cycles between the plas ma membrane and the cytosol. MARCKS bi nds to the inner leaf of the plasma membrane via its N-terminal myristoyl group and its PSD. Upon PSD phosphorylation, de-m yristoylation, F-actin or Ca2+-calmodulin binding, this in teraction is abolished and MARCKS translocat es to the cytoplasm. Sticks with hexagon heads represent phenylalanine residues in the PSD.
41 MURINE MG*AQFSK TAAKGEATAERPGEAAVASSPSKANGQENGHVKVNGDASPAAAEPGAKEELQA 60 RAT MG*AQFSK TAAKGEAAAERPGEAAVASSPSKANGQENGHVKVNGDASPAAAEPGAKEELQA 60 BOVINE MG*AQFSK TAAKGEATAERPGEAAVASSPSKANGQENGHVKVNGDASPAAAEPGAKEELQA 60 HUMAN MG*AQFSK T AAKGEAAAERPGEAAVASSPSKANGQENGHVKVNGDASPAAAESGAKEELQA 60 Unknown protease cleavage site MURINE NGSAPAADKEEPASG-S-AATPAAAEKDE-AAAA-TEPGAGAADKEA-AEAEPAEPS S P114 RAT NGSAPAADKEEPASG--GAATPAAADKDE-AAAA-PEPGAATADKEA-AEAEPAEPGSP114 BOVINE NGSAPAADKEEPAAAGSGAASPAAAEKDEPAAAA-PDAGASPVEKEAPVEGEAAEPG S PT 119 HUMAN NGSAPAADKEEPAAAGSGAASPSAAEKGEPAAAAAPEAGASPVEKEAPAEGEAAEPGSPT 120 Calpain cleavage site MURINE AAEAEGASA-SSTS S PKAEDGAAPSPSS ETPKKKKKRFSFKKSFKLSGFSFKKSKKESGE 173 RAT SAETEGASA-SSTS SPKAEDGAAPSPSS ETPKKKKKRFSFKKSFKLSGFSFKKSKKEAGE 173 BOVINE AAEGEAASAASSTS SPKAEDGATPSPSN E TPKKKKKRFSFKKSFKLSGFSFKKNKKEAGE 179 HUMAN AAEGEAASAASSTS SPKAEDGATPSPSN E TPKKKKKRFSFKKSFKLSGFSFKKNKKEAGE 180 Unknown cysteine protease cleavage site MURINE GAEAEGAT---AEGAKDEAA----AAAGGEGAAAPGEQAGG------AGAEGAAGGEPRE 220 RAT GAEAEGAT---ADGAKDEAA----AAAGGDAAAAPGEQAGG------AGAEGAEGGESRE 220 BOVINE GGEAEGAAGASAEGGKDEASGGA-AAAAGEAGAAPGEPTAAPGEEAAAGEEGAAGGDPQE 238 HUMAN GGEAEAPA---AEGGKDEAAGGA-AAAAAEAGAASGEQAAAPGEEAAAGEEGAAGGDPQE 236 MURINE AEAAEPEQPEQPEQPAAEEPQAEEQSEAAGEK-AEEPAPGATAG--DASSAAGP----EQ 273 RAT AEAAEPEQPEQPEQPAAEEPRAEEPSEAVGEK-AEEPAPGATAD--DAPSAAGP----EQ 273 BOVINE AKPEEAAVAPEKPPASEEAKAVEEPS-KAEEK-AEEAGV--SAAGCEAPSAAGPGVPPEQ 294 HUMAN AKPQEAAVAPEKPPASDETKAAEEPS-KVEEKKAEEAGA--SAAACEAPSAAGPGAPPEQ 293 MURINE EA-PAATDEAAASAAPAAS--P--EPQPECSPEAPPAPTAE 309 RAT EA-PAATDEPAASAAPSAS--P--EPQPECSPEAPPAPVAE 309 BOVINE EAAPA--EEAAAAPASSACAAPSQEAQPECSPEAPPAEAAE 333 HUMAN EAAPA--EEPAAAAASSACAAPSQEAQPECSPEAPPAEAAE 332 Figure1-2. Known post-translati onal modifications of MARCKS. Myristoylated re sidues are marked by an asterisk, cleavage sites are underlined and residues separate d by a green vertical bar, residues expe rimentally shown to be phosphorylated are highlighted in yellow, and the N-glycosylation consensus sequ ence is boxed in red. The PSD is also reported to be ADPribosylated, but the exact positi on of this PTM is unknown. Ser27 and Thr150 highlighted in green are phosphorylated by E-Cdk2, Ser113 in pink by p42MAP Kinase, Ser in red are phosphorylated by PKC and PRK1; Ser159 in blue is also phosphorylated by PKA and RhoA Kinase. Serines in cyan are phosphorylated by proline-di rected protein kinases.
42 CHAPTER 2 INVESTIGATING INTRAMOLECULAR INTERACTIONS OF MARCKS Introduction MARCKS is a highly acidic protein (pI 4.2-4.6, [71, 208, 209]) with a central extrem ely basic domain (pI 11.61) called the Phosphorylation S ite Domain (PSD). The PSD is composed of 25 amino acids, 13 of which are positively charged (12 lysines and 1 arginine) and does not contain any acidic residues. In c ontrast, the rest of the protein is 21% acidic with 51 glutamic acids and 8 aspartic acids out of 309 amino acids in murine MARCKS. In this chapter, we will test the hypothesis th at intramolecular inter actions occur between the phosphorylation site domain (PSD, also known as effector domain, ED) of MARCKS and the rest of the protein. This hypot hesis is based on our observation that recombinant, full length non-myristoylated MARCKS doe s not bind F-actin and Ca2+-calmodulin in vitro as previously reported, [5, 17] and on the fact that MARCKS is an unstructured protein with a unique charge distribution (Figure 21). Why is the PSD unavailable to interact with MARCKS binding proteins in an unfolded, unstructu red protein? While this coul d simply be due to continued failure to produce active protein over the past few years using various different purification protocols, it is most likely mo re complicated than that. We hypothesize that ionic interactions betw een the acidic termini of MARCKS and the oppositely charged PSD, which is the site of mo st known interactions of MARCKS, mask the PSD thus making it unavailable to interact with MARCKS binding partners such as actin and calmodulin (Figure 2-2). Various experiments done in our lab have s hown that a synthetic rhodamine-labeled PSD peptide can bind to th e full-length protein, and competition assays suggest a single binding site fo r the PSD on the protein .
43 We will test our hypothesis in two ways; firs t, we will do a chemical neutralization of acidic charges on MARCKS (pulished work: Tapp H, Al-Naggar IM et al. JBC (2005) 280: 9946-9956); then, we will make truncations of the pr otein which will be missing either terminus but retain the full PSD; We will then test whethe r either of these modifications will render the protein more active in bi ochemical binding assays. Neutralization of Acidic Residues on MARCKS Neutralization of acidic resi dues on MARCKS was done as pr eviously described  using 1-Ethyl-3-(3-Dimethylaminopropyl) ca rbodiim ide Hydrochloride (EDC, MW 191.7, Figure 2-3, A), to crosslink the negatively ch arged carboxyl groups on ac idic residues to ethanolamine, resulting in a loss of negative charge on those resi dues (Figure 2-3, B). EDC is a heterobifunctional cross-linker that possesses two different reactive groups (a carboxyl and amine functional groups) that allo w for sequential (two-stage) conj ugations, helping to minimize undesirable polymerization or self-conjugation. EDC couples car boxyls to primary amines or hydrazides resulting in the formation of am ide or hydrazone bonds (Figure 2-3, D). Carboxy termini of proteins can be targeted, as well as glutamic and aspartic acid side-chains. In the presence of excess cross-linker, polymerization is likely to occur because all proteins contain carboxyls and amines. The bond that re sults is a peptide bond, so re versal of the conjugation is impossible without destroyi ng the protein [212-214]. EDC reacts with carboxylic acid groups and ac tivates the carboxyl group to form an active O -acylisourea intermediate, allowing it to be co upled to the amino group in the reaction mixture (ethanolamine was used in our experiments). An EDC by-product is released as a soluble urea derivative after displaceme nt by the nucleophile. The O -acylisourea intermediate is unstable in aqueous solutions, making it ineffective in twostep conjugation procedur es without increasing the stability of the intermedia te using N-hydroxysuccinimide. This intermediate reacts with a
44 primary amine to form an amide derivative. Inabi lity to react with an am ine results in hydrolysis of the intermediate, regeneration of the carboxyl s, and the release of an N-unsubstituted urea. The cross-linking reaction is usually performe d between pH 4.5 to 5 and requires only a few minutes for many applications. However, the yield of the reaction is similar at pH from 4.5 to 7.5. In this experiment, we performed a simple charge neutralization protocol, whereby negative residues on MARCKS (namely glutamic and aspartic acids, found only on either terminus on MARCKS and not in the PSD) were chemically modified to neutral amides. Once modified in that manner, MARCKS was used to perform biochemical assays, including actin polymerization assays, high speed pelleting a ssays, actin bundling assays, binding assays and fluorescence anisotropy assays with reporte d MARCKS binding partners, F-actin and Ca2+calmodulin, which we have so far been unsuccessf ul at reproducing in ou r lab with full length non-myristoylated MARCKS. Neutralization of the acidic residues on the te rmini of MARCKS is expected to prevent hypothesized ionic intramolecular interactions with the basic PSD of MARCKS, which is the known site of interaction between MARCKS an d its binding partners such as actin and calmodulin. We believe that these interactions ma sk the PSD and make it unavailable to interact with these proteins, and therefore expect that neutralizing the charge s responsible for these interactions while leaving the PSD intact would ma ke the latter available to interact with actin and calmodulin and would produce positive results in our biochemical assays that measure interactions and binding.
45 Methods Preparation of EDC-modified MARCKS As previously described for modification of the acidic residue s of caldesmon,  MARCKS (28 M) in 100 m M MES, 400 mM ethanol amine pH 5.5 was incubated for 10 minutes at room temperature. Freshly prepar ed EDC in water (200 mM) was added to give varying final EDC concentrations (0 mM, 4 mM, 12 mM and 20 mM ). Reactions were incubated at room temperature for 1 hour, then stopped by addition of -mercaptoethanol (100 mM), and then dialyzed into 5 mM Tris-HCl, 5 mM -mercaptoethanol, pH 7.9. Aliquots of treated MARCKS from each reaction mixture were run on 12% SDS-PAGE to conf irm modification by EDC as detected by an expected shift in electrophoretic mobility. Unmodified MARCKS runs anomalously as an ~80 kDa protein because of its unusual pI, and modification of the acidic residues shifts it towards its expected position at 30 kDa . Modificati on of the acidic but not basic residues was confirmed by amino acid analysis. Actin-binding assays Time course of actin polymerization. The effect of EDC-treated MARCKS on the rate of actin polym erization was determined using a time-course of actin pol ymerization assay. Three M 4% pyrenyl-actin (in G-buffer; 5 mM Tris, 0.1 mM CaCl2, 0.01 mM NaN3, 0.2 mM ATP, 0.2 mM DTT, pH7.9) and 6 M EDCtreated MARCKS (in 10 mM Tris, 5 mM mercaptoethanol, pH7.9) were used. Actin polym erization was initiated by the addition of 195 M MgCl2 to a final Mg2+ of 200 M and pyrenyl-actin fluo rescence intensity was immediately measured at 21C in a Photon Technology Inte rnational (South Brunswi ck, NJ) photon-counting spectrofluorimeter with excita tion at 366 nm and emission at 386 nm. MARCKS buffer alone (10 mM Tris, 5 mM -mercaptoethanol, pH7.9) wa s used as a control.
46 Time course of actin depolymerization. The effect of EDC-trea ted MARCKS on the rate of actin depolymerization was measured in a tim e course of actin filament depolymerization assay by dilution of 10% pyrenyl-labeled F-actin (10 M) polymerized with 2 mM MgCl2 to 0.1 M in F-buffer (5 mM Tris, 2 mM MgCl2, 0.125 mM EGTA, 40 mM KCl, 0.1 mM CaCl2, 0.01 mM NaN3, 0.2 mM ATP, 0.2 mM DTT, pH 7.9) in the absence or pres ence of 2 M EDC-treated MARCKS (0, 4, 12 and 20 mM EDC-treated). Pyrenyl-actin fluorescence intensity was followed at 21C in a Photon Technology Inte rnational (South Brunswi ck, NJ) photon-counting spectrofluorimeter with excitation at 366 nm and emission at 386 nm. High speed F-actin binding. To find out whether EDC-treated MARCKS binds to Factin, a high speed F-actin bindi ng assay was performed. In this assay, G-actin was allowed to polymerize to F-actin, then 10 M F-actin was incubated for 1 hour with 6 M EDC-treated MARCKS (0, 4, 12 or 20 mM EDC-treated MARCKS). Reactions were then centrifuged at high speed (65,000 rpm) for 1 hour at 12C to allow F-actin and anything bou nd to it to pellet. Supernatants were loaded onto a 12% SDS ge l and stained with SYPRO Ruby protein stain (Molecular Probes). MIANS-calmodulin binding assay To determine whether E DC-treated MARCKS bound more efficiently to calmodulin, we performed a MIANS-calmodulin binding assay. MIANS-calmodulin is a fluorescently labeled calmodulin whose fluorescence increases upon bindi ng to other molecules. To calmodulin equilibration buffer (10 mM Tr is, 50 mM KCl, 0.6 mM CaCl2, pH 7.9) increasing concentrations of EDC-treated MARCKS (0, 0.2, 0.5, 1, 3, 6 M, etc) were added to 0.2 M spinach MIANS-calmodulin, until a plateau in MIANS-cal modulin fluorescence was reached. Samples were excited at 322 nm and emission read at 438 nm in a Photon T echnology International
47 (South Brunswick, NJ) photon-coun ting spectrofluorimeter. For ne gative controls, MARCKS is replaced with MARCKS buffer ( 10 mM Tris, 40 mM KCl, 5 mM -mercaptoethanol, pH7.9). Results Neutralizing charges on MARCKS by EDC causes a shif t in MA RCKS motility on SDSPAGE: 30kDa MARCKS runs anomalously as a 65-87kDa protein on an SDS gel due to its highly negative charge, which prev ents binding of SDS molecules due to same charge repulsion, so MARCKS does not run based only on its mass as it is supposed to in SDS-PAGE. This charge neutralization was shown by amino acid anal ysis to be very mild, i.e. only a few amino acids were actually neutralized overall, e.g, at the highest EDC concentration used, only 6 amino acids were shown to be neutralized. However, this modification still seems to have a great effect on MARCKS activation making it more reactive with its binding partners (F-actin and calmodulin) in our biochemical assays. EDC neutralization of MARCKS increased the ra te of actin polymerization, especially at 20 mM EDC treatment (Figure 24). Neutralized MARCKS also had the greatest effect on the rate of actin depolymerization, slowing it down at 20 mM EDC (Figure 2-5), an effect attributed to the barbed end capping activity of MARCKS. In an actin high speed pelleting assay, only 12 and 20 mM EDC-treated MARCKS pelleted with F-actin following centrifugation (Figure 2-6), meaning that MARCKS can only bind the negative ly charged binding site on F-actin if it loses some of its negative charges. The binding of MARCKS to MIANS-calmodulin was also very much enhanced by the EDC-charge neutralizatio n of MARCKS (Figure 2-7) All these effects were consistent with the hypothe sis that EDC-neutralized MARCKS would behave like a peptide that corresponds to the PSD.
48 MARCKS Truncations Deleting either end of MARCKS is the second way we will tes t our hypothesis that the MARCKS termini are somehow interacting with th e PSD. In addition, the EDC negative charge neutralization experiment does not provide us with any information about which one of the termini of MARCKS actually inte racts with and blocks the PSD, or whether they both do. In order to answer this question, we made truncations of MARCKS wh ere deleted either terminus at a time while leaving the PSD intact (Figures 2-8 and 2-9). We then purified the resulting truncated proteins, MARCKS N-te rminus and MARCKS C-terminus, and used them for assays routinely performed to test for MARCKS-protein interactions (i.e., poly merization assays, high speed pelleting assays, actin bundling assays, bind ing assays and fluoresce nce anisotropy assays with known MARCKS binding partners, F-actin and Ca2+-calmodulin). Either one or both of the acidi c termini of the protein could be interacting with the PSD and rendering it inactive (Figure 22). If the former is true, de leting one terminus would make one of our constructs active. If the latter is true, deleting onl y one terminus may only weakly activate the protein, if at all, suggesting that more complicated post-translational modifications exist intracellularly that lead to the activation of MARCKS. Methods Generating truncations of MARCKS Cloning. Primers containing NdeI and Ba mHI restri ction sites were designed to amplify the entire N-terminus of MARCKS starting at the beginning of MARCKS up to and including the PSD (Fwd Primer: agg gaa ttc cat atg ggt gcc cag ttc tcc, Rev Primer: cgc gga tcc tta gcc cga ctc ctt ctt gct). A primer set was also designed to amplify the C-terminus of MARCKS, starting 4 residues before the PSD, including the PSD a nd ending at the STOP codon of MARCKS (Fwd Primer: agg gaa ttc cat atg agc agc gag acc ccg, Re v Primer: tta gga tcc tgg agc tta ctc ggc cgt).
49 PCR was carried out using as a template the complete murine MARCKS sequence in a pET19b vector (Novagen) using Roche s High Fidelity Taq Polymerase and PCR reagents and the following parameters: an initial denaturation of 95C for 10 minutes, 30 cycles of 94C for 1 min (denaturation), 55C for 1 min (annealing), 72C fo r 1 min (extension), a nd a final extension at 72C for 10 minutes. Either 20% sterile glycer ol or 10% DMSO was also required for the success of the PCR reaction with both primer sets The resulting PCR fragment was TA cloned into a TOPO pCR2.1 vector (Invitrogen), double digested with NdeI and BamHI (New England Biolabs) and cloned into the NdeI and BamHI sites of pET9a vector (Novagen, Figure 2-10) under control of the T7 promoter using T4 DNA Li gase (New England Biolabs). Ligations were carried out overnight in a 16C water bath and were then transformed into E. coli Nova Blue competent cells (Novagen) using a heat shock protocol (add 1 l of purified plasmid to 20 l of competent cells, place on ice for 30 min, heat shock at 42C for 45 seconds and place back on ice for 5 minutes, add 80 l of fresh SOC media, shake for 1 hour at 37C) and plated on Kanamycin-containing LB agar plat es. Colonies were picked th e following day, grown in 10 mL LB in a shaker at 37C for 16 hours, plasmid DNA isolated using Qiagens Plasmid Miniprep Kit and sent for DNA sequencing for sequence veri fication. Correct cons tructs were then transformed into E. coli BL21(DE3) expression cells using th e heat shock protocol described above, and grown in large scale in LB to an optical density at 600 nm of 0.5-0.6 at 37C and induced for 3 hours by addition of 1 mM IPTG. After 3 hours, cells were spun down and frozen at -80C and proteins were subsequently purified. Purification. Frozen cells were resuspended in re suspension buffer (5 mL buffer for every 500 mL of cell culture pellet, 10 mM Tris, 5 mM -mercaptoethanol, 2 mM EDTA, 100 M EGTA, pH 7.9 and protease inhibi tors 0.1 mM PMSF and 1 mM DFP). Resuspended cells were
50 sonicated four times for 30 seconds each with 30second intervals on ice, heated to 85C for 10 min, and centrifuged at 38,000 rpm for 1 hour. The supernatant was loaded onto a DEAE anion exchange column and fractions collected with a 0-400 mM KCl gradient in 5 mM Tris buffer, pH 7.9. Fractions containing protein were iden tified by running a 12% SDS gel. Fractions containing our protein of intere st were pooled, dialyzed into 25 mM Sodium Phosphate buffer, pH6.2 and loaded onto an HAP column. Proteins we re eluted from the HAP column with a step gradient of 100, 200 and 300 mM Sodium Phosph ate buffer, pH 6.2. Fractions containing protein were identified by running a 12% SDS gel and UV scans between 240 and 300nm (look for phenylalanine peaks between 258nm and 263nm). Fractions c ontaining protein were then dialyzed into MARCKS gel filtration buffer (5 mM Tris, 40 mM KCl, 5 mM -mercaptoethanol, pH7.9) and loaded onto a 100 cm Sephacryl HR300 gel filtration column. Purity of eluted MARCKS truncation was confirmed by SDS-page, amino acid analysis and UV absorption. UV scans are particularly useful with MARCKS an d its truncations, because phenylalanine (Phe, F) is the only aromatic amino acid in the protein (MARCKS contains no tyro sines or tryptophans), and in pure MARCKS there is characteristic phenylalanine fine stru cture in the 240-300nm region of the UV spectrum, and lack of absorban ce at 280nm. If further purification was needed, the protein was then loaded onto a Mono-Q ani on exchange column, and eluted with a 100-400 KCl gradient in the pres ence of 10 mM Tris, pH7.9. Testing activity of MARCKS truncations Low speed actin bundling. This assay tests whether the PSD on MARCKS truncations is available to bundle F-actin. In this assay, G-actin was allowed to polymerize to F-actin (1% pyrenyl-labeled) by addition of 2 mM MgCl2, then 3 M F-actin was incubate d overnight with 3 or 6 M of either MARCKS trucation or PSD as a positive control. Pyrene fluorescence was measured before and after a low speed centr ifugation at 14,000 rpm for 15 minutes at room
51 temperature in a tabletop microcentrifuge. Pyre nyl-actin fluorescence intensity was followed at 21C in a Photon Technology International (South Brunswick, NJ) photon-counting spectrofluorimeter with excitation at 366nm and emission at 386nm. Actin-binding in native gels. To look for interactions between actin and MARCKS truncations, we mixed 4 M actin with MARCKS (5 M), MARCKS C-terminus (5 M), and MARCKS N-terminus (14 M). Samples were in cubated for 30 minutes at room temperature, mixed with native sample buffer and loaded on a 7% Tris-Tricine native gel (25 mM TrisTricine, 0.1 mM CaCl2, 0.01 mM NaN3, 0.2 mM ATP and 0.2 mM DTT, pH 8.3). Gel electrophoresis was carried at a constant 100V. F-actin high speed pelleting assay. Previously described. Time course of actin polymerization. Previously described. Results By deleting either end of MARCKS se parately, we expected to activate the PSD by unmasking it, and making it inter act with F-actin and calmodulin. However, this would not be the case if both ends of MARCKS were interac ting with and masking th e PSD. All our actinbinding and interaction biochemical assays were negative with MARCKS truncations, indicating the presence of PSD-interacting sites at either end of MARCKS or an incorrect hypothesis. Neither Nnor C-terminus of MARCKS bundled F-actin in a low speed bundling assay whereas PSD did successfully (Figure 2-11). Neither the Nnor C-terminus formed complexes with actin in a native gel (Figure 2-12). Neither protein pelleted with F-actin in an F-actin high speed pelleting assay (Figure 2-13). Bo th MARCKS truncation proteins on ly slightly slowed down the rate of actin polymerization (Fi gure 2-14). In this same assa y, lower concentrations of PSD slowed down the rate of actin polymerization wh ile higher concentrations greatly increased the rate of actin polymerization.
52 Conclusions The EDC charge neut raliza tion experiments which activ ated MARCKS in all our biochemical assays provide strong evidence that there is an intramolecu lar interaction between the PSD of MARCKS and the rest of the protein, and that this interaction has a large ionic component to it. The negative results obtained from experi ments performed with the MARCKS truncation proteins suggest that there is more than one binding site for the PSD on MARCKS and that they are found at both N-terminus and C-terminus.
53 *G AQFS K TAA K G E ATAER PG E AAVASSPS K ANGQ E NGHV K VNG D ASPAAA E PGA K EEL QANGSAPAA D K EEPASGSAATPAAA E K DE AAAAT E PGAGAA D K E AA E A E PA E PSSPAA E A E GASASSTSSP K A ED GAAPSPSS E TP KKKKKR FS p F KKS p F K LS p GFS p F KK S KKE SG E G A E A E GATAEGA K DE AAAAAGG E GAAAPG E QAGGAGA E GAAGG E P R E A E AA E P E QP E Q P E QPAA EE PQAEE QS E AAG E K A EE PAPGATAG D ASS AAGP E Q E APAAT DE AAASAAPA ASP E PQP E CSP E APPAPTA E Figure 2-1. Amino acid sequence of muri ne MARCKS (Accession # NP_032564). Acidic residues are shown in red, basic residues shown in blue, PSD is in italic, potentially phosphorylated residues in the PSD are followed by a lower case green p, the myristoylation sequence is underlined, and th e myristoylated residue is marked by an asterisk.
54 Figure 2-2. The basic PSD of MARCKS may be mask ed by interacting with the acidic termini of MARCKS. A) PSD interacting with the N-terminus only. B) PSD interacting with the C-terminus only. C) PSD interacting with both the Nand C-termini of MARCKS simultaneously. Sticks with hexagon heads represent phenylalanine residues in the PSD. C-terminus N-terminus -++PSD++ N-terminus -++PSD++ C-terminus ++PSD++ N-terminus C-terminus A B C
55 A B C D Figure 2-3. EDC coupling reaction for negative charge neutraliza tion on MARCKS. A) Structure of 1-Ethyl-3-(3-Dimethylaminopr opyl) Carbodiimide Hydrochloride. (MW 191.7 Da), B) EDC coupling reaction in the presence of excess ethanolamine (HOCH2CH2NH2). The specific nucleophile (NH2R2) provided in excess in our reaction is ethanolamine, and results in th e covalent modification hereby illustrated, C) EDC reacts with carboxylic acid group and activates the carboxyl group, allowing it to be coupled to the amino group in the r eaction mixture, D) EDC is released as a soluble urea derivative after displacement by the nucleophile. 32 2 3 3CHCHNCNCHNCH HCl 3CH EDC HOCH2CH2NH2Protein-COO-Protein-CONHCH2CH2OH O13 22 33RCOHCHCHNCN(CH)NCH HCl 3CH H 32 23 3CHCHNHCN(CH)NCH 22NHR HCl 3CH 1RCO O 123 2 2 3RCNRCHCHNCN(CH)N OO H H H 3CH3CH
56 Figure 2-4. Effect of EDC neutralization of negative charges on MARCKS on actin polymerization. Untreated, 0 mM ED C and 4 m EDC-treat ed MARCKS do not accelerate the rate of actin polymerization as the PSD would alone. At 12 mM EDC treatment a slight acceleration in actin polym erization is observed and much more at 20 mM EDC treatment.
57 Figure 2-5. Effect of EDC neutralization of negative charges on MARCKS on actin depolymerization. 2 M of 0 EDC and 4 m EDC-treated MARCKS do not affect the rate of actin depolymerization as the PSD does at both 0.2 and 2 M. At 12 mM EDC treatment a slight slowi ng in actin polymerization is observed and much more at 20 mM EDC treatment. At 2 M, 20 mM EDC-treated MARCKS affects depolymerization similarly to 0.2 M PSD.
58 Figure 2-6. Effect of EDC neut ralization of negative charges on MARCKS on F-actin binding. Increasing concentrations of EDC (4, 12 and 20 mM) in the presence of ethanolamine were used to neutralize some acidic residues on MARCKS by conjugating them with ethanolamine. At 0 and 4 mM EDC, MARCKS does not bind to and pellet with F-actin in a high speed F-actin pelleting assay. However, at 12 and 20 mM EDC, MARCKS disappears from the s upernatant and pellets with F-actin. EDC (mM) 0 4 12 20 ----------------------------------------------------------------------Spun + + + + + + + + Actin + + + + EDC-treated MARCKS Actin
59 Figure 2-7 Effect of EDC neutralization of negative charges on MARCKS on calmodulin binding. Without EDC charge neutraliza tion, MARCKS binds minimally to MIANSlabeled calmodulin. However, EDC treated MARCKS binds to MIANS-calmodulin increasingly with increasing EDC concentrations used to neutralize the charges.
60 A GAQFSKTAAKGEATAERPGE AAVASSPSKANGQENGHVK VNGDASPAAAEPGAKEEL QANGSAPAADKEEPASGSAATPAAAEKDEAAAATEPGAGAADKEAAEAEPAEPSSPAA EAEGASASSTSSPKAEDGAAPSPSSETPKKKKKRFSFKKSFKLSGFSFKKSKK B SETPKKKKKRFSFKKSFKLSGFSFKKSKK ESGEGAEAEGATAEGAKDEAAAAAGGEGA AAPGEQAGGAGAEGAAGGEPREAEAAEPEQP EQPEQPAAEEPQAEEQSEAAGEKAEEP APGATAGDASSAAGPEQEAPAATDEAAASAAPAASPEPQPECSPEAPPAPTAE Figure 2-8. Amino acid sequence of MARCKS truncation proteins. A) MARCKS N-terminus + PSD. B) MARCKS C-terminus + PSD.
61 Figure 2-9. Schematic diagram of MARCKS truncations. A) Full length MARCKS. B) PSD+MARCKS N-terminus protein. C) PSD+MARCKS C-terminus protein. + + + PSD N-terminus C-terminus + + + PSD N-terminus + + + PSD C-terminus
62 Figure 2-10. Novagens pET-9a bacterial expression vector. MARCKS N-and C-terminus truncations were cloned using the NdeI and BamHI restriction sites under the control of the T7 promoter. pET-9a 4341 bp Blp I 458 Bam HI 510 Nde I 550 Xba I 588 Bsa I 613 T7 Promoter(615-631) 615 Bgl II 646 Sgr AI 687 Sph I 843 Sal I 928 Hin cII 930 Psh AI 993 Eag I 1216 Bst API 1328 Bsp MI 1331 Msc I 1723 Bsg I 1912 Xmn I 2310 Pvu II 2343 Pfo I 2394 Tth 111I 2497 Bsa AI 2504 Bst Z17I 2523 Tat I 2556 Sap I 2636 Bsr BI 2685 Afl III 2752 Pci I 2752 Ori (2814) 2814 Bss SI 2925 Bci VI 2955 Alw NI 3168 Acu I 3300 Ppi I 3435 Ppi I 3467 Psp XI 3552 Xho I 3552 Xma I 3826 Sma I 3828 Ssp I 3879 Asi SI 3954 Pvu I 3954 Kanamycin MARCKS N or C
63 Figure 2-11.Effect of MARCKS truncations and PSD on F-actin bund ling. Fluorescence of pyrenyl actin is measured in the supernatants before and after a low speed spin. The PSD shows a dose-dependent e ffect on F-actin bundling at 1, 2, 4 and 6 M, but neither Nnor C-termini show any bundling at 3 or 6 M concentrations. 0 50000 100000 150000 200000 250000Control 6 M N-term 6 M C-term 6 M PSD 4 M PSD 2 M PSD 1 M PSD Control Control 3 M N-term 3 M C-term SamplePyrene Fluorescence (cps) Before low speed pelleting After low speed pelleting
64 1 2 3 4 5 6 7 8 9 10 Actin MARCKS C-terminus N-terminus Figure 2-12. Coomassie stained native gel of MARCKS proteins with actin and rhodaminelabeled PSD. 1) Actin, 2) MARCKS, 3) MARCKS and Rh-PSD, 4) MARCKS and actin, 5) C-terminus, 6) C-terminus and Rh-PSD, 7) C-terminus and actin, 8) Nterminus, 9) N-terminus and Rh-PSD 10) N-terminus and actin.
65 Figure 2-13. Actin high speed pelleting a ssay with MARCKS truncations. A, 1) 6 M N-term. + 3 M F-actin, 2) 6 M C-term. + 3 M F-actin, 3) 3 M F-actin, 4) 6 M N-term, 5) 6 M C-term., 6) 3 M F-actin, 7) 3 M N-term. + 3 M F-actin, 8) 3 M C-term. + 3 M F-actin, 9) 6 M N-term. + 3 M F-actin, 10) 6 M C-term. + 3 M F-actin, 11) 3 M F-actin, 12) 6 M N-term, 13) 6 M C-term., 14) 3 M F-actin, 15) 3 M N-term. + 3 M F-actin, 16) 3 M C-term. + 3 M F-actin. B, invert of A. In both A and B, 18, unspun samples, 9-16, samples after high-speed spin: 1 hour at 65,000rpm at 12C. See text for details. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 B
66 Figure 2-14. Effect of MARCKS tr uncations on the time course of actin polymerization. At 3 and 6 M concentrations, both the Nand Ctermini of MARCKS very slightly slow down the rate of actin polymerization. PSD is shown as a control.
67 CHAPTER 3 SEARCHING FOR AN INTERNAL BINDING SITE ON M A RCKS Introduction Experiments described in Chapter 2 support our hypothesis that ionic intramolecular interactions are occurring within MARCKS mol ecules, rendering the protein inactive in our biochemical assays. The nature of these interactions is still unclear: are we looking at a single high affinity internal binding site for the PSD on the rest of the protei n, or non-specific ionic interactions? In the former case, can such a binding site be identified? In the latter case, are these occurring with both ends of the protein or just one? Could th ere be more than one site of interaction for the PSD with each terminus of MARCKS? We attempted to answer these questions using two approaches. In the first approach, we used mass spectrometry to sequence the PSD bindi ng site on MARCKS. In the second approach, we employed a direct binding assay based on fluorescence anisotropy using the MARCKS truncations created and described in Chapter 2 and a rhodamine-label ed synthetic external PSD. Mass Spectrometric Determination of Internal Binding Site of MARCKS Mass Spectrometry Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of ions. MS has been an estab lished technique in organic chemis try for many years. It is an enormously sensitive technique that requires very little material. Masses can be obtained with great accuracy, often with an error of less than one part in a million. Until recently, however, the very low volatility of proteins made mass spectrometry useless for the investigation of these molecules. This difficulty has been circum vented by the introduction of techniques for effectively dispersing proteins and other macromolecules into the gas phase (namely matrixassisted laser desorption-ionization (MALDI) [2 15-217] and electrospray ionization (ESI) [218-
68 221]). Mass spectrometry allows one to determine the precise ma ss of intact proteins and of peptides derived from them by enzymatic or chem ical cleavage. This information can then be used to search genomic databases, in which the masses of all proteins and of all their predicted peptide fragments have been tabulated. A match to a particular open reading frame can often be made knowing the mass of only a few peptides deri ved from a given protein. Mass spectrometry has permitted the development of peptide mass fingerprinting. Following two-dimensional gel electrophoresis, the sample of inte rest is extracted and chemically or enzymatically cleaved. The masses of the protein fragments are then determined by MS. Fi nally, the peptide masses, or fingerprint, are matched against the fingerprint fo und in databases of proteins that have been electronically cleaved by a computer simulatin g the same fragmentation technique used for the experimental sample. Mass spectrometry is also used to determine the sequence of amino acids of individual peptide fragments. Peptide sequenci ng is also important if proteins contain posttranslational modifications, such as attached carbohydrates, phosphates, or methyl groups. In this case, the precise amino acids that are the sites of modifications can usually be determined. To obtain such peptide sequence information, two mass spectrometer s are required in tandem. The first separates peptides obtained af ter digestion of the protein of in terest and allows one to zoom in on one peptide at a time. This peptide is then further fragmented by collision with high-energy inert gas atoms (e.g., argon, xenon), which prefer entially cleave the peptide bonds, generating a ladder of fragments, each differing by a single am ino acid. The second mass spectrometer then separates these fragments and displays their masses. The amino acid sequence can be deduced from the differences in mass between the pept ides. Post-translational modifications are identified when the amino acid to which they ar e attached show a characteristically increased mass.
69 Methods Biotin-labeled PSD was synthesized at the Univ ersity of Floridas Protein Core, having the following sequence KKKKKRFSFKKSFKLSGFSFKKSKKSK-biotin. Experiments to elucidate the internal binding site of the PSD on MARCKS were done by immobilizing the biotin-PSD on a streptavidin column, followed by addition of MARCKS to those columns either before or after digestion with an endoproteinase as described in detail in the followi ng protocols. MARCKS fragments were then eluted from the column us ing a salt gradient, desalted and sent for mass spectrometric analysis. AspN pre-column digest Three milliliters of MARCKS (3 mL of 10 M MARCKS =0.9 mg MARCKS (0.3 mg/mL) = 30 nmoles) were dialyzed into Asp-N digestion buffer (50 mM sodium phosphate buffer, pH 7.9). MARCKS was then digested with endoproteinase As pN (4 g AspN, 1:225 ratio) overnight at 37C in a waterbath. A 0.5 mL streptavidin column (Ultralink Immobilized Streptavidin Plus, Product #53117 Lot # HI106344) wa s equilibrated with 1X PBS and 0.75 mL of 1 mg/mL (220 nmoles) MARCKS PSD-EK-bio tin (diluted in 100 mM MES, 50 mM NaCl, pH 4.9) were bound to the streptavidin column fo r 1 hour at room temperature on a rotator. The biotin-PSD column was equilibrated with 10 mM Tris, pH 7.9. The AspN digest was boiled for 10 minutes to inactivate AspN, and then kept on ice. It was then diluted 1:20 with 10 mM Tris, pH 7.9, no KCl before applying to biotin-PSD column. The AspN digested material was applied to the biotin PSD column and allowed to bind by reapplying flow through to the column at RT multiple times. The column was washed with 10 mM Tris, pH 7.9 (No KCl), followed by a 1 mL step gradient of 10 mM Tris, pH 7.9 with 50 mM KCl increments (50,100,150,200,250,300,400,500,1000 mM KCl). One milliliter fractions were collected. Fractions were desalted using a MacroSpin column packed with Vydac C18 with a loading
70 capacity of 300 g (Catalog number SMM SS 18V, Nest Group, Southborough, MA). The column was rinsed with acetonitrile, equilibrated and washed with 0.1% TFA, and fragments el uted with 80% acetoni trile and 20% TFA. Fragments were then run on the QSTAR XL H ybrid LC/MS/MS system (a high-performance, hybrid quadrupole time-of-flight mass spectrometer) mass spectrometer at the University of Floridas Protein core with an LC Pa ckings HPLC to obtain sequence data. Glu-C on column digest Ten milliliters of MARCKS (10 mL of 6 M MARCKS= 60 nmoles MARCKS) were dialyzed into 10 mM Tris-HCl, pH 7.9. A 1 mL streptavidin column (Ultralink Immobilized Streptavidin Plus, Product #53117 Lot # HI106344) wa s equilibrated with 1X PBS and 2 mL of 1 mg/mL (570 nmoles) MARCKS PSD-SK-biotin (diluted in 1XPBS) were bound to the streptavidin column for 1 hour at room temperature on a rotator. The streptavidin-biotin-PSD column was then equilibrated with 10 mM Tr is-HCl, pH 7.9 and 10 mL MARCKS bound to the equilibrated column in 10 mM Tris-HCl, pH 7.9 on a rotator at 4C overnight. The resin was allowed to settle and the buffer to pass th rough, flow through collected and UV scan (240300nm) showed it contained some MARCKS. The column was then equilibrated with endoproteinase GluC digestion buffer (25 mM a mmonium bicarbonate, pH 8) and 100 g GluC dissolved in 500 L ammonium bicarbonate buffer, pH 8 added to the column. Digestion was allowed to take place at RT fo r 7 hours on a rotator. Followi ng the digestion, the resin was allowed to settle for 30 minutes then a step gr adient of 10 mM Tris, pH 7.9 with 50 mM KCl increments was carried out (1 mL of each salt co ncentration was added and fractions collected): 50, 100, 150, 200, 250, 300, 400, 1000 mM KCl. Fractions were boiled for 10 minutes to inactivate GluC then frozen at -80C until sent for mass spec analysis. Fractions were desalted using a MacroSpin column packed with Vydac C18 with a loading capacity of 300 g (Catalog
71 number SMM SS18V, Nest Gr oup, Southborough, MA). The column was rinsed with acetonitrile, equilibrated and washed with 0.1% TFA, and fragments eluted with 80% acetonitrile and 20% TFA. Fragments were run on the QSTAR XL Hybrid LC/MS/MS system (a highperformance, hybrid quadrupole time-of-flight mass spectrometer) mass spectrometer at the University of Floridas Protein core with th e LC Packings HPLC to obtain sequence data. Results We used the observation that the intramolecular interaction between the PSD and MARCKS has a strong ionic component to elute MARCKS fragments from a biotinylated PSD immobilized on a streptavidin column using a KCl salt gradient. Resulting fragments were subjected to mass spectrometry to determine which fragments of MARCKS had the highest affinity to the PSD. When AspN was used to digest MARCKS, we obt ained poor coverage of MARCKS (about 30%), mostly in the N-terminus of the protein. AspN generated MARCKS fragments that were too large for the mass spect rometer we were employing. AspN did not cut MARCKS often enough and generated larger frag ments of MARCKS, especially at the Cterminus. The majority of the N-terminus seemed to come off in the washes (Figure 3-1), suggesting that it did not bind to the PSD. However, a few fragments at the N-terminus were not present and thus we cannot eliminate the possibilit y that those are interac ting with the PSD. The fact that we were not seeing mo st fragments of the C-terminus could simply be due to the previously mentioned problem th at AspN fragments of MARCKS were too large for detection by electrospray mass spectrometry and we may need to use MALDI-TOF which can tolerate larger fragment sizes, or that they were indeed binding to PSD. This problem was solved by using GluC instead of AspN in our digestion, sin ce GluC cuts MARCKS about 50 times and generates fragments of optimal size for de tection by electrospray LCMS/MS. These fragments were then run onto a mass spectrometer (Table 3-1) and as expected we found most of the protein in the
72 flow through and washes (Figure 3-2, A and B), but some peptides did not elute below 200 mM KCl concentration (Figure 3-2, E). The sequen ce for these peptides could be obtained to 95% probability and we obtained nice spectra for these pe ptides. Only 3 fragments seemed to persist at the higher salt concentration (150 mM KCl), one of them located at the N-terminus of MARCKS and two at the C-terminus. Their sequences are: AAEAEPAEPSSPAAEAEG, AAAAAGGEGAAAPGEQAGGAGAEGAAGGEPR EAE and AAASAAPAASPEPQPE. Interestingly, the identified N-terminus fr agment was missing from the AspN washes (DKEAAEAEPAEPSSPAAEAEGASASSTSSPKAE) and may either mean that it was binding to PSD or that it was too large to detect (33 amino acids, monoisotopic MW 3159.4084Da). Use of MARCKS Truncations to Det ermine PSD-Interacting Terminus In order to narrow down the location of the PSD binding site on MARCKS, we decided to use our truncated MARCKS proteins (described in Chapter 2) in direct binding assays with a rhodamine-labeled synthetic PSD. We hypothesize that if a single binding site for the PSD was present on one of the termini of MARCKS or if PSD bound to multiple sites on a single terminus, then we would only see binding of our exte rnal PSD to that terminus. Similarly, if the PSD interacts with both termini of MARCKS, then we would observe interaction of our rhodamine-labeled PSD with both truncations of MARCKS. In the first set of experiments, we used a technique based on the change in fluorescence anisotropy of the rhodamine-labeled PSD upon binding to other proteins, i.e. MARCKS truncations. In the second set of experiments, we used native ge l electrophoresis to detect PSDMARCKS truncations interactions. Fluorescence Anisotropy Assays Fluorescence anisotropy is a technique for m easuring the binding interaction between two molecules and can be used to meas ure the binding or dissociation cons tants for the interaction. It
73 is based on the fact that when a fluorophore is excited by polarized light, it will also emit polarized light. However, if a molecule is moving, it will scramble the polarization of the light by radiating at a different direc tion from the incident light. Th e scrambling effect is greatest when the fluorophores are tumbling freely in solu tion and decreases with decreased rate of tumbling. We can measure protein interactions by labeling a protein (preferably a small protein or peptide) with a fluorophore; once that labeled protein binds to another protein or molecule forming a larger, more stable complex in solution it will tumble more slowly, resulting in an increase in the polarization of the emitted light and reducing the scrambling effect. The extent of polarization is proportional to the amount of protein complex formed and that is also proportional to the amount of the binding partners in solution. Thus, we can titrate the amount of one of the proteins and ge nerate a binding curve. The anisotropy is defined as the ratio of the difference between the emission intensity parallel to the polarization of th e electric vector of the exciting light and that perpendicular to that vector divided by the total in tensity. Because the anisotropy of emission (A) is related to the correlation time of the fluorophor e through the Perrin equation A0/A-1 = / c where A0, is the limiting anisotropy of the probe, which depends on the angle between the absorption and emission transition dipoles, and is the fluorescence lifetime, these measurements can be used to obtain hydrodynamic information concerning m acromolecules and macromolecular complexes [222, 223]. For our anisotropy assays, we synthesized a tetramethylrhodamine-labeled PSD [4, 224, 225]. This peptide was used in both a dire ct binding assay with our truncated MARCKS proteins, which allowed us to calculate a bindi ng constant for each of the proteins, and in a competition assay with unlabeled synthetic PSD as the competitor in the presence of our
74 MARCKS truncations . The purpose of the competition assay was to determine the stoichiometry of the interaction between MARCKS trun cation proteins and extern al PSD. Direct binding anisotropy assay In this assay, 0.15 M Tetramethylrhodami ne-5-maleimide-labeled PSD (Rh-PSD) was added to increasing concentrati ons of MARCKS truncation proteins (N-terminus or C-terminus) in MARCKS gel filtration buffer ( 10 mM Tris, 40 mM KCl, 5 mM -mercaptoethanol, pH 7.9). Data were collected on a Photon Technol ogy International (South Brunswick, NJ) spectrofluorimeter. Tetramethylrhodamine-5-maleimi de-labeled PSD was excited with vertically polarized light at 546 nm. The horizontal and vertical components of the emitted light were detected at 568 nm. Competition anisotropy assay In this assay, a constant amount of Tetramethylrhodamine-5-maleimide-labeled PSD (0.2 M) and a constant amount of e ither MARCKS truncation proteins (0.5 M, chosen from direct binding anisotropy data) were adde d to increasing concentrations of unlabeled PSD (0-100 M). Reactions were carried out in MARCKS gel f iltration buffer (10 mM Tris, 40 mM KCl, 5mM mercaptoethanol, pH 7.9). Data were collected on a Photon Technology International (South Brunswick, NJ) spectrofluorimeter. Tetramet hylrhodamine-5-maleimide-labeled PSD was excited with vertically polarized light at 546 nm The horizontal and vertical components of the emitted light were detected at 568 nm. Native Gel Electrophoresis with Fluorescently Labeled PSD "Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS or any denaturing agents. While in SDS-PAGE the el ectrophoretic mobility of proteins depends primarily on their molecular mass, in native PAGE the mobility depends on both the protein's charge and its hydrodynamic size [226-229].
75 The electric charge driving th e electrophoresis is governed by the intrinsic charge on the protein at the pH of the running buffer. This charge depends on the amino acid composition of the protein as well as post-translational modifications. Since the protein remains folded, its hydrodyna mic size and mobility on the gel will also vary with its conformation (higher mobility for more compact conformations, lower for larger structures like oligomers). If na tive PAGE is carried out near neut ral pH to avoid acid or alkaline denaturation, then it can be used to study confor mation, self-association or aggregation, and the binding of other proteins or compounds. For native gel electrophoresis of proteins, either polyacrylamide or agarose gels can be used. We used both types of gels to look for complex formation between the rhodamine-labeled PSD and MARCKS truncation proteins. Native PAGE of rhodamine-labe led PSD and MARCKS truncations To look for interactions between Rh-PSD and MARCKS proteins, we mixed 11 M RhPSD with MARCKS (5 M), MARCKS C-terminus (5 M), and MARCKS N-terminus (14 M). Samples were incubated for 30 minutes at room temperature, mixed with native sample buffer and loaded on a 7% Tris-Tricine na tive gel (25 mM Tris-Tricine, 0.1 mM CaCl2, 0.01 mM NaN3, 0.2 mM ATP and 0.2 mM DTT, pH 8.3) . Gel electrophoresis was carried out at 100V constant. The gel was first visualized unde r UV to look for fluorescence of Rh-PSD then stained with Coomassie Brilliant Blue G-250 protein staining dye. Native agarose gel electrophoresis of rhod amine-labeled PSD and MARCKS truncations Samples were prepared exactly as described for Native PAGE and run in a 2% agarose gel made in Tris-Tricine native buffe r (25 mM Tris-Tricine, 0.1 mM CaCl2, 0.01 mM NaN3, 0.2 mM ATP and 0.2 mM DTT, pH 8.3). Gel electrophoresis was carried out at a constant 100 V.
76 Results Both the N-and C-termini of MARCKS bound with high affinity to an external rhodaminelabeled PSD (Figures 3-3 and 3-4), which is consistent with th em being inactive in our actinbinding assays as shown in chapter 2. The N-terminus has a lower Kd than the C-terminus (0.07 M versus 0.28 M). A competition assay with both rhodamine-labeled and unlabeled external PSD to determine specificity of the interaction showed that the N-terminus only has one binding site for the PSD, whereas it takes about 5 times more unlabeled PSD to compete off the labeled PSD off of the C-terminus, suggesting multiple binding sites for the PSD on the Cterminus (Figures 3-5 and 3-6). Some may be specific whereas others may be non-specific and their affinities for the PSD may vary. An ove rlay of direct binding and competition anisotropy data for the N-and C-termini of MARCKS is shown in Figure 3-7. Both agarose and polyacrylamide native gels suggest that the N-terminus interacts more strongly with the rhodamine-labeled PSD (Figures 3-8 and 3-9). The smear formed by Rh-PSD interacting with N-terminus was expected given that the N-terminus itself runs as a smear or forms a ladder on a native gel (Figure 3-10) further s uggesting that it has th e ability to form oligomers due to an intramolecular binding site that can become intermolecular. Conclusions The PSD can interact with multiple sites on MARCKS. Mass spectrometry has identified three possible sites, one at the N-terminus and two at the C-terminus. This is consistent with the data in Chapter 2 which showed that removi ng either end does not render MARCKS active and with binding and competition data showing that both Nand C-terminus of MARCKS can interact with external PSD. It is important to note that our experiment s were carried out in 40 mM KCl, which is about three times lower than the intracellular salt c oncentration. Under these conditions, the C-
77 terminus of MARCKS may not be interacting at all with its phosphorylati on site domain, and the N-terminus, which has a higher affi nity binding site for the PSD as shown in our assays may. This can easily be determined by repeati ng the binding and competition assays using 100-150 mM KCl, which is more physiological. Specific proteolysis N-terminal to the PSD, post-translational modifications, changes in ionic strength or binding by other proteins are all mechan isms by which MARCKS may be activated in vivo. Interestingly, three cleavage sites for MARCKS have been described (Figure 1-2), all cutting the protein N-terminal to th e PSD, and two of which would remove the PSD binding site we have identified at the N-term inus (residues 103-119 in murine MARCKS). One cut site is between Asn 147 and Glu 148 (found only in human and bovine MARCKS) and the other between Ser 126 and Ser127 (present in all sp ecies). In addition, se rine residues in our identified PSD binding sites both at the Nand C-termini were reported to be phosphorylated: at the N-terminus binding site, residues 103-119, Se r 113 was reported to be phosphorylated. At the C-terminus, where we identified residue s 189-222 and 282-297 as two potential PSD binding site, Ser 291 was reported to be phosphorylated in rat MARCKS. These post-translational modifications and proteolysis may account for the activity of MARCKS in vivo and MARCKS purified from brain compared to the recombinant, bacterially expressed pr otein we used in our actin and calmodulin-binding assays with little to no success.
78 Table 3-1. Mass spectrometry results of GluC MARCKS fra gments bound to biotin-PSD. Fraction Peptide sequence Best peptide identification probability Best Mascot Ion score Best Mascot identity score Best X! Tandem log(e) score Calculated peptide mass (AMU) FT AAAATEPGAGAADKE 95.00% 59.50 55.50 6.510 1329.6288 FT AAASAAPAASPEPQPE 95.00% 39.40 55.30 2.060 1464.6973 FT AAGEKAEEPAPGATAGDASSAAGPEQE 95.00% 00.00 00.00 5.960 2469.1018 FT AAVASSPSKANGQENGHVKVNGDASPAAA E 93.40% 00.00 00.00 2.820 2837.3193 FT AEPAEPSSPAAE 95.00% 46.10 55.00 4.920 1155.517 FT AEPAEPSSPAAEAE 95.00% 42.30 55.30 3.620 1355.5967 FT ATAERPGE 95.00% 50.10 56.60 0.921 830.4009 FT GAAAPGEQAGGAGAE 95.00% 36.00 55.40 3.440 1213.545 FT GAQFSKTAAKGE 95.00% 42.70 55.30 3.960 1194.6121 FT GASASSTSSPKAE 95.00% 52.90 55.40 3.800 1179.5493 FT KAEEPAPGATAGDASSAAGPE 95.00% 00.00 00.00 3.820 1883.8625 FT KAEEPAPGATAGDASSAAGPEQE 95.00% 00.00 00.00 3.620 2140.9636 FT LQANGSAPAADKEE 67.40% 00.00 00.00 1.660 1400.6659 FT LQANGSAPAADKEEPASGS AATPAAAE 93.40% 00.00 00.00 2.820 2482.1697 FT LQANGSAPAADKEEPASGSAATPAAAEKD E 95.00% 00.00 00.00 3.800 2854.3342 FT NGHVKVNGDASPAAAE 95.00% 47.80 55.30 5.070 1536.7408 FT NGHVKVNGDASPAAAEPG 95.00% 00.00 00.00 3.920 1690.8152 FT NGHVKVNGDASPAAAEPGAKE 95.00% 42.10 54.70 2.960 2018.9899 FT NGHVKVNGDASPAAAEPGAKEE 95.00% 00.00 00.00 5.920 2148.0325 FT PAPGATAGDASSAAGPEQE 95.00% 00.00 00.00 3.260 1683.7463 FT PGAGAADKE 67.40% 00.00 00.00 1.660 815.3901
79 Table 3-1. Continued 1. Fraction Peptide sequence Best peptide identification probability Best Mascot Ion score Best Mascot identity score Best X! Tandem log(e) score Calculated peptide mass (AMU) FT QPEQPAAEEPQAE 95.00% 00.00 00.00 3.420 1406.6079 FT QPEQPAAEEPQAEE 89.20% 00.00 00.00 2.460 1552.677 FT QPEQPEQPAAEEPQAE 95.00% 00.00 00.00 4.430 1760.7618 FT QPEQPEQPAAEEPQAEE 95.00% 00.00 00.00 7.080 1906.8309 Wash AAAATEPGAGAADKE 95.00% 70.90 55.50 6.000 1329.6288 Wash AAASAAPAASPEPQPE 51.70% 35.10 55.30 0.362 1464.6973 Wash AAGEKAEEPAPGATAGDASSAAGPE 95.00% 00.00 00.00 7.320 2212.0007 Wash AAGEKAEEPAPGATAGDASSAAGPEQE 95.00% 00.00 00.00 4.440 2469.1018 Wash AAVASSPSKANGQE 95.00% 46.30 55.70 3.220 1317.6287 Wash AAVASSPSKANGQENGHVKV NGDASPAAAE 95.00% 00.00 00.00 4.600 2835.3511 Wash AEPAEPSSPAAEAE 95.00% 55.40 55.20 5.060 1355.5967 Wash ATAERPGE 95.00% 45.20 56.60 9.300 830.4009 Wash GAAGGEPREAE 95.00% 45.40 55.60 1.720 1043.476 Wash GASASSTSSPKAE 95.00% 57.40 55.40 3.920 1179.5493 Wash KAEEPAPGATAGDASSAAGPE 87.10% 00.00 00.00 2.600 1883.8625 Wash KAEEPAPGATAGDASSAAGPEQE 95.00% 00.00 00.00 6.420 2140.9636 Wash LQANGSAPAADKEEPASGSAATPAAAE 95.00% 00.00 00.00 3.520 2482.1697 Wash LQANGSAPAADKEEPASGSAATPAAAEKDE 95.00% 00.00 00.00 4.390 2854.3342 Wash NGHVKVNGDASPAAAE 95.00% 45.40 54.90 0.495 1538.7089 Wash NGHVKVNGDASPAAAEPGAKEE 95.00% 00.00 00.00 4.340 2148.0325 Wash QPEQPEQPAAEEPQAE 95.00% 00.00 00.00 3.770 1777.7883
80 Table 3-1. Continued 2. Fraction Peptide sequence Best peptide identification probability Best Mascot Ion score Best Mascot identity score Best X! Tandem log(e) score Calculated peptide mass (AMU) Wash QPEQPEQPAAEEPQAEE 95.00% 00.00 00.00 4.020 1906.8309 50 mM KCl AAAATEPGAGAADKE 95.00% 73.40 55.50 6.000 1329.6288 50 mM KCl AAEAEPAEPSSPAAEAE 95.00% 00.00 00.00 4.320 1626.7136 50 mM KCl AAGEKAEEPAPGATAGDASSAAGPE 95.00% 00.00 00.00 3.280 2212.0007 50 mM KCl AAGEKAEEPAPGATAGDASSAAGPEQE 95.00% 00.00 00.00 5.100 2469.1018 50 mM KCl AAVASSPSKANGQE 95.00% 41.60 55.70 0.854 1317.6287 50 mM KCl AEPAEPSSPAAE 93.30% 00.00 00.00 2.680 1155.517 50 mM KCl AEPAEPSSPAAEAE 95.00% 00.00 00.00 4.960 1355.5967 50 mM KCl GAAAPGEQAGGAGAE 95.00% 00.00 00.00 2.920 1213.545 50 mM KCl GAAAPGEQAGGAGAEGAAGGEPR EAE 95.00% 00.00 00.00 7.960 2238.0022 50 mM KCl GASASSTSSPKAE 95.00% 42.10 55.40 3.150 1179.5493 50 mM KCl KAEEPAPGATAGDASSAAGPE 95.00% 00.00 00.00 3.200 1883.8625 50 mM KCl KAEEPAPGATAGDASSAAGPEQE 95.00% 00.00 00.00 3.410 2140.9636 50 mM KCl LQANGSAPAADKEEPASGSAA TPAAAE 95.00% 00.00 00.00 3.750 2482.1697 50 mM KCl LQANGSAPAADKEEPASGSAA TPAAAEKDE 95.00% 00.00 00.00 9.960 2854.3342 50 mM KCl NGHVKVNGDASPAAAE 67.10% 23.00 55.30 1.500 1536.7408 50 mM KCl PAPGATAGDASSAAGPEQE 95.00% 00.00 00.00 4.390 1683.7463 50 mM KCl QPEQPAAEEPQAEE 85.30% 00.00 00.00 2.170 1552.677 50 mM KCl QPEQPEQPAAEEPQAE 95.00% 00.00 00.00 4.390 1777.7883 50 mM KCl QPEQPEQPAAEEPQAEE 95.00% 00.00 00.00 3.770 1906.8309 100 mM KCl AAAAAGGEGAAAPGEQAGGAGAE 73.40% 00.00 00.00 1.800 1811.8162
81 Table 3-1. Continued 3. Fraction Peptide sequence Best peptide identification probability Best Mascot Ion score Best Mascot identity score Best X! Tandem log(e) score Calculated peptide mass (AMU) 100 mM KCl AAAAAGGEGAAAPGEQAGGAGAEGAAGGEPREAE 95.00% 00.00 00.00 5.460 2836.2732 100 mM KCl AAAATEPGAGAADKE 95.00% 77.40 55.50 6.000 1329.6288 100 mM KCl AAASAAPAASPEPQPE 95.00% 46.60 55.30 2.570 1464.6973 100 mM KCl AAEAEPAEPSSPAAEAE 95.00% 00.00 00.00 5.750 1626.7136 100 mM KCl AEPAEPSSPAAEAE 95.00% 44.70 55.20 4.050 1355.5967 100 mM KCl ATAERPGE 95.00% 60.10 56.60 1.140 830.4009 100 mM KCl GAAAPGEQAGGAGAEGAAGGEPR EAE 95.00% 00.00 00.00 3.700 2238.0022 100 mM KCl GAAGGEPREAE 95.00% 33.50 55.60 1.820 1043.476 100 mM KCl LQANGSAPAADKEEPASGSAA TPAAAE 94.00% 00.00 00.00 2.770 2482.1697 100 mM KCl LQANGSAPAADKEEPASGSAA TPAAAEKDE 95.00% 00.00 00.00 3.770 2854.3342 150 mM KCl AAAAAGGEGAAAPGEQAGGAGAE 91.00% 00.00 00.00 2.600 1811.8162 150 mM KCl AAAAAGGEGAAAPGEQAGGAGAEGAAGGEPREAE 95.00% 00.00 00.00 3.570 2836.2732 150 mM KCl AAASAAPAASPEPQPE 95.00% 29.40 55.30 2.080 1464.6973 150 mM KCl AAEAEPAEPSSPAAEAE 95.00% 00.00 00.00 6.620 1626.7136 150 mM KCl AEPAEPSSPAAEAE 95.00% 39.50 55.30 1.020 1355.5967 150 mM KCl ATAERPGEAAVASSPSKANGQE 95.00% 00.00 00.00 10.800 2128.0271 150 mM KCl GAAAPGEQAGGAGAEGAAGGEPR EAE 95.00% 00.00 00.00 3.570 2238.0022 150 mM KCl KANGQENGHVKVNGDASPAAAEPGAKEE 82.40% 00.00 00.00 2.150 2757.3193 150 mM KCl LQANGSAPAADKEEPASGSAA TPAAAE 95.00% 00.00 00.00 5.310 2482.1697 150 mM KCl LQANGSAPAADKEEPASGSAA TPAAAEKDE 95.00% 00.00 00.00 12.800 2854.3342 150 mM KCl NGHVKVNGDASPAAAEPGAKEE 95.00% 00.00 00.00 4.340 2148.0325
82 Table 3-1. Continued 4. Fraction Peptide sequence Best peptide identification probability Best Mascot Ion score Best Mascot identity score Best X! Tandem log(e) score Calculated peptide mass (AMU) 200 mM KCl AAAAAGGEGAAAPGEQAGGAGAE 90.40% 00.00 00.00 2.750 1811.8162 200 mM KCl AAAAAGGEGAAAPGEQAGGAGAEGAAGGEPREAE 95.00% 00.00 00.00 7.770 2836.2732 200 mM KCl AAASAAPAASPEPQPE 95.00% 28.80 55.30 3.820 1464.6973 200 mM KCl AAEAEPAEPSSPAAEAE 95.00% 00.00 00.00 6.280 1626.7136 200 mM KCl AEPAEPSSPAAEAE 95.00% 37.40 55.30 3.220 1355.5967 200 mM KCl GAAAPGEQAGGAGAEGAAGGEPR EAE 95.00% 00.00 00.00 5.430 2238.0022 200 mM KCl LQANGSAPAADKEEPASGSAA TPAAAEKDE 87.00% 00.00 00.00 2.540 2854.3342
83 A B C Figure 3-1. Peptide coverage for salt fractions of Asp-N digested MARCKS eluted from biotinPSD column. A) Wash 1: 6 unique peptides 6 unique spectra, 7 total spectra, 92/309 amino acids (30% coverage). B) Wash 2: 3 unique peptides, 5 uni que spectra, 5 total spectra, 49/309 amino acids (16% coverage). C) Fraction 1: 50 mM KCl: 2 unique peptides, 2 unique spectra, 2 total spec tra, 40/309 amino acids (13% coverage).
84 A B C D Figure 3-2. Peptide coverage for salt fractions of GluC digested MARCKS eluted from biotinPSD column. A) Flow through: 22 unique peptides, 23 unique spectra, 27 total spectra, 203/309 amino acids (66% coverage ). B) Wash: 16 unique peptides, 17 unique spectra, 20 total spectra, 171/309 amino acids (55% coverage). C) Fraction 1: 50 mM KCl: 17 unique peptides, 19 unique spectra, 20 total sp ectra, 159/309 amino acids (51% coverage). D) Fraction 2: 100 mM KCl: 10 unique peptides, 10 unique spectra, 10 total spectra, 120/309 amino acids (39% coverage). E) Fraction 3: 150 mM KCl: 10 unique peptides, 10 unique sp ectra, 10 total spec tra, 141/309 amino acids (46% coverage). F) Fraction 4: 200 mM KCl: 6 unique peptides, 6 unique spectra, 6 total spectra, 67/309 amino acids (22% coverage).
85 E F Figure 3-2. Continued.
86 Figure 3-3. Direct bi nding fluorescence anisotropy of MARCKS N-terminus to Rh-PSD. A) 1 minute after addition of Rh-PSD to sample, where Kd=72nM. B) 15 minutes after addition of Rh-PSD to sample, where Kd=56nM. A B
87 Figure 3-4. Direct bi nding fluorescence anisotropy of MARCKS C-terminus to Rh-PSD. A) 1 minute after addition of Rh-PSD to sample, where Kd=287nM. B) 15 minutes after addition of Rh-PSD to sample, where Kd=288nM. A B
88 Figure 3-5. Fluorescence anisotropy comp etition with MARCKS N-terminus, Rh-PSD and unlabeled PSD. 0.5 M N-terminus, 0.2 M Rh-PSD and increasing concentrations of unlabeled PSD were used. Kl=0.072, Ku=0.0715, rf=0.068 and rb=0.148.
89 Figure 3-6. Fluorescence anisotropy comp etition with MARCKS C-terminus, Rh-PSD and unlabeled PSD. 0.5 M C-terminus, 0.2 M Rh-PSD and increasing concentrations of unlabeled PSD were used. Kl=0.287, Ku=0.990, rf=0.069 and rb=0.164.
90 Figure 3-7. Overlay of N-and C-terminus anis otropy and competition data. A) Anisotropy, B) Competition. The inset in B shows the first 4 data points. 0123 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 N-terminus, Kd 72nM C-terminus, Kd 290nMFluorescence Anisotropy (cps)Concentration of Nor C-terminus (M) A 020406080100 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 0.14 0246810 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 0.14 Fluorescence Anisotropy (cps)Concentration of Unlabeled PSD (M)Fluorescence Anisotropy (cps)Concentration of Unlabeled PSD (M) N-terminus C-terminus B
91 1 2 3 4 5 6 7 8 9 10 Figure 3-8. UV photo of 7% nati ve Tris-tricine polyacrylamide gel of MARCKS proteins with actin and rhodamine-labeled PSD. 1) Ac tin, 2) MARCKS, 3) MARCKS and Rh-PSD, 4) MARCKS and actin, 5) C-terminus, 6) Cterminus and Rh-PSD, 7) C-terminus and actin, 8) N-terminus, 9) N-terminus and RhPSD, 10) N-terminus and actin. Only UV fluorescence from the tetramethylrhodamine labeled PSD appears on this gel.
92 12 11 10 9 8 7 6 5 4 3 2 1 Figure 3-9. UV photo of 1% native Tris-tricine ag arose gel of MARCKS proteins with actin and rhodamine-labeled PSD. 1) Actin, 2) MARCKS, 3) MARCKS and Rh-PSD, 4) MARCKS and actin, 5) C-terminus, 6) C-te rminus and Rh-PSD, 7) C-terminus and actin, 8) N-terminus, 9) N-terminus and Rh-PSD, 10) N-terminus and actin, 11) RhPSD, 12) Rh-PSD. Only UV fluorescence fr om the tetramethylrhodamine labeled PSD appears on this gel.
93 1 2 3 4 5 Actin N-terminus Possible complex Figure 3-10. MARCKS N-terminus forms oligomers in a native gel. 7% Tris-Tricine native gel with 1) 4 M G-actin, 2) 15 M N-terminus, 3) 18.5 M N-terminus, 4) 15 M Nterminus + 4 M G-actin 5) 18.5 M N-terminus + 4 M G-actin. Lanes 4 and 5 contain an extra band that may represen t a complex between MARKCS N-terminus and actin. .
94 CHAPTER 4 MARCKS AND TNF Introduction As previously described, TNF induces reorganization of the ac tin cytoskeleton in different cell types [75, 111, 112, 118, 121, 122], resulting in filopodia and lamellopodia formation, stress fibers, F-actin polymerization or depolymerization, actin synthesis, membrane ruffles, chemotaxis and migration. In endothelial cells these effects were f ound to be mediated by members of the Rho family of small GTPases, Rho, Rac and Cdc42 . In addition, TNFinduced apoptosis was shown to require rearrangement of the actin cytoskeleton, which was mediated by Rho-kinase . In contrast, TNF was found to elicit antiapoptotic effects in opossum kidney cells also via redistribution of the actin cytoskeleton through inhibition of caspase-3; this was governed by the phosphatid ylinositol-3 kinase, Cdc42/Rac1, and phospholipase1 . The actin cytoskeleton plays an important role in TNF-induced apoptosis, via multiple mechanisms, including delivering apoptosis proteins to where they need to be and causing morphological changes typical of apoptosis such as membrane blebbing [231, 232]. As mentioned previously, MARCK S was shown to constitute 90% of proteins synthesized de novo in response to stimulation with TNF in neutrophils and macrophages [71, 72]. This induction of MARCKS suggests it plays an important role in the signal transduction pathway of this cytokine. MARCKS may play a role in mediating the effects observed on the actin cytoskeleton in response to TNF. MARCKS phosphorylation by both PK C and Rho-kinase [8, 70], two kinases found downstream of TNF and important for act in reorganization, further supports this hypothesis.
95 To elucidate the role of MARCKS in th e TNF signaling pathway, we used RNA interference to knock down MARC KS in the human promyelocytic leukemia cell line HL-60, followed by treatment with TNF, RNA isolation and use of a PCR microarray of TNF ligands and receptors that profiles the expression of 84 genes whose expression is controlled by or involved in the TNF ligand and TNF receptor signaling pathways. Members of the TNF Superfamily and TNF Receptor Superfamily are re presented on this array (Tables 4-1 and 4-2). Methods Culture conditions and nucleofection HL-60 cells were grown in Iscove's Modi fied DMEM (ATCC Cat. No. 30-2005), 100 g/ml streptomycin, 100 U/ml penicillin and 20% fetal bovine serum (FBS) in a humidified 37C incubator with 6%CO2. Medium was replaced 2-3 times per week (30 mL per 162 cm2 flask). Cells were seeded at a density of 1x105 cells/mL and passaged at a density of 7-8x105 cells/mL. Cells were passaged 3 days before nucleofect ion, and density for nucleofection was 5-7x105 cells/mL. Each sample required 2x106 cells. Once cells reached the requied density for nucleofection, 2x106 cells were centrifuged at 90xg for 10 mi nutes at room temperature. The supernatant was discarded completely and the pellet was resuspended in 100 L room temperature Cell Line Nucleofect or Solution V. Cells should not be stored longer than 15 minutes in this solution. Two and a half g of siRNA (MARCKS or Non-Targeting) were pipetted into the 100 L cell suspension and the nucleofectio n sample was transfered into an amaxa certified cuvette, making sure the sample covered the bottom and that there were no air bubbles. Cuvettes were placed in Nucloefector II cuvette holder and program T-019 was run. Immediately following nucleofection, cuvettes are taken out and 500 L pre-warmed media were added. Samples were removed from cuvette usin g an Amaxa certified pipette and placed in a
96 well with 1.5 mL pre-warmed media in a 12-well plate. The plate was placed in a humidified 37C incubator with 6%CO2. TNF treatment and qRT-PCR Twenty four hours after nucleofection, cells were scraped an d split into 2 wells of a 12well plate. One mL fresh culture medium was a dded to each well. Cells were counted and their viability determined using Trypan blue. Forty eight hours after nucleofection, one well of each sample was treated with 200 U/mL TNF. The ot her well was an untreated control. This was done for both MARCKS and Non-targeting siR NA treated cells. Four hours after TNF treatment, reactions were inhibited by addition of 1 mM EGTA and placing flasks on ice. Cells were scraped and wells washed with 1 mL PB S + 1 mM EDTA. Cells were pelleted mRNA isolated using QIAGENs RNeasy RNA purification kit. To verify MARCKS knockdown, qRTPCR was carried out as described in Chapter 4. Briefly, following DNase I treatment of mRNA, concentrations were determined, 2 g of RNA were reverse transcribed into cDNA and used in qRT-PCR using TaqMan probes for MARCKS a nd GAPDH. After verification of knockdown as determined by qRT-PCR, the Human Tumor Necrosis Factor (TNF ) Ligand and Receptor Signaling Pathways RT Profiler PCR Array wa s used (SuperArray, Cat. No. APHS-063) by following the kits instructions. Results Nucleofection of MARCKS (Dharmacon, human MARCKS smart pool reagent Cat. No. M-00477200-0050) and non-target ing siRNAs (Dharmacon, Non-Targeting siRNA Pool #2 Cat. No. NC9554221) into HL-60 cells using Amaxas Nucleofector II machine and the HL-60 Cell Line Nucleofector Kit V (Amaxa, Cat. No. VC A-1003) successfully silenced MARCKS message in HL-60 cells 48 hours after nucleofection. Cell s had a viability of 75-78% as determined by a Trypan blue exclusion test of cell viability test. To determine whether MARCKS was
97 successfully silenced, we used qRT-PCR with TaqMan probes for MARCKS and GAPDH as described in Chapter 4. As determined by qRT-PCR using the Ct method and GAPDH as a housekeeping control gene, MARCKS was found to be knocked down to about 18% of control non-targeting siRNA-transfected HL-60 cells (Table 4-4). Four TNF SuperArrays were r un: non-targeting siRNA transf ected HL-60 cells with and without TNF treatment and MARCKS siRNA transfected HL-60 cells with and without TNF treatment. For each RNAi sample, the non-TNF treated sample was used as a control for quantifying fold change in each gene in respons e to TNF treatment using the delta delta Ct method. Fold changes thus obtained were then compared to determine the effect of silencing MARCKS on genes involved in TNF signaling (Table 4-5). Listed below are TNF SuperArra y genes grouped functionally; genes that are up-regulated more than 1 fold in HL-60 cells where MARCKS was silenced by RNA interference following treatment with TNF are shown in red, those that were down-regulated more than 1 fold following TNF treatment are shown in blue. Of 71 genes assayed, 31 had less than a 1 fold difference between TNF-treated MARCKS silenced or unsilenced cells, 33 were more than 1 fold upregulated in MARCKS knock down cells following TNF treatment, and only 7 were more than 1 fold downregulated compared to TNF-treat ed non silenced HL-60 cells. Knocking down MARCKS expression seems to affect expression of genes from all pathways of TNF, including those that are important to the inflammatory response and apoptosis. TNF Superfamily Members: Induction of Apoptosis: FASLG, LTA TNFSF10 TNFSF14, TNFSF8. Caspase Activation: TNFSF15. Caspase Inhibition: TNFSF14. Anti-apoptosis Genes: CD40LG TNF TNFSF18. Other Apoptosis-Related Ge nes: CD70 (TNFSF7), TNFSF9. Inflammatory Response: CD40LG TNF
98 NF B Signaling Pathway: FASLG, TNF TNFSF10, TNFSF14, TNFSF15. Other TNF Superfamily Members: LTB PGLYRP1 TNFSF11 TNFSF12, TNFSF13 TNFSF13B, TNFSF4, TNFSF5IP1. TNF Receptor Superfamily Members: Induction of Apoptosis: FAS, TNFRSF10A TNFRSF10B, TNFRSF19 TNFRSF25, CD27 (TNFRSF7), TNFRSF9, TRADD. Caspase Activation: TNFRSF10A TNFRSF10B. Caspase Inhibition: CD27 (TNFRSF7). Anti-apoptosis Genes: FAS, TNFRSF10D TNFRSF18 TNFRSF6B CD27 (TNFRSF7). Other Apoptosis Genes: CD40, LTBR, NGFR, TNFRSF10C, TNFRSF11B TNFRSF12A, TNFRSF14, TNFRSF1A TNFRSF1B, TNFRSF21. Inflammatory Response: CD40, TNFRSF1A NF B Signaling Pathway: CD40, EDA2R, LTBR, TNFRSF10A TNFRSF10B, TNFRSF1A CD27 (TNFRSF7), TRADD. JNK Signaling Pathway: EDA2R, TNFRSF19 CD27 (TNFRSF7). Other TNF Receptor Superfamily Members: TNFRSF11A TNFRSF13B TNFRSF13C TNFRSF17, TNFRSF19L TNFRSF4, TNFRSF8. TNFR1 Signaling Pathway: Induction of Apoptosis: CASP3 CRADD, FADD, TRADD. Caspases: CASP2, CASP3 CASP8. Anti-apoptosis Genes: BAG4 CASP2, TNF Other Apoptosis Genes: DFFA, PAK1, TNFRSF1A, TRAF2. Inflammatory Response: TNF TNFRSF1A NF B Signaling Pathway: CASP8, FADD, TNF TNFRSF1A TRADD. JNK Signaling Pathway: MAP2K4, MAPK8 PAK1. Transcription Regulators: JUN, PARP1, RB1, TNF TNFRSF1A TNFR1 Signaling Pathway: ARHGDIB, CAD HRB, LMNA LMNB1, LMNB2, MADD MAP3K1 MAP3K7, PAK2, PRKDC, SPTAN1 TNFR2 Signaling Pathway: Induction of Apoptosis: IKBKG, LTA TRAF3. Anti-apoptosis Genes: NFKB1, TNFAIP3 Other Apoptosis Genes: NFKBIA TNFRSF1B, TRAF1, TRAF2. Inflammatory Response: NFKB1. NF B Signaling Pathway: CHUK IKBKB IKBKG, NFKBIA TNFAIP3. Transcription Regulators: IKBKB IKBKG, NFKB1, NFKBIA TNFR2 Signaling Pathway: DUSP1 HRB, IKBKAP, MAP3K1 MAP3K14 TANK
99 Interestingly, there seemed to be an upregulation of anti-a poptotic genes (5: BAG4/SODD, CD154/CD40L, DIF/TNF-alpha, AITR/GITR, DCR3/M68) and a down-regulation of pro-apoptotic genes (2: CPP32/CP P32B, APO2L/Apo-2L); of course, there were exceptions: 3 up-regulated pro-apoptotic genes (APT1LG1 /CD178,LT/TNFB,TAJ/TAJ-alpha) and 1 downregulated anti-apoptotic gene (CD264/DCR2; Table 4-6). In genes marked as no change between MARCKS RNAi and non-targeting RNAi TNF-treated samples, there was also upregulation of 3 anti-apoptotic genes, down-regulation of 5 pro-apoptotic genes and upregulation of 2 pro-apoptotic genes (Table 4-6). In addition, knocking down MARCKS seemed to cause a general down-regulation of the expression of genes involved in TNF signaling (Tab les 4-7 to 4-9, last column). This was measured by using results for non-targeting siRNA treated HL-60 cells without TNF treatment as a control and comparing it to MARCKS siRNA tr eated HL-60 cells also wihoout TNF treatment using the Ct method. When MARCKS was knocked down, 58 genes were downregulated and 22 were up-regulated. Following TNF treat ment, genes that were up-regulated due to MARCKS RNAi, end up being le ss up-regulated and sometimes down-regulated compared to non-targeting RNAi treated samples. Similarl y, genes that were downregulated due to MARCKS RNAi are up-regulated in response to TNF treatment. Curiously, MARCKS RNAi treated HL-60 cells behaved similarly to non-targeting siRNA treated HL-60 cells that ha ve been treated with TNF. Of 73 genes, 27 behaved the same way in non-TNF treated MARCKS silenced HL60 cells and TNF-treated non-targeting siRNA cells, 23 genes were unchanged due to any treatme nt or RNA interference, and 23 were the same in TNF-treated MARCKS silenced and un-silenced HL -60 cells. In all, 50 out of 73 genes acted the same way in MARCKS RNAi treated HL-60 cells and non-targeti ng siRNA treated HL-60
100 cells that have been treated with TNF. Alt hough many more experiments would have to be done to test this hypothesis, these preliminary data suggest that MARCKS may play a role in a negative feedback loop initiated by TNF in HL-60 cells to silence itself. Conclusion MARCKS has many reported functions, many of which appear to be related to its interactions with actin. It binds and cross-links actin filaments, resulting in the remodeling of the actin cytoskeleton and cytoskeletal processes, i.e. phagocytosis, endocytosis, cell morphology, motility, and adhesion to the extracellular matrix. Many of these processes are also important for apoptosis. For example, the actin cytoskeleton is required for delivering al l the proteins required to the death receptors to activate caspases and tr ansduce the apoptotic signal from the receptor. For instance, the cytoskeletal protein ezrin wh ich provides a link between the plasma membrane and the actin cytoskeleton, was shown to be requ ired for mediating Fas-induced apoptosis . As previously described, the binding of TNF to its receptors ca uses an immediate polymerization and/or depolymerization of the actin cytoskeleton, depending on ce ll type. Ruffles, filopodia, lamelopodia and stress fibers have all been de scribed to occur following TNF treatment (see Chapter 1). As the phosphorylation site domain of MARCKS has been shown to influence actin polymerization and depolymerization, it can potentia lly play a role in these observed phenomena. During apoptosis, other cytoskeletal processe s such as endocytosis and phagocytosis also take place. Coincidentally, these two important cellular processes were reported to require MARCKS activity [29-33]. One to a few hours after the TNF receptor is bound by TNF molecules, the TNFR is internalized by endocytosis, a process that is essen tial for the rest of the apoptotic pathway to successfully resume as we ll as to down-regulate th e TNF signal [234, 235]. Interestingly, if cells are pretreated with PMA, an activator of PKC, this endocytosis is inhibited, and TNF is not down-regulated. As the major substrate for PKC and having such an
101 important role in endocytosis, MARCKS is certainly a likely player in the process of TNF receptor internalization. Finally, phagocytosis is an esse ntial step in apoptosis which en sures that dying cells do not spill their content, causing inflammation and autoi mmunity. Debris from dying cells is packaged and phagocytosed by neighboring cells. Phagocyt osis has also been shown to require MARCKS to take place.
102 Table 4-1. TNF Superarray arra y layout. Cat. No. APHS-063. 1 2 3 4 5 6 7 8 9 10 11 12 ARHGDIB BAG4 CAD CASP2 CASP3 CASP8 CD40 CD40LG CHUK CRADD DFFA DUSP1 EDA2R FADD FAS FASLG HRB IKBKAP IKBKB IKBKG JUN LMNA LMNB1 LMNB2 LTA LTB LTBR MADD MAP2K4 MAP3K1 MAP3K7 MAPK8 NFKB1 NFKBIA NGFR PAK1 PAK2 PARP1 PGLYRP1 PRKDC RB1 SPTAN1 TNF TNFAIP3 TNFRSF10ATNFRSF10BTNFRSF10CTNFRSF10D TNFRSF11A TNFRSF11B TNFRSF12A TNFRSF13BTNFRSF13CTNFRSF14 TNFRSF17 TNFRSF18 TNFRSF19 RELT TNFRSF1A TNFRSF1B TNFRSF 21 TNFRSF25 TNFRSF4 TNFRSF6B CD27 TNFRSF8 TNFRSF9 TNFSF10 TNFSF11 TNFSF12 TNFSF13 TNFSF13B TNFSF14 TNFSF15 TNFSF18 TNFSF4 PSMG2 CD70 TNFSF8 TNFSF9 TRADD TRAF1 TRAF2 TRAF3 B2M HPRT1 RPL13A GAPDH ACTB HGDC RTC RTC RTC PPC PPC PPC
103 Table 4-2. TNF SuperArray gene table. Cat. No. APHS-063. Well UniGene RefSeq Symbol Description Gene Name A01 Hs.504877 NM_001175 ARHGDIB Rho GDP dissociation inhibitor (GDI) beta D4/GDIA2 A02 Hs.194726 NM_004874 BAG4 BCL2-associated athanogene 4 BAG-4/SODD A03 Hs.377010 NM_004341 CAD Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase CAD A04 Hs.368982 NM_032982 CASP2 Caspase 2, apoptosis-related cystei ne peptidase (neural precursor cell expressed, developmentally down-regulated 2) CASP-2/ICH-1L A05 Hs.141125 NM_004346 CASP3 Caspase 3, apoptosis-related cysteine peptidase CPP32/CPP32B A06 Hs.591630 NM_001228 CASP8 Caspase 8, apoptosis-related cysteine peptidase CAP4/FLICE A07 Hs.472860 NM_001250 CD40 CD40 molecule, TNF receptor superfamily member 5 Bp50/CDW40 A08 Hs.592244 NM_000074 CD40LG CD40 ligand (TNF superfamily, member 5, hyper-IgM syndrome) CD154/CD40L A09 Hs.198998 NM_001278 CHUK Conserved helix-loop-helix ubiquitous kinase IKBKA/IKK-alpha A10 Hs.38533 NM_003805 CRADD CASP2 and RIPK1 domain containing adaptor with death domain RAIDD A11 Hs.484782 NM_004401 DFFA DNA fragmentation factor, 45kDa, alpha polypeptide DFF-45/DFF1 A12 Hs.171695 NM_004417 DUSP1 Dual specificity phosphatase 1 CL100/HVH1 B01 Hs.302017 NM_021783 EDA2R Ectodysplasin A2 receptor EDA-A2R/EDAA2R B02 Hs.86131 NM_003824 FADD Fas (TNFRSF6)-associated via death domain GIG3/MORT1 B03 Hs.244139 NM_000043 FAS Fas (TNF receptor superfamily, member 6) ALPS1A/APO-1 B04 Hs.2007 NM_000639 FASLG Fas ligand (TNF superfamily, member 6) APT1LG1/CD178 B05 Hs.591619 NM_004504 HRB HIV-1 Rev binding protein RAB/RIP
104 Table 4-2. Continued 1. Well UniGene RefSeq Symbol Description Gene Name B06 Hs.494738 NM_003640 IKBKAP Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein DKFZp781H1425/ DYS B07 Hs.413513 NM_001556 IKBKB Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta IKK-beta/IKK2 B08 Hs.43505 NM_003639 IKBKG Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma FIP-3/FIP3 B09 Hs.525704 NM_002228 JUN Jun oncogene AP1/c-Jun B10 Hs.594444 NM_005572 LMNA Lamin A/C CDCD1/CDDC B11 Hs.89497 NM_005573 LMNB1 Lamin B1 LMN/LMN2 B12 Hs.538286 NM_032737 LMNB2 Lamin B2 LAMB2/LMN2 C01 Hs.36 NM_000595 LTA Lymphotoxin alpha (TNF superfamily, member 1) LT/TNFB C02 Hs.376208 NM_002341 LTB Lymphotoxin beta (TNF superfamily, member 3) TNFC/TNFSF3 C03 Hs.1116 NM_002342 LTBR Lymphotoxin beta receptor (TNFR superfamily, member 3) CD18/D12S370 C04 Hs.82548 NM_003682 MADD MAP-kinase activating death domain DENN/IG20 C05 Hs.514681 NM_003010 MAP2K4 Mitogen-activated protein kinase kinase 4 JNKK/JNKK1 C06 Hs.634810 XM_042066 MAP3K1 Mitogen-activated protein kinase kinase kinase 1 MAPKKK1/ MEKK C07 Hs.652105 NM_003188 MAP3K7 Mitogen-activated protein kinase kinase kinase 7 TAK1/TGF1a C08 Hs.138211 NM_002750 MAPK8 Mitogen-activated protein kinase 8 JNK/JNK1 C09 Hs.431926 NM_003998 NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105) DKFZp686C01211/ EBP-1 C10 Hs.81328 NM_020529 NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha IKBA/MAD-3
105 Table 4-2. Continued 2. Well UniGene RefSeq Symbol Description Gene Name C11 Hs.415768 NM_002507 NGFR Nerve growth factor receptor (TNFR superfamily, member 16) CD271/ TNFRSF16 C12 Hs.435714 NM_002576 PAK1 P21/Cdc42/Rac1-activated kinase 1 (STE20 homolog, yeast) PAKalpha D01 Hs.518530 NM_002577 PAK2 P21 (CDKN1A)-activated kinase 2 PAK65/ PAKgamma D02 Hs.177766 NM_001618 PARP1 Poly (ADP-ribose) polymerase family, member 1 ADPRT/ ADPRT1 D03 Hs.137583 NM_005091 PGLYRP1 Peptidoglycan recognition protein 1 PGLYRP/PGRP D04 Hs.491682 NM_006904 PRKDC Protein kinase, DNA-activat ed, catalytic polypeptide DNAPK/DNPK1 D05 Hs.408528 NM_000321 RB1 Retinoblastoma 1 (including osteosarcoma) OSRC/RB D06 Hs.372331 NM_003127 SPTAN1 Spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) II-SPECTRIN D07 Hs.241570 NM_000594 TNF Tumor necrosis factor (TNF superfamily, member 2) DIF/TNF-alpha D08 Hs.211600 NM_006290 TNFAIP3 Tumor necrosis factor, alpha-induced protein 3 A20/OTUD7C D09 Hs.591834 NM_003844 TNFRSF10A Tumor necrosis factor receptor superfamily, member 10a APO2/CD261 D10 Hs.521456 NM_003842 TNFRSF10B Tumor necrosis factor receptor superfamily, member 10b CD262/DR5 D11 Hs.655801 NM_003841 TNFRSF10C Tumor necrosis factor receptor superfa mily, member 10c, decoy without an intracellular domain CD263/DCR1 D12 Hs.213467 NM_003840 TNFRSF10D Tumor necrosis factor receptor supe rfamily, member 10d, decoy with truncated d eath domain CD264/DCR2 E01 Hs.204044 NM_003839 TNFRSF11A Tumor necrosis factor receptor supe rfamily, member 11a, NFKB activator CD265/ODFR E02 Hs.81791 NM_002546 TNFRSF11B Tumor necrosis factor receptor superfa mily, member 11b (osteoprotegerin) OCIF/OPG E03 Hs.355899 NM_016639 TNFRSF12A Tumor necrosis factor receptor superfamily, member 12A CD266/FN14
106 Table 4-2. Continued 3. Well UniGene RefSeq Symbol Description Gene Name E04 Hs.158341 NM_012452 TNFRSF13B Tumor necrosis factor receptor superfamily, member 13B CD267/CVID E05 Hs.344088 NM_052945 TNFRSF13C Tumor necrosis factor receptor superfamily, member 13C BAFF-R/BAFFR E06 Hs.512898 NM_003820 TNFRSF14 Tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry mediator) ATAR/HVEA E07 Hs.2556 NM_001192 TNFRSF17 Tumor necrosis factor receptor superfamily, member 17 BCM/BCMA E08 Hs.212680 NM_004195 TNFRSF18 Tumor necrosis factor receptor superfamily, member 18 AITR/GITR E09 Hs.149168 NM_018647 TNFRSF19 Tumor necrosis factor receptor superfamily, member 19 TAJ/TAJ-alpha E10 Hs.533720 NM_032871 TNFRSF19L Tumor necrosis factor receptor superfamily, member 19-like RELT E11 Hs.279594 NM_001065 TNFRSF1A Tumor necrosis factor receptor superfamily, member 1A CD120a/FPF E12 Hs.256278 NM_001066 TNFRSF1B Tumor necrosis factor receptor superfamily, member 1B CD120b/TBPII F01 Hs.443577 NM_014452 TNFRSF21 Tumor necrosis factor receptor superfamily, member 21 BM-018/DR6 F02 Hs.462529 NM_003790 TNFRSF25 Tumor necrosis factor receptor superfamily, member 25 APO-3/DDR3 F03 Hs.129780 NM_003327 TNFRSF4 Tumor necrosis factor receptor superfamily, member 4 ACT35/CD134 F04 Hs.554786 NM_003823 TNFRSF6B Tumor necrosis factor receptor superfamily, member 6b, decoy DCR3/M68 F05 Hs.355307 NM_001242 CD27 CD27 molecule S152/T14 F06 Hs.1314 NM_001243 TNFRSF8 Tumor necrosis factor receptor superfamily, member 8 CD30/D1S166E F07 Hs.193418 NM_001561 TNFRSF9 Tumor necrosis factor receptor superfamily, member 9 4-1BB/CD137 F08 Hs.478275 NM_003810 TNFSF10 Tumor necrosis factor (ligand) superfamily, member 10 APO2L/Apo-2L F09 Hs.333791 NM_003701 TNFSF11 Tumor necrosis factor (ligand) superfamily, member 11 CD254/ODF
107 Table 4-2. Continued 4. Well UniGene RefSeq Symbol Description Gene Name F10 Hs.415839 NM_003809 TNFSF12 Tumor necrosis factor (ligand) superfamily, member 12 APO3L/DR3LG F11 Hs.54673 NM_003808 TNFSF13 Tumor necrosis factor (ligand) superfamily, member 13 APRIL/CD256 F12 Hs.525157 NM_006573 TNFSF13B Tumor necrosis factor (ligand) superfamily, member 13b BAFF/BLYS G01 Hs.129708 NM_003807 TNFSF14 Tumor necrosis factor (ligand) superfamily, member 14 CD258/HVEML G02 Hs.241382 NM_005118 TNFSF15 Tumor necrosis factor (ligand) superfamily, member 15 TL1/TL1A G03 Hs.248197 NM_005092 TNFSF18 Tumor necrosis factor (ligand) superfamily, member 18 AITRL/GITRL G04 Hs.181097 NM_003326 TNFSF4 Tumor necrosis factor (ligand) superfamily, member 4 (tax-transcriptionally activated glycoprotein 1, 34kDa) CD134L/CD252 G05 Hs.464652 NM_020232 TNFSF5IP1 Tumor necrosis factor superfamily, member 5-induced protein 1 CLAST3/HCCA3 G06 Hs.501497 NM_001252 CD70 CD70 molecule CD27L/CD27LG G07 Hs.494901 NM_001244 TNFSF8 Tumor necrosis factor (ligand) superfamily, member 8 CD153/CD30L G08 Hs.1524 NM_003811 TNFSF9 Tumor necrosis factor (ligand) superfamily, member 9 4-1BB-L G09 Hs.460996 NM_003789 TRADD TNFRSF1A-associated via death domain Hs.89862 G10 Hs.531251 NM_005658 TRAF1 TNF receptor-associated factor 1 EBI6/MGC:10353 G11 Hs.522506 NM_021138 TRAF2 TNF receptor-associated factor 2 MGC:45012/ TRAP G12 Hs.510528 NM_003300 TRAF3 TNF receptor-associated factor 3 CAP-1/CD40bp H01 Hs.534255 NM_004048 B2M Beta-2-microglobulin B2M H02 Hs.412707 NM_000194 HPRT1 Hypoxanthine phosphoribosyltransferase 1 (Lesch-Nyhan syndrome) HGPRT/HPRT H03 Hs.546356 NM_012423 RPL13A Ribosomal protein L13a RPL13A
108 Table 4-2. Continued 5. Well UniGene RefSeq Symbol Description Gene Name H04 Hs.544577 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase G3PD/GAPD H05 Hs.520640 NM_001101 ACTB Actin, beta PS1TP5BP1 H06 N/A X66290 HGDC Human Genomic DNA Contamination HGDC H07 N/A SA_00104 RTC Reverse Transcription Control RTC H08 N/A SA_00104 RTC Reverse Transcription Control RTC H09 N/A SA_00104 RTC Reverse Transcription Control RTC H10 N/A SA_00103 PPC Positive PCR Control PPC H11 N/A SA_00103 PPC Positive PCR Control PPC H12 N/A SA_00103 PPC Positive PCR Control PPC
109 Table 4-3. Cell viability 48 hours after nucleofection. MARCKS siRNA Non-Targeting siRNA Total # dead Total # dead count 1 18 5 36 8 count 2 26 5 34 13 count 3 36 10 24 5 count 4 23 3 24 4 Total 103 23 118 30 % Viability 77.7 74.6
110 Table 4-4. Results of qRT-PCR to measur e MARCKS knockdown by RNA interference. Delt a delta Ct method was used to calculate MARCKS knockdown, using sample nucleofected with non-targeting siRNA as control. Sample Ct GAPDH Ct TNF Ct MARCKS Ct MARCKSCt GAPDH % of Control HL-60, 48hr MARCKS siRNA, no TNF20.116 24.686 24.642 4.5 17.8 HL-60, 48hr Non-Targeting siRNA, no TNF24.55 27.323 26.589 2.0 100.0
111 Table 4-5. TNF SuperArray results following RNA interference in response to TNF treatment. Well Symbol Gene Name Non-Targeting RNAi fold change MARCKS RNAi fold change Absolute Difference A01 ARHGDIB D4/GDIA2 1.25 1.40 0.14 A02 BAG4 BAG-4/SODD 1.24 1.13 2.37 A03 CAD CAD 5.51 -1.07 4.44 A04 CASP2 CASP-2/ICH-1L N/A 1.04 N/A A05 CASP3 CPP32/CPP32B 1.71 -1.14 2.85 A06 CASP8 CAP4/FLICE 1.24 -1.82 0.58 A07 CD40 Bp50/CDW40 11.61 5.95 5.66 A08 CD40LG CD154/CD40L 1.43 1.02 2.45 A09 CHUK IKBKA/IKK-alpha 2.07 1.10 3.18 A10 CRADD RAIDD 1.45 -1.41 0.03 A11 DFFA DFF-45/DFF1 1.16 -1.20 0.05 A12 DUSP1 CL100/HVH1 3.17 -2.02 1.14 B01 EDA2R EDA-A2R/EDAA2R 1.43 1.02 2.45 B02 FADD GIG3/MORT1 1.21 -1.01 0.20 B03 FAS ALPS1A/APO-1 2.17 2.87 0.70 B04 FASLG APT1LG1/CD178 1.43 1.02 2.45 B05 HRB RAB/RIP 1.31 -1.24 0.08 B06 IKBKAP DKFZp781H1425/DYS 1.08 1.10 0.03 B07 IKBKB IKK-beta/IKK2 1.11 1.24 2.35 B08 IKBKG FIP-3/FIP3 1.55 -1.02 0.53 B09 JUN AP1/c-Jun 2.15 -1.43 0.72 B10 LMNA CDCD1/CDDC 1.37 1.17 2.54 B11 LMNB1 LMN/LMN2 1.48 -1.30 0.18 B12 LMNB2 LAMB2/LMN2 1.72 -1.08 0.64 C01 LTA LT/TNFB 2.33 1.99 4.32 C02 LTB TNFC/TNFSF3 1.6 2.68 1.08 C03 LTBR CD18/D12S370 1.06 -1.06 0.00 C04 MADD DENN/IG20 1.18 1.03 2.21 C05 MAP2K4 JNKK/JNKK1 1.32 1.13 0.19 C06 MAP3K1 MAPKKK1/MEKK 1.04 1.26 2.30 C07 MAP3K7 TAK1/TGF1a 1.48 -1.59 0.11 C08 MAPK8 JNK/JNK1 3.01 1.04 1.97 C09 NFKB1 DKFZp686C01211/EBP-1 2.07 2.52 0.45 C10 NFKBIA IKBA/MAD-3 4.85 9.60 4.75 C11 NGFR CD271/TNFRSF16 2.02 -1.15 0.86 C12 PAK1 PAKalpha 1.51 -1.34 0.16
112 Table 4-5. Continued 1. Well Symbol Gene Name Non-Targeting RNAi fold change MARCKS RNAi fold change Absolute Difference D01 PAK2 PAK65/PAKgamma 1.04 -1.01 0.03 D02 PARP1 ADPRT/ADPRT1 1.17 -1.13 0.04 D03 PGLYRP1 PGLYRP/PGRP 1.14 1.09 2.23 D04 PRKDC DNAPK/DNPK1 1.67 1.59 0.07 D05 RB1 OSRC/RB 2.62 2.38 0.23 D06 SPTAN1 (ALPHA)II-SPECTRIN 1.43 1.02 2.45 D07 TNF DIF/TNF-alpha 1.07 1.37 2.44 D08 TNFAIP3 A20/OTUD7C 8.68 7.12 1.56 D09 TNFRSF10A APO2/CD261 2.48 -1.45 1.03 D10 TNFRSF10B CD262/DR5 1.38 1.50 0.11 D11 TNFRSF10C CD263/DCR1 1.6 -1.17 0.43 D12 TNFRSF10D CD264/DCR2 2.54 1.02 1.52 E01 TNFRSF11A CD265/ODFR 1.04 2.85 1.81 E02 TNFRSF11B OCIF/OPG 1.08 -3.38 4.47 E03 TNFRSF12A CD266/FN14 1.99 -1.30 0.69 E04 TNFRSF13B CD267/CVID 1.43 1.02 2.45 E05 TNFRSF13C BAFF-R/BAFFR 5.29 1.16 6.45 E06 TNFRSF14 ATAR/HVEA 1.26 1.44 2.70 E07 TNFRSF17 BCM/BCMA 1.74 9.66 7.93 E08 TNFRSF18 AITR/GITR 1.62 1.21 2.82 E09 TNFRSF19 TAJ/TAJ-alpha 1.43 1.48 2.90 E10 TNFRSF19L RELT 1.42 1.87 3.29 E11 TNFRSF1A CD120a/FPF 2.87 1.24 4.11 E12 TNFRSF1B CD120b/TBPII 1.02 1.03 0.01 F01 TNFRSF21 BM-018/DR6 1.18 -1.73 0.54 F02 TNFRSF25 APO-3/DDR3 1.43 -1.90 0.48 F03 TNFRSF4 ACT35/CD134 1.51 8.53 7.02 F04 TNFRSF6B DCR3/M68 1.86 2.70 4.56 F05 CD27 S152/T14 1.23 2.02 0.79 F06 TNFRSF8 CD30/D1S166E 1.19 -1.94 0.75 F07 TNFRSF9 4-1BB/CD137 6.18 5.29 0.89 F08 TNFSF10 APO2L/Apo-2L 4.22 1.86 2.36 F09 TNFSF11 CD254/ODF 1.43 1.02 2.45 F10 TNFSF12 APO3L/DR3LG 1.06 2.74 1.68 F11 TNFSF13 APRIL/CD256 1.99 1.00 2.99 F12 TNFSF13B BAFF/BLYS 91.61 234.35 142.73
113 Table 4-5. Continued 2. Well Symbol Gene Name Non-Targeting RNAi fold change MARCKS RNAi fold change Absolute Difference G01 TNFSF14 CD258/HVEML N/A N/A N/A G02 TNFSF15 TL1/TL1A N/A N/A N/A G03 TNFSF18 AITRL/GITRL N/A N/A N/A G04 TNFSF4 CD134L/CD252 N/A N/A N/A G05 TNFSF5IP1 CLAST3/HCCA3 N/A N/A N/A G06 CD70 CD27L/CD27LG N/A N/A N/A G07 TNFSF8 CD153/CD30L N/A N/A N/A G08 TNFSF9 4-1BB-L N/A N/A N/A G09 TRADD Hs.89862 N/A N/A N/A G10 TRAF1 EBI6/MGC:10353 N/A N/A N/A G11 TRAF2 MGC:45012/TRAP N/A N/A N/A G12 TRAF3 CAP-1/CD40bp N/A N/A N/A H01 B2M B2M 1.06 1.19 0.13 H02 HPRT1 HGPRT/HPRT 8.04 1.09 6.95 H03 RPL13A RPL13A 1.19 -1.04 2.23 H04 GAPDH G3PD/GAPD 1.34 -1.24 0.10 H05 ACTB PS1TP5BP1 1.06 1.08 0.02 Genes with <1 fold change between MARCKS and non-targeting RNAi samples following TNF treatment shown in green; >1 fold higher in MARCKS RNAi samples shown in red and >1 fold lower than non-targeting in blue. N/A, not available.
114 Table 4-6. Differential expression of apoptosis genes in TNFtreated HL-60 cells following MARCKS and non-targeting RNAi. Gene Name Apoptosis Non-targeting RNAi fold change MARCKS RNAi fold change Effect BAG-4/SODD anti -1.24 1.13 up-regulated CPP32/CPP32B pro 1.71 -1.14 down-regulated CD154/CD40L anti -1.43 1.02 up-regulated RAIDD pro -1.45 -1.41 no change GIG3/MORT1 pro -1.21 -1.01 no change ALPS1A/APO-1 pro? anti? 2.17 2.87 no change APT1LG1/CD178 pro -1.43 1.02 up-regulated FIP-3/FIP3 pro -1.55 -1.02 no change LT/TNFB pro -2.33 1.99 up-regulated DKFZp686C01211/EBP-1 anti 2.07 2.52 no change DIF/TNF-alpha anti -1.07 1.37 up-regulated A20/OTUD7C anti 8.68 7.12 no change APO2/CD261 pro -2.48 -1.45 no change CD262/DR5 pro 1.38 1.50 no change CD264/DCR2 anti 2.54 1.02 down-regulated AITR/GITR anti -1.62 1.21 up-regulated TAJ/TAJ-alpha pro -1.43 1.48 up-regulated APO-3/DDR3 pro -1.43 -1.90 no change DCR3/M68 anti -1.86 2.70 up-regulated S152/T14 pro? anti? 1.23 2.02 no change 4-1BB/CD137 pro 6.18 5.29 no change APO2L/Apo-2L pro 4.22 1.86 down-regulated CD258/HVEML pro? anti? N/A N/A N/A TL1/TL1A pro N/A N/A N/A AITRL/GITRL anti N/A N/A N/A CD153/CD30L pro N/A N/A N/A Hs.89862 pro N/A N/A N/A CAP-1/CD40bp pro N/A N/A N/A
115 Table 4-7. Genes in TNF SuperArray whose expression was similar in TNF-treated non-targeting siRNA transfected cells and nonTNF-treated MARCKS siRNA transfected cells. Well Symbol Gene Name Non-Targeting RNAi fold change--with TNF MARCKS RNAi fold change--with TNF MARCKS RNAi fold change--No TNF A02 BAG4 BAG-4/SODD -1.24 1.13 -1.24 A03 CAD CAD -5.51 -1.07 -7.94 A05 CASP3 CPP32/CPP32B 1.71 -1.14 1.31 A08 CD40LG CD154/CD40L -1.43 1.02 -1.88 A09 CHUK IKBKA/IKK-alpha -2.07 1.10 -1.31 B01 EDA2R EDA-A2R/EDAA2R -1.43 1.02 -1.88 B04 FASLG APT1LG1/CD178 -1.43 1.02 -1.88 B07 IKBKB IKK-beta/IKK2 -1.11 1.24 -1.36 B10 LMNA CDCD1/CDDC -1.37 1.17 -1.59 C01 LTA LT/TNFB -2.33 1.99 -7.73 C04 MADD DENN/IG20 -1.18 1.03 -1.74 D03 PGLYRP1 PGLYRP/PGRP -1.14 1.09 -2.03 D06 SPTAN1 (ALPHA)II-SPECTRIN -1.43 1.02 -1.88 D07 TNF DIF/TNF-alpha -1.07 1.37 -1.88 E02 TNFRSF11B OCIF/OPG 1.08 -3.38 1.84 E04 TNFRSF13B CD267/CVID -1.43 1.02 -1.88 E05 TNFRSF13C BAFF-R/BAFFR -5.29 1.16 -6.77 E06 TNFRSF14 ATAR/HVEA -1.26 1.44 -1.61 E08 TNFRSF18 AITR/GITR -1.62 1.21 -4.11 E09 TNFRSF19 TAJ/TAJ-alpha -1.43 1.48 -1.55 E10 TNFRSF19L RELT -1.42 1.87 -2.13 E11 TNFRSF1A CD120a/FPF -2.87 1.24 -2.77 F04 TNFRSF6B DCR3/M68 -1.86 2.70 -4.11 F09 TNFSF11 CD254/ODF -1.43 1.02 -1.88 F11 TNFSF13 APRIL/CD256 -1.99 1.00 -2.55
116 Table 4-7. Continued. Well Symbol Gene Name Non-Targeting RNAi fold change--with TNF MARCKS RNAi fold change--with TNF MARCKS RNAi fold change--No TNF H02 HPRT1 HGPRT/HPRT 8.04 1.09 8.69 H03 RPL13A RPL13A 1.19 -1.04 1.43
117 Table 4-8. Genes in TNF SuperArray whose expressi on was unchanged in RNAi a nd TNF treated HL-60 cells. Well Symbol Gene Name Non-Targeting RNAi fold change--with TNF MARCKS RNAi fold change--with TNF MARCKS RNAi fold change--No TNF B05 HRB RAB/RIP -1.31 -1.24 -1.37 B08 IKBKG FIP-3/FIP3 -1.55 -1.02 -1.78 B09 JUN AP1/c-Jun -2.15 -1.43 -1.55 B11 LMNB1 LMN/LMN2 -1.48 -1.30 -1.30 B12 LMNB2 LAMB2/LMN2 -1.72 -1.08 -1.47 C03 LTBR CD18/D12S370 -1.06 -1.06 -1.16 C05 MAP2K4 JNKK/JNKK1 1.32 1.13 1.21 C07 MAP3K7 TAK1/TGF1a -1.48 -1.59 -1.45 C08 MAPK8 JNK/JNK1 3.01 1.04 2.89 C09 NFKB1 DKFZp686C01211/EBP-1 2.07 2.52 1.12 C11 NGFR CD271/TNFRSF16 -2.02 -1.15 -2.25 C12 PAK1 PAKalpha -1.51 -1.34 -1.59 D01 PAK2 PAK65/PAKgamma -1.04 -1.01 -1.01 D05 RB1 OSRC/RB 2.62 2.38 1.79 D08 TNFAIP3 A20/OTUD7C 8.68 7.12 1.32 D09 TNFRSF10A APO2/CD261 -2.48 -1.45 -1.74 D11 TNFRSF10C CD263/DCR1 -1.60 -1.17 -1.96 D12 TNFRSF10D CD264/DCR2 2.54 1.02 3.25 E03 TNFRSF12A CD266/FN14 -1.99 -1.30 -1.97 F06 TNFRSF8 CD30/D1S166E -1.19 -1.94 -1.27 F07 TNFRSF9 4-1BB/CD137 6.18 5.29 1.23 F08 TNFSF10 APO2L/Apo-2L 4.22 1.86 1.85 H04 GAPDH G3PD/GAPD -1.34 -1.24 -1.15
118 Table 4-9. Genes in TNF SuperArray whose expression was unchanged following TNF treatment of both MARCKS and nontargeting siRNA transfected HL-60 cells. Well Symbol Gene Name Non-Targeting RNAi fold change--with TNF MARCKS RNAi fold change--with TNF MARCKS RNAi fold change--No TNF A01 ARHGDIB D4/GDIA2 1.25 1.40 -1.38 A06 CASP8 CAP4/FLICE -1.24 -1.82 2.85 A10 CRADD RAIDD -1.45 -1.41 1.07 A11 DFFA DFF-45/DFF1 -1.16 -1.20 1.11 B02 FADD GIG3/MORT1 -1.21 -1.01 1.02 B03 FAS ALPS1A/APO-1 2.17 2.87 -1.28 B06 IKBKAP DKFZp781H1425/DYS 1.08 1.10 -1.11 C10 NFKBIA IKBA/MAD-3 4.85 9.60 -2.04 C02 LTB TNFC/TNFSF3 1.60 2.68 -1.93 D02 PARP1 ADPRT/ADPRT1 -1.17 -1.13 1.08 D04 PRKDC DNAPK/DNPK1 1.67 1.59 -1.16 D10 TNFRSF10B CD262/DR5 1.38 1.50 -1.15 E01 TNFRSF11A CD265/ODFR 1.04 2.85 -2.08 E07 TNFRSF17 BCM/BCMA 1.74 9.66 -7.62 E12 TNFRSF1B CD120b/TBPII 1.02 1.03 -1.53 F01 TNFRSF21 BM-018/DR6 -1.18 -1.73 1.18 F02 TNFRSF25 APO-3/DDR3 -1.43 -1.90 1.04 F03 TNFRSF4 ACT35/CD134 1.51 8.53 -1.68 F05 CD27 S152/T14 1.23 2.02 -1.72 F10 TNFSF12 APO3L/DR3LG 1.06 2.74 -1.58 F12 TNFSF13B BAFF/BLYS 91.61 234.35 -2.39 H01 B2M B2M 1.06 1.19 -1.14 H05 ACTB PS1TP5BP1 1.06 1.08 -1.09
119 CHAPTER 5 MARCKS IN RHEUMATOID ARTHRITIS Introduction Recent reports highlight the early onset of stru ctural damage in patients with rheumatoid arthritis (RA) [236, 237]. There is a lack of a distinct clinical, laborator y or radiological marker to support an early diagnosis of RA: diagnosis is defined by the 1987 criteria of the American College of Rheumatology, which have low sensitivity in early RA. Therefore, a more specific and sensitive marker for progressive RA is need ed which ideally should be present at an early stage of the disease. Standard immunosuppressive treatment regime ns have multiple potential complications, limiting their use in the absence of a definite diagnosis. Recently, TNFblockade therapy has emerged as a successful therapy for the treatment of this disease. However, these drugs are used only after the failure of standard drug regimens because of cost and lack of data for long-term safety . Overall, this leads to under-treatment of RA, resulting in deformity and disability that may have been avoided with early use of TNFblockade. Although a very strong tool, TNFblockade is not always successful. Due to its high cost and potentially harmful side effects, it would be useful to ha ve a test that allows differen tiation between cases where this therapy will be successful, versus those where it will not [239, 240]. An increase in the protein MA RCKS accounts for 90% or more of the cellular response to treatment with TNFin macrophages and monocytes [71, 72]. Based on its known function as a regulator of the actin cytoskeleton, [17, 241] we hypothesize th at MARCKS has a major functional role in the cellular response to TNFin RA and therefore MARCKS levels may have specificity for the diagnosis of RA . We r eason that MARCKS levels may be indicative of not only the extent of cu mulative stimulation by TNFin patients with RA, thus indicating
120 prognosis, but also that MARCKS le vels may correlate with the cap acity of these patients to respond to TNFblockade, thus predicting whether TNFblockade will be successful. Higher MARCKS mRNA or protein level in patients blood may predic t a better response to TNFblockade, and if MARCKS levels are comparable to normal constitutive levels, then therapy may be less effective. Even if MARCKS does not have a ma jor functional role in TNFsignaling, the striking increase in this protein ob served after chroni c stimulation by TNFmakes MARCKS an attractive candidate as a marker for RA disease, for prognosis, and for response to TNFblockade. One percent of the population has RA, with a female predominance. Disability and mortality are common in this disease. Recently the most common cause of death in RA was shown to be due to cardiovascular disease  Effective treatments begun early in the course of disease are the only known way to prevent disa bility . Factors associated with a bad outcome include delays in diagnosis and treatment. Therapy with TNFblockade has been shown to decrease both the occurren ce of disability and death  Presumably these therapies decrease the inflammation that is responsible for an increase in car diovascular events in RA. Yet the cost of TNFblockade is high and in the range of $20,000 per year when all associated costs are included. Our work has the potential to alter several factors related to disability and death, including improvements in early diagnosis and the potential to identify RA patients most likely to benefit from treatment with TNFblockade. If an individuals TNFis causally related to disease it may be the cellular responses to TNFrather than the time-dependent average level of TNFsecretion that is responsible for disease severity or activity. MARCKS or maybe levels of MARCKS metabolites (e.g., phosphorylated MARCKS, proteolyzed MARCKS, etc) may best reflect the level of
121 activated cellular response and therefore may be bett er predictive of disease. Specific findings related to MARCKS could tell us that a patient has severe RA or that the disease, in this particular patient, is caused by the cellular response to TNFand therefore this patient is a good candidate to respond to TNFinhibition. Study Subjects Patients and controls were recruited for th is study following IRB approval. We were aiming to collect blood samples from the four following subject groups: Group 1: patients presenti ng with early synovitis; Group 2: RA patients at different disease stages : untreated, treated with classical RA drugs, treated with TNF-inhibitors; Group 3: patients with other inflammatory polyar thritides (e.g. SLE, gout ); this is a control group; Group 4: healthy controls with similar age, sex and race characterist ics as study subjects; this is another control group. Initially, we were hoping to collect at le ast 10-15 blood samples from each of the four subject groups described above; however, we were unable to reach our goal in the time period of the study. In addition, some of our subjects did not return for follow up, making it impossible to track their progress and changes in MARCKS. Levels of MARCKS parameters were compared for early synovitis patients who later developed RA versus those who did not to asse ss the usefulness of MARCKS as a specific marker for diagnosis. We also compared MARCKS parameters in RA patients who responded to TNF blockade therapy versus those who failed th erapy to test the usef ulness of MARCKS as predictor of response to TNF blockade therapy. We looked for a correlation between a MARCKS-related parameter or a change in a MARCKS-related parameter, preand post-TNF blockade therapy, which would measure ac tivated MARCKS and thus indicate long-term
122 outcome, i.e. prognosis: pre-treatm ent levels may help determine whether a patient should be treated more or less aggressive ly, whereas post-treatment levels may help determine whether therapy has changed prognosis. Cell Isolation from Whole Blood Both mononuclear cells and granulocytes were isolated from whole blood of study subjects. Leukocytes were sepa rated using Histopaque-1119 (pol ysucrose, 6.0g/dl and sodium diatrizoate, 16.7g/dl, density=1.119) and Hist opaque-1077 (polysucrose, 5.7g/dl and sodium diatrizoate, 9.0g/dl, density=1.077) (Sigma-Aldri ch, St. Louis, MO). According to the SigmaAldrich procedure, a double grad ient is formed by layering an equal volume of Histopaque-1077 and Histopaque-1119. Whole blood is careful ly layered onto the upper Histopaque-1077 medium. The tubes are then centrifuged at 700x g for 30 minutes. Cells of the granulocytic series are found at the 1077/1119 interphase wh ereas lymphocytes, other mononuclear cells and platelets are found at the plasma/ 1077 interphase (Figure 5-1). Detailed Cell Isolation Protocol Whole blood was diluted 1:2 in room temperature 1xPBS. To 17 mL blood isolated in Heparin vaccutainers, 34 mL of 1xPBS were added. Two Histopaque-1077/Histopaque-1119 double gradients were made by layering 12.5 mL of Histopaque-1077 over 12.5 mL Histopaque1119 in a 50 mL conical tube. Twenty five mL of diluted blood was then carefully layered onto the upper gradient of the tube and centrifuged at 700xg for 30 minutes at room temperature. Two distinct opaque layers were formed followi ng centrifugation, corresp onding to leukocytes (A and B in Figure 5-1). Plasma was aspirated to within 0.5 cm of layer A and frozen. Cells from layer A were transfered to a tube marked mononuclear. Remaining fluid was aspirated to within 0.5cm of layer B and discarded. Cells from this layer were transferred to a tube labeled granulocytes. Cells were washed three times with cold 10 mL of isotonic PBS, centrifuged for
123 10 minutes at 200xg and the supernat ant discarded. Cells were counted using a hemacytometer. At the final wash, after resuspension, cells we re divided into two 5 mL aliquots. RNA was immediately isolated RNA from the first aliquot using QIAGENs RNeasy Kit for RT-PCR analysis and pellets of second aliquot were frozen for future protein isolation for use in ELISA. Quantitative Real Time RT-PCR for Determination of mRNA Concentration Quantitative real time RT-PCR using TaqMan probes (Applied Biosystems Gene Expression Assays) was performed on mRNA isol ated from patient blood cells. MARCKS, TNF, IL-1 MCP-1 and IL-17 mRNA levels were measured to compare healthy and patient samples. Detailed qRT-PCR Protocol DNAse I treatment. Frozen RNA was thawed and samples spun to bring all to the bottom. Volume in all tubes was brought up to 100 L w ith 40 L RNAse-free sterile water. Ten L 10X DNase I reaction buffer ( 10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2, pH 7.6 @ 25C) were added to each sample. Five Kunits (5 L) of DNase I (bovine pancreas Dnase I, USB, Amersham Cat # 14345, lyophilized powder dissolve d in storage buffer, 50% glycerol+10 mM Tris-HCl+2 mM CaCl2, pH 7.6 @ 25C, powder was 2,974 Kunits/mg, I made a 1,000 Kunits/mL stock and froze at -20C) were added to each sample, mixed by pipetting and incubated at 37C for 15 minutes. Two L of 0.27 M EDTA (protects RNA from being degraded during heat inactivation) were added to a final concentration of ~ 5 mM and samples mixed. DNase I was heat inactivated at 75-80C for 10 minutes. To quantify the RNA, 10 L of each RNA sample were added to 90 L RN ase-free water (10X dilution) and OD 260 and 280 measured to determine RNA concentration and purity following DNase I digestion.
124 Reverse transcribing RNA into cDNA for RT-PCR. Two micrograms of RNA were brought to a final volume of 60 L with nuclease-fr ee water. Sixty microl iters of a 2X reverse transcription master mix (H igh Capacity cDNA Archive kit (Cat. # 4322171, Applied Biosystems), 12 L 10X reverse transcription buffer, 4.8 L 25X dNTPs, 12 L 10X random primers, 6 L 50 U/L MultiScribe Reverse Tr ansciptase, 25.2 L nuclease-free water) were added. Samples were incubated at 25C for 10 minutes, then at 37C for 2 hours in a water bath. cDNA was then frozen until used. Real-time PCR of cDNA samples. Real time PCR was carried out in 96-well plates in an Applied Biosystems 7500 Standard 96-well block thermal cycler. Samples were prepared by mixing 1.25 L 20X TaqMan Gene Expression Assay (catalog number varies with each gene, for MARCKS (Hs00158992_m1), TNF(Hs00174128_m1), GAPDH (Hs99999905_m1), 18S (Hs99999901_s1), IL-1 (Hs00174097), MCP-1 (Hs002344140_m1) and IL-17 (Hs00174383_m1)), 11.25 L cDNA template and 12.5 L 2X TaqMan Universal PCR Master Mix (Applied Biosystems Cat. # 4304437). The PCR parameters used were as follows: Stage 1: 1 rep at 50C for 2 minutes; Stage 2: 1 rep at 95C for 10 minutes; Stage 3: 50 reps of: a. 95C for 15 seconds; b. 60C for 1 minute. Quantitative real time RT-PCR data were analyzed using the comparative Ct method (also called the 2 Ct method). This involves comparing the Ct values (cycle at which the fluorescence of our sample crosses the threshold, the cycle number at which the increase in fluorescence (and therefore cDNA) is exponential) of the samples of interest with a control or calibrator such as a non-treated sample or RNA from normal tissue or a healthy control. The Ct values of both the calibrator and the sample s of interest are normalized to an appropriate endogenous housekeeping
125 gene (GAPDH was our choice). So, Ct = Ct,sample Ct,reference, where Ct,sample is the Ct value for any sample (e.g., cDNA from patie nt samples) normalized to the endogenous housekeeping gene (GAPDH) and Ct,reference is the Ct value for the calibrator (e.g., cDNA from healthy controls) also normalized to the endogenous housekeeping gene (GAPDH). Results RT-PCR data (Tables 5-1 to 5-6) from mRNA isolated from white blood cells of patients with inflammatory diseases, mainly RA, show a strong correlation betwee n the levels of TNF mRNA and MARCKS mRNA (Figures 5-2 and 5-3). This correlation is similar to that observed for other cytokines known to be impor tant for TNF signaling such as IL-1 and MCP-1 (Figure 5-2 and 5-3). The highest corr elation with TNF was for IL-1 in mononuclear cells (F-statistic 216), followed by MARCKS in mononuclear cells (F -statistic 179), MCP-1 in mononuclear cells (F-statistic 135), MCP-1 in granulocytes (F-statistic = 52), IL-1 in granulocytes (F-statistic = 36) and finally MARCKS in granuloc ytes (F-statistic = 29). IL-17 had no correlation with TNF in mononuclear cells, with an F-statistic of 0.1; to be significant a correlation, the F-statistic should be much larger than 1. We noticed th at there were a few outliers in each graph that seemed to skew the data and weigh heavily into the analysis. To overcome this problem, we threw out the most obvious outlier in each case and re-calculated F-values. In mononuclear cells, the values became 31 for IL-1 18 for MARCKS and 9 for MCP-1, but the order was retained. However, in granulocytes, the order was changed and became similar to that observed in mononuclear cells, where IL-1 had the highest corre lation with TNF with an F-value of 35, followed by MARCKS whose F-value was 29 and finally MCP-1 whose F-value was 17. IL-17 could not be detected in healt hy controls granulocytes and so a fold change could not be calculated using the Ct method, which requires a control sample.
126 Quantitative Sandwich ELISA of MARCKS and TNFEnzyme-linked immunosorbent assays (ELISAs) allow the detection and quantification of substances such as peptides, proteins, antibodie s and hormones [244, 245]. In a standard ELISA, the antigen is first immobilized to a solid surface. It is then complexed with an antibody that is linked to an enzyme. Detection is accomplishe d by incubating this enzyme-complex with a substrate that produces a detectab le product. The most crucial element of the detection is a highly specific antibody-a ntigen interaction. A more common type of ELISA is the sandwich assay. This is a more sensitive and robust type of ELISA which allows better quantification of the antigen of interest . It is called a sandwich ELISA because the antigen is bound betw een two antibodies, the capture antibody and detection antibody. The capture and detec tion antibody ideally r ecognize different nonoverlapping epitopes. Binding by the capture an tibody should in no way alter or obscure the epitope recognized by the detection antibody, an d both antibodies shoul d be able to bind simultaneously to the analyte to be measured. Either monoclonal or polyclonal antibodies can be used as an ELISA pair. A detection enzyme (m ost commonly horseradish peroxidase or alkaline phosphatase) may be linked directly to the dete ction antibody or introduced through a secondary antibody that recognizes the detection antibody. We were interested in measuring the amounts of TNFand MARCKS in our patient samples. TNFcan be assayed in plasma, whereas MARCKS can be measured in leukocytes. Commercially available sandwich ELISA kits ar e widespread for commonl y measured cytokines such as TNF. We used the Human TNF OptEIA ELISA Set (BD Biosciences, Cat. # 555212) to measure TNFin the plasma of our patient and control samples.
127 To measure MARCKS concentrations in our samples, we had to develop and test a sandwich ELISA, as neither a comm ercially available kit exists, nor has one been described in the literature. This was done by testing different commercially available MARCKS antibodies for coating and detection, as well as testing diffe rent coating buffers and blocking buffers until a useful assay was reached, as determined by use of purified recombinant MARCKS as an antigen. Quantification of Plasma TNFUsing a Commercial Sandwich ELISA Plasma was collected duri ng the Histopaque-1077/Histopaque-1119 double gradient leukocyte isolation described prev iously. Following centrifugation, plasma was kept on ice and then frozen at -80C. Plasma was thawed qui ckly under running water and used as is in our ELISA. In most cases, this plasma had been dilu ted 1:2 with 1X PBS for the leukocyte isolation protocol. A TNFstandard ranging from 5000 pg/mL to 2. 5 pg/mL was loaded on the plates for construction of a standard curve. The recommended assay procedure from the kit was followed exactly. Microwells were coated with 100 L per well of capture antibody d iluted in coating buffer. Seal plate and incubate overnight at 4 C. Wells were aspirated and washed 3 times with 300 L/well Wash Buffer. After the last wash, plat es were inverted and blotted on absorbent paper to remove any residual buffe r. Wells were blocked with 200 L/well assay diluent and incubated at RT for 1 hour. Wells were aspira ted and washed 3 times. Standards and sample dilutions were prepared in assay diluent. One hundred L of each standard, sample, and control were pipetted into appropriate we lls, plate sealed a nd incubated for 2 hours at RT. Wells were aspirated and washed 5 times. One hundred L of working detector (detection Antibody + SAvHRP reagent) were added to each well, plate sealed and incubated for 1 hour at RT. Wells were aspirated and washed 7 times. NOTE: In this final wash step, wells were soaked in wash buffer for 30 seconds to 1 minute for each wash. One hundred L of substrate solution were added to
128 each well and the plate was incubated (without plat e sealer) for 30 minutes at room temperature in the dark. Fifty L of stop solution were added to each we ll. Absorbance was read at 450 nm within 30 minutes of stopping reaction. Quantification of Cellular MARCKS Using Optimized Sandwich ELISA MARCKS Serotec antibody (Goat anti-human MARCKS polyclonal antibody, raised against synthetic peptide ECSPE APPAEAAE derived from C-terminus of MARCKS protein, immunoaffinity purified against the MARCKS synthetic peptide, Cat. # AHP695) was diluted to 2 g/mL in coating buffer (40 L of 0.5 mg /mL antibody into 10 mL coating buffer=0.1 M sodium carbonate, pH 9.5). Microwells were co ated with 100 L per well of diluted coating (=capture) antibody, the plate was sealed and incubated at 4C overnight. Wells were aspirated and washed 3X with 300 L/well of wash buffe r=TBS with 0.05%Tween-20. After washing, the plate was inverted and blotted on paper towels. Wells were bl ocked with 200 L blocking buffer (0.5%BSA, 150 mM NacCl, 2 mM EDTA, 50 mM Tris, pH7.5, 0.3% Nonidet P-40) for 1.5 hours at RT. Standard and samples were prepared in lysis buffer (0.5 M NaCl, 50 mM Tris, 2 mM EDTA, 1% NP-40, pH 7.5). Wells were aspirated and washed 2X with 300 L/well of wash buffer=TBS with 0.05%Tween-20. After washing, the plate was inverted and blotted on paper towels. One hundred L of each standard or sample were pipetted into appropriate wells, plate sealed and incubated overnight at 4C. Wells were aspirated and washed 3X with 300 L/well of wash buffer=TBS with 0.05%Tween-20. After wa shing, the plate was i nverted and blotted on paper towels. Calbiochem antibody (=dete ction antibody, Rabbit anti-mouse MARCKS antibody, purified against native MARCKS from mouse brain, polycl onal antibody, total IgG immunoaffinity purification, Ca t. # 442707) was diluted to 2.7 g/mL (40 L in 10 mL of blocking buffer) and 100 L added per well. The plate was sealed and in cubated for 2 hours at RT. Wells were aspirated and washed 3X with 300 L/well of wash buffer=TBS with
129 0.05%Tween-20. After washing, the plate was inverted and blotted on paper towels. One hundred L of AP-conjugated monoclonal mouse an ti-rabbit IgG were pipetted into each well (secondary detection antibody, diluted 1:1000 in blocking buffer, Sigma, Cat. No. A2556) and the plate was incubated at RT for 1 hour. We lls were aspirated and washed 5 times. NOTE: In this final wash step, wells were soaked in wash buffer for 30 seconds for each wash. One hundred L of substrate solution (two 5 mg tablets of p-nitrophe nyl phosphate (AP substrate, Sigma-Aldrich, Cat. No. N 9389), 5 L 1 M MgCl2, 10 L diethanolamine and 10 mL ddH2O) were added to each well and the plate was incubated (without plate sealer) for 30 minutes at room temperature in the dark. Absorbance was read at 450 nm at 30 minutes and read again every 30 minutes until we got good development in all wells without overde veloping the highest standard concentration. Standard Preparation: Recombinant murine MARCKS was purified from E. coli as described previously. The concentration of MARCKS was determined by amino acid analysis to be 4.65 M. To make a serial dilution of stock MARCKS to make ELISA standards, 20 ELISA dilution tubes were placed on a used ELISA plate. Five hundred L of diluted MARCKS stock (250 L into 3.75 mL lysis buffer) were pipetted into the first tube and 250 L of ly sis buffer into tubes 2-20. Two hundred and fifty L were transfered from tube 1 to tube 2 and mixed well by pipetting. Two hundred and fifty L were transfered from tube 2 to tube 3 and mixed well by pipetting. This was repeated until tube 20. Sample Preparation: Frozen cell pellets of leukocytes isolated from human bl ood were resuspended in 500 L lysis buffer containing 5 mM DFP, 0.25 mM leupeptin, 1 mM PMSF, and 0.125 mg trypsin
130 inhibitor. Samples were vortexed briefly to re suspend cells and kept on ice for 1 hour. Samples were sonicated for 30 seconds to break the ce lls and placed back on ice. Samples were centrifuged for 15 minutes at 4C at 4,000 rpm. Supernatants were taken and used for ELISA. The remaining samples were frozen. Results The serum levels of TNF measured by quant itative sandwich ELISA in patient samples were within the range of what is expected (Tab le 5-10). There appeared to be no statistically significant difference between healthy controls and RA patients, either untreated or on any of the treatments shown (Figure 5-4). The only statistical significance in terms of TNF serum levels was found between healthy controls and the MCTD group (p=0.03). However, this group only had two subjects and therefore no co nclusions could be drawn. MARCKS levels appeared to be reduced overall in RA patients (Table 5-10). With the small number of samples we had in each gr oup, it was difficult to determine whether the observed differences were meaningful. In a few cases, it appeared that MARCKS levels were much lower in patients with active RA versus th ose with inactive RA (Figure 5-5). For example, patients 19 and 38 both have RA and are taking Humi ra. At the particular visit where blood was drawn for this analysis, patient 19 had active RA whereas 38 had inactive RA. MARCKS levels for patient 19 were very low (0.03ng/100,000 cells) whereas patient 38 had higher MARCKS levels (0.51ng/100,000 cells). However, as te sted by a Mann-Whitney 2-tailed U test, only statistically significant differ ences were found between health y controls and patients with juvenile rheumatoid arthritis (JRA, p=0.02). There was also a nearly statistical significant (p=0.08) that patients whom had untreated early synovitis that later develo ped into RA had lower levels of MARCKS. Again, in both these goups (JRA and untreat ed early synovitis) we only had 3 samples and therefore it was hard to determine at this point whether it is meaningful.
131 Conclusions The RT-PCR correlation data were interesting in further establishing MARCKS as an important signaling molecule downstream of TNF in the TNF signal transduction pathway. Due to the limited number of samples obtained in each category, it was not possible to draw meaningful conclusions from this study. However, we can come up with a few hypotheses and our data can be used in a pow er analysis to determine the number of patients that would be needed to yield statistica lly significant results in a future followup study. As previously described, TNF se rum levels in our analysis did not appear to be sensitive or specific for diagnosis or prognosis of early synov itis, RA or response to treatment. They were also highly variable, and this has been previously reported presumably because they can be affected by a simple bacteria l infection or a common cold. MARCKS data appeared a little more promising than TNF for diagnosis and/or prognosis of RA. Opposite to what we had expected based on the finding that TNF induces a large increase in MARCKS protein levels in macropha ges and neutrophils, it appeared that MARCKS levels are reduced in RA patients. This may point to an aberrant TNF signaling transduction in those patients or that their cells are somehow no longer responsive to TNF. An analysis of parameters related to disease activity (as measur ed for example by CRP or an activity score) may be more revealing. Increasing evidence suggests that the chronic pe rsistence of the inflamed condition of joint tissue in RA can be attributed to mechanisms that inhibit programmed cell death of activated cells within the joint tissues in RA . Because the cells th at drive the pathology of RA are resistant to pathways that would otherwise enable their natural clearance, the inflamed condition is maintained indefinitely. Nu merous histologic studies of jo int tissues recovered from RA paitents suggest that the occurren ce of apoptosis in the synovium of the RA joint is particularly
132 rare, and that proteins that block apoptosis are prevalent in cells and tissues of the afflicted joints [248-252]. Thus, it has been suggested that therap ies that can amplify apoptotic signals or block inhibitors of apoptosis may be successful in down-regulating the chronic inflammation and work in synergy with existing therap ies to cure RA [247, 253, 254]. If MARCKS is important for normal signaling by TNF, specifically for TNF-indu ced apoptosis as our preliminary data in Chapter 4 point to, having lower levels of MA RCKS may cause this resistance to apoptosis found in those cells and finding stra tegies to activate MARCKS or in crease its levels in synovial joint tissues may aid in the treatment of RA. In addition, it has been reporte d that RA synovial fi broblasts acquire many characteristics of transformed cells, becoming more invasive . This is also quiet interesting in terms of MARCKS involvement because reduced levels of MARCKS are found to cau se cell proliferation and tumor progression in many cell types, and ag ain may play a role in the transformed phenotype of RASFs [53-56]. As previously described (C hapters 1-3), MARCKS is stri ctly regulated by various mechanisms including at least a dozen post-tran slational modifications and proteolysis which may activate or inactivate the protein dependi ng on their location, as well as change its distribution from membrane-bound to cytosolic a nd vice versa. MARCKS is also regulated at the transcription level by different promoters; and the MARCKS transcript contains CU-rich elements making it unstable unless bound by RN A binding proteins whose presence is cell specific and variable. All these regulatory requirements make it su ch that if any of them should go wrong (for example, by a mutation in a phosp horylation site, cleavage site or site of intramolecular interaction), it could have a de trimental effect on MARCKS function. Given MARCKSs important roles this can potentially cause disease.
133 Table 5-1. Ct values obtained by quantitati ve real time RT-PCR fo r mononuclear cells from patient samples. Sample # MCP-1 IL-17A GAPDH IL-1 MARCKS TNFGAPDH 1A 27.66 31.73 19.07 18.69 21.35 21.89 19.24 2A 26.52 32.48 20.14 16.79 21.00 20.22 20.69 3A 28.05 30.69 19.77 17.07 21.89 20.39 20.25 4A 30.34 40.52 19.41 20.42 23.13 20.87 19.69 5A 27.90 32.38 18.90 22.67 22.97 23.59 19.31 7A 31.09 40.23 20.16 21.47 24.13 23.44 20.44 8A 25.04 34.54 21.13 21.18 23.39 22.60 21.44 9A 29.80 34.00 20.10 20.06 22.51 21.16 20.37 10A 29.31 40.49 19.17 19.85 23.22 21.13 19.58 11A 29.24 30.20 18.34 20.67 21.69 22.64 18.95 12A 31.71 34.18 21.85 22.72 24.11 24.38 22.23 13A 29.00 36.10 19.22 23.75 23.54 25.08 19.69 14A 28.84 36.29 20.33 25.28 23.98 25.64 20.70 15A 29.10 37.38 19.84 24.69 23.12 24.39 20.17 16A 28.35 und 21.69 26.13 23.92 25.00 20.49 17A 28.96 34.92 21.88 19.12 22.68 22.23 20.59 18A 24.47 31.73 20.75 18.48 21.31 19.82 19.25 19A 28.42 33.33 21.10 18.52 21.44 20.60 20.48 20A 30.11 33.31 22.86 18.69 22.23 21.13 21.32 21A 32.72 37.31 22.65 20.34 23.55 23.10 21.96 22A 26.87 34.38 22.51 16.37 20.61 20.27 21.19 24A 25.79 33.90 22.43 17.68 21.39 20.55 21.55 25A 27.56 34.84 22.07 17.48 21.33 20.54 21.15 26A 28.52 34.84 22.00 18.22 21.59 20.97 20.99 27A 29.53 34.34 23.44 18.03 21.88 21.00 22.00 28A 25.21 37.49 22.12 18.85 22.90 21.50 21.63 29A 29.13 34.29 21.47 21.65 22.59 23.02 20.86 30A 27.78 34.06 22.00 20.65 22.34 21.04 21.31 31A 34.00 35.49 26.29 22.45 23.00 23.06 22.80 32A 33.29 36.81 22.80 21.30 22.72 23.96 21.56 33A 34.65 36.07 26.45 22.30 23.46 23.21 23.91 34A 29.26 34.29 23.65 19.13 21.76 21.10 22.01 35A 25.73 35.00 23.29 19.23 21.44 22.14 21.50 36A 28.44 und 22.23 20.95 23.10 21.43 21.00 37A 27.23 36.09 23.41 21.64 22.55 22.54 20.48 38A 30.70 31.03 23.70 26.36 22.85 24.35 21.05 39A 31.98 und 25.68 29.00 24.18 28.96 25.68
134 Table 5-1. Continued. Sample # MCP-1 IL-17A GAPDH IL-1 MARCKS TNFGAPDH 40A 30.60 und 23.33 19.54 22.82 23.64 23.33 41A 27.29 und 24.25 23.35 24.08 25.03 24.25 42A 25.41 42.74 27.61 20.08 23.55 24.46 27.61 43A 28.38 und 23.29 20.59 23.70 22.06 23.29 44A 26.05 und 22.98 20.51 23.27 21.57 22.98 45A 26.95 und 23.49 20.52 22.29 22.00 23.49 46A 30.92 und 24.47 27.49 23.51 26.00 24.47 47A 29.84 und 23.60 23.14 24.00 25.89 23.60 48A 26.36 und 19.43 24.41 22.23 24.79 19.43 49A 28.73 37.94 20.16 25.28 23.40 24.66 20.16 50A 29.29 und 20.57 24.83 23.27 24.33 20.57 51A 28.83 und 20.63 23.43 23.11 23.07 20.63 52A 27.03 und 20.32 24.10 23.43 23.44 20.32 53A 27.40 41.70 20.26 21.00 21.59 21.30 20.26 54A 27.38 und 20.92 23.33 23.82 23.24 20.92 55A 28.52 40.56 20.23 25.93 23.00 25.00 20.23 56A 27.91 und 26.11 23.86 24.44 25.81 26.11 57A 28.13 und 19.99 24.89 22.32 24.31 19.99 und=undetermined
135 Table 5-2. Ct values obtained by quantitative re al time RT-PCR for granulocytes from patient samples. Sample # MCP-1 IL-17A GAPDH IL-1 MARCKS TNFGAPDH 1B 33.78 39.73 21.82 23.82 21.16 27.00 22.00 2B 31.59 33.28 22.04 18.12 20.23 22.65 22.30 3B 33.07 33.88 23.68 21.00 22.76 25.27 24.28 4B 33.15 und 21.20 21.58 21.12 24.74 22.02 5B 32.46 und 24.58 23.27 23.66 26.24 24.91 9B 34.72 40.48 21.50 24.23 22.70 26.02 22.06 10B 31.01 und 21.08 22.24 22.55 24.32 21.47 11B 34.48 und 20.58 21.23 21.36 25.38 21.04 14B 33.24 und 23.42 23.95 23.15 26.65 23.89 15B 34.00 und 26.00 25.41 24.19 27.77 26.59 16B 32.47 und 26.09 24.01 22.95 25.62 24.09 18B 31.78 und 25.46 24.29 24.28 26.21 24.29 19B 36.05 36.00 26.49 25.52 24.35 26.78 25.42 21B 35.95 35.76 25.37 24.26 22.97 26.86 23.47 22B 32.67 35.90 24.86 22.59 22.46 25.35 22.95 25B 32.13 38.76 27.58 24.95 24.57 27.12 26.27 26B 34.81 39.21 25.17 24.18 23.00 26.14 24.13 27B 37.14 und 26.02 24.66 23.33 27.43 26.02 29B 36.00 43.08 24.17 24.79 22.51 26.91 23.54 30B 32.62 und 24.73 24.00 22.07 25.56 24.17 32B 39.89 und 26.45 26.21 22.13 27.89 23.92 34B 34.93 36.21 26.14 23.41 21.22 25.85 24.17 35B 35.36 und 29.00 24.84 24.30 27.26 26.83 37B 35.66 37.78 29.42 27.00 25.21 28.00 24.66 40B 35.28 und 26.50 26.51 24.23 29.51 26.50 41B 34.21 und 28.13 25.69 25.05 29.30 28.13 42B 30.30 und 28.18 24.09 24.38 27.28 28.18 43B 36.00 und 29.30 27.46 26.00 31.87 29.30 44B 30.66 und 27.00 24.75 24.39 26.76 27.00 48B 34.27 und 23.53 25.78 23.83 28.63 23.53 49B 37.00 und 24.48 27.11 23.28 29.15 24.48 54B 36.20 und 28.00 26.04 23.40 27.82 28.00 55B 39.43 und 29.41 31.34 27.77 32.18 29.41 57B 36.14 und 25.54 27.59 24.03 29.54 25.54
136 136 Table 5-3. Ct and fold change calculations for monocytic cells qRT-PCR fr om patient samples. Results shown in this table are for MCP-1, IL-1 and IL-17A. Sample # Ct MCP-1Ct GAPDH % of control MCP-1 fold change Ct IL-1 Ct GAPDH % of control IL-1 fold change Ct IL-17ACt GAPDH % of control IL-17A fold change 1A 8.60 135.50 1.36 0.38 1508.63 15.09 12.66 241.59 2.42 2A 6.38 627.38 6.27 3.35 11845.27 118.45 12.34 300.96 3.01 3A 8.28 168.69 1.69 2.69 7507.01 75.07 10.92 806.44 8.06 4A 10.94 26.76 0.27 1.02 574.05 5.74 21.11 0.69 0.01 5A 9.01 101.98 1.02 3.78 84.63 0.85 13.49 136.00 1.36 7A 10.93 26.82 0.27 1.31 467.24 4.67 20.07 1.41 0.01 8A 3.91 3478.36 34.78 0.06 1115.92 11.16 13.41 142.96 1.43 9A 9.70 62.87 0.63 0.04 1195.19 11.95 13.90 102.07 1.02 10A 10.14 46.50 0.47 0.68 721.58 7.22 21.32 0.60 0.01 11A 10.90 27.50 0.27 2.33 230.24 2.30 11.86 418.60 4.19 12A 9.86 56.30 0.56 0.87 635.18 6.35 12.33 303.47 3.03 13A 9.78 59.68 0.60 4.53 50.39 0.50 16.88 12.97 0.13 14A 8.51 143.33 1.43 4.95 37.61 0.38 15.96 24.43 0.24 15A 9.26 85.58 0.86 4.85 40.25 0.40 17.54 8.19 0.08 16A 6.67 516.34 5.16 4.44 53.37 0.53 und und und 17A 7.07 388.88 3.89 2.77 7896.64 78.97 13.04 185.26 1.85 18A 3.72 3970.73 39.71 2.27 5583.77 55.84 10.98 770.91 7.71 19A 7.32 327.46 3.27 2.58 6927.04 69.27 12.23 324.80 3.25 20A 7.2 4 345.89 3.46 4.18 20998.85 209.99 10.45 1115.46 11.15 21A 10.07 48.61 0.49 2.31 5744.73 57.45 14.66 60.27 0.60 22A 4.36 2553.37 25.53 6.14 81811.89 818.12 11.87 415.42 4.15 24A 3.36 5099.67 51.00 4.75 31238.18 312.38 11.46 552.34 5.52 25A 5.49 1163.45 11.63 4.58 27823.63 278.24 12.77 223.08 2.23 26A 6.51 573.32 5.73 3.79 15991.56 159.92 12.83 213.70 2.14
136 137 Table 5-3. Continued 1. Sample # Ct MCP-1Ct GAPDH % of control MCP-1 fold change Ct IL-1 Ct GAPDH % of control IL-1 fold change Ct IL-17ACt GAPDH % of control IL-17A fold change 27A 6.09 769.72 7.70 5.42 49495.95 494.96 10.90 816.56 8.17 28A 3.09 6162.03 61.62 3.27 11183.02 111.83 15.37 36.72 0.37 29A 7.66 259.43 2.59 0.18 1022.60 10.23 12.82 216.38 2.16 30A 5.78 950.93 9.51 1.35 2959.26 29.59 12.06 366.44 3.66 31A 7.71 250.76 2.51 3.85 16670.65 166.71 9.20 2654.87 26.55 32A 10.49 36.46 0.36 1.51 3301.77 33.02 14.01 94.58 0.95 33A 8.20 178.43 1.78 4.16 20695.40 206.95 9.61 1991.20 19.91 34A 5.62 1066.15 10.66 4.51 26469.12 264.69 10.64 977.14 9.77 35A 2.44 9682.67 96.83 4.06 19376.53 193.77 11.71 466.72 4.67 36A 6.22 704.86 7.05 1.27 2805.46 28.05 und und und 37A 3.82 3709.96 37.10 1.76 3937.39 39.37 12.69 236.29 2.36 38A 7.00 409.07 4.09 2.66 183.80 1.84 7.33 9697.67 96.98 39A 6.30 663.61 6.64 3.32 116.16 1.16 und und und 40A 7.27 340.42 3.40 3.79 16058.20 160.58 und und und 41A 3.04 6374.91 63.75 0.90 2158.81 21.59 und und und 42A 2.20 240919.15 2409.19 7.53 214411.51 2144.12 15.13 43.36 0.43 43A 5.09 1538.37 15.38 2.70 7553.99 75.54 und und und 44A 3.08 6217.80 62.18 2.47 6427.41 64.27 und und und 45A 3.4 6 4761.46 47.61 2.97 9102.35 91.02 und und und 46A 6.46 597.25 5.97 3.02 142.62 1.43 und und und 47A 6.24 692.27 6.92 0.46 1600.18 16.00 und und und 48A 6.92 432.39 4.32 4.98 36.81 0.37 und und und 49A 8.58 137.01 1.37 5.12 33.31 0.33 17.78 6.91 0.07 50A 8.72 124.00 1.24 4.26 60.51 0.61 und und und
136 138 Table 5-3. Continued 2. Sample # Ct MCP-1Ct GAPDH % of control MCP-1 fold change Ct IL-1 Ct GAPDH % of control IL-1 fold change Ct IL-17ACt GAPDH % of control IL-17A fold change 51A 8.21 177.19 1.77 2.80 166.57 1.67 und und und 52A 6.71 500.14 5.00 3.79 84.16 0.84 und und und 53A 7.14 371.75 3.72 0.74 694.59 6.95 21.44 0.55 0.01 54A 6.47 592.30 5.92 2.42 217.22 2.17 und und und 55A 8.29 167.29 1.67 5.71 22.24 0.22 20.33 1.18 0.01 56A 1.80 15088.79 150.89 2.26 5545.20 55.45 und und und 57A 8.14 185.23 1.85 4.90 38.77 0.39 und und und
136 139 Table 5-4. Ct and fold change calculations for monocytic cells qRT-PCR from patient samples. Results shown in this table are for MARCKS and TNF. Sample # Ct MARCKSCt GAPDH % of control MARCKS fold change Ct TNFCt GAPDH % of control TNFfold change 1A 2.11 184.59 1.85 2.65 248.08 2.48 2A 0.31 640.56 6.41 0.47 2161.31 21.61 3A 1.65 254.09 2.54 0.15 1412.16 14.12 4A 3.43 73.73 0.74 1.17 692.99 6.93 5A 3.67 62.65 0.63 4.28 80.32 0.80 7A 3.69 61.57 0.62 3.00 195.05 1.95 8A 1.94 206.96 2.07 1.16 700.72 7.01 9A 2.14 180.04 1.80 0.79 901.19 9.01 10A 3.64 63.83 0.64 1.55 532.52 5.33 11A 2.74 119.28 1.19 3.68 121.57 1.22 12A 1.88 215.75 2.16 2.15 352.55 3.53 13A 3.85 55.30 0.55 5.39 37.34 0.37 14A 3.29 81.53 0.82 4.95 50.69 0.51 15A 2.95 103.12 1.03 4.23 83.44 0.83 16A 3.43 73.78 0.74 4.51 68.67 0.69 17A 2.09 186.91 1.87 1.64 499.97 5.00 18A 2.07 190.04 1.90 0.57 1051.10 10.51 19A 0.96 408.78 4.09 0.12 1436.85 14.37 20A 0.91 422.32 4.22 0.19 1784.98 17.85 21A 1.59 263.96 2.64 1.14 708.53 7.09 22A 0.58 1192.02 11.92 0.92 2960.63 29.61 24A 0.16 892.18 8.92 1.00 3129.44 31.29 25A 0.18 703.88 7.04 0.61 2384.86 23.85 26A 0.60 525.01 5.25 0.02 1583.27 15.83
136 140 Table 5-4. Continued 1. Sample # Ct MARCKSCt GAPDH % of control MARCKS fold change Ct TNFCt GAPDH % of control TNFfold change 27A 0.12 862.98 8.63 1.00 3131.61 31.32 28A 1.28 328.83 3.29 0.12 1701.62 17.02 29A 1.73 239.39 2.39 2.16 348.90 3.49 30A 1.03 389.96 3.90 0.27 1884.14 18.84 31A 0.20 690.83 6.91 0.27 1297.65 12.98 32A 1.16 356.85 3.57 2.39 297.49 2.97 33A 0.46 1090.81 10.91 0.70 2531.35 25.31 34A 0.25 945.67 9.46 0.91 2925.95 29.26 35A 0.06 828.98 8.29 0.64 1003.41 10.03 36A 2.10 185.62 1.86 0.43 1163.04 11.63 37A 2.06 190.44 1.90 2.06 375.77 3.76 38A 1.80 228.52 2.29 3.29 159.20 1.59 39A 1.50 2255.43 22.55 3.28 160.97 1.61 40A 0.51 1137.14 11.37 0.31 1259.55 12.60 41A 0.17 892.80 8.93 0.78 910.61 9.11 42A 4.06 13282.04 132.82 3.15 13850.88 138.51 43A 0.41 597.25 5.97 1.23 3657.62 36.58 44A 0.29 650.85 6.51 1.40 4132.19 41.32 45A 1.20 1833.25 18.33 1.49 4398.18 43.98 46A 0.96 1550.15 15.50 1.53 540.33 5.40 47A 0.40 603.49 6.03 2.28 320.61 3.21 48A 2.79 114.82 1.15 5.36 38.10 0.38 49A 3.25 83.93 0.84 4.51 68.67 0.69 50A 2.71 121.95 1.22 3.76 114.94 1.15
136 141 Table 5-4. Continued 2. Sample # Ct MARCKSCt GAPDH % of control MARCKS fold change Ct TNFCt GAPDH % of control TNFfold change 51A 2.48 142.34 1.42 2.45 286.16 2.86 52A 3.11 92.23 0.92 3.12 179.98 1.80 53A 1.33 316.75 3.17 1.04 760.44 7.60 54A 2.91 106.24 1.06 2.33 311.20 3.11 55A 2.77 116.34 1.16 4.77 57.07 0.57 56A 1.67 2539.25 25.39 0.31 1930.41 19.30 57A 2.34 157.61 1.58 4.33 77.80 0.78
136 142 Table 5-5. Ct and fold change calculations for gra nulocyte qRT-PCR from patient samples. Results shown in this table are for MCP-1, IL-1 and IL-17A. Sample # Ct MCP-1Ct GAPDH % of control MCP-1 fold change Ct IL-1 Ct GAPDH % of control IL-1 fold change Ct IL-17ACt GAPDH % of control IL-17A fold change 1B 11.96 14.69 0.15 2.00 16.86 0.17 17.91 und und 2B 9.55 78.18 0.78 3.91 1018.09 10.18 11.24 und und 3B 9.39 87.53 0.88 2.68 432.83 4.33 10.20 und und 4B 11.95 14.79 0.15 0.37 52.19 0.52 und und und 5B 7.88 249.29 2.49 1.31 167.81 1.68 und und und 9B 13.23 6.11 0.06 2.74 10.16 0.10 18.98 und und 10B 9.93 59.99 0.60 1.16 30.35 0.30 und und und 11B 13.90 3.84 0.04 0.64 43.31 0.43 und und und 14B 9.82 65.01 0.65 0.52 47.07 0.47 und und und 15B 8.00 228.91 2.29 0.59 102.16 1.02 und und und 16B 6.38 702.65 7.03 2.08 286.16 2.86 und und und 18B 6.32 733.50 7.34 1.18 152.71 1.53 und und und 19B 9.56 77.64 0.78 0.97 132.39 1.32 9.51 und und 21B 10.57 38.50 0.38 1.11 146.19 1.46 10.39 und und 22B 7.81 260.77 2.61 2.27 325.31 3.25 11.04 und und 25B 4.55 2508.58 25.09 2.64 420.99 4.21 11.18 und und 26B 9.63 73.75 0.74 0.99 134.24 1.34 14.04 und und 27B 11.12 26.28 0.26 1.36 173.97 1.74 und und und 29B 11.83 16.09 0.16 0.62 43.98 0.44 18.91 und und 30B 7.89 247.74 2.48 0.73 112.49 1.12 und und und 32B 13.44 5.27 0.05 0.24 80.04 0.80 und und und 34B 8.78 133.03 1.33 2.74 450.27 4.50 10.07 und und 35B 6.36 713.94 7.14 4.16 1209.89 12.10 und und und 37B 6.25 771.04 7.71 2.42 360.70 3.61 8.36 und und
136 143 Table 5-5. Continued. Sample # Ct MCP-1Ct GAPDH % of control MCP-1 fold change Ct IL-1 Ct GAPDH % of control IL-1 fold change Ct IL-17ACt GAPDH % of control IL-17A fold change 40B 8.78 132.94 1.33 0.01 67.12 0.67 und und und 41B 6.08 868.06 8.68 2.45 368.79 3.69 und und und 42B 2.12 13509.21 135.09 4.10 1157.39 11.57 und und und 43B 6.70 565.22 5.65 1.84 242.30 2.42 und und und 44B 3.66 4623.14 46.23 2.25 321.94 3.22 und und und 48B 10.74 34.31 0.34 2.25 14.20 0.14 und und und 49B 12.52 9.98 0.10 2.63 10.92 0.11 und und und 54B 8.20 199.42 1.99 1.96 262.95 2.63 und und und 55B 10.02 56.64 0.57 1.92 17.84 0.18 und und und 57B 10.60 37.81 0.38 2.05 16.29 0.16 und und und
136 144 Table 5-6. Ct and fold change calculations for gra nulocyte qRT-PCR from patient samples. Results shown in this table are for MARCKS and TNF. Sample # Ct MARCKSCt GAPDH % of control MARCKS fold change Ct TNFCt GAPDH % of control TNF-fold change 1B 0.84 87.03 0.87 5.00 14.64 0.15 2B 2.07 204.29 2.04 0.35 367.62 3.68 3B 1.53 140.11 1.40 0.98 236.89 2.37 4B 0.90 90.60 0.91 2.73 70.82 0.71 5B 1.25 115.64 1.16 1.33 186.24 1.86 9B 0.64 31.11 0.31 3.96 30.13 0.30 10B 1.08 23.03 0.23 2.85 65.08 0.65 11B 0.32 38.95 0.39 4.34 23.10 0.23 14B 0.74 81.43 0.81 2.75 69.51 0.70 15B 2.39 255.19 2.55 1.18 206.37 2.06 16B 1.14 106.85 1.07 1.53 162.02 1.62 18B 0.01 48.86 0.49 1.92 123.56 1.24 19B 1.07 101.93 1.02 1.36 182.54 1.83 21B 0.50 68.95 0.69 3.38 44.88 0.45 22B 0.49 68.09 0.68 2.40 88.65 0.89 25B 1.71 158.95 1.59 0.84 261.57 2.62 26B 1.13 106.41 1.06 2.01 116.73 1.17 27B 2.69 313.53 3.14 1.41 176.32 1.76 29B 1.03 99.28 0.99 3.37 45.35 0.45 30B 2.10 208.15 2.08 1.39 179.03 1.79 32B 1.78 167.09 1.67 3.97 29.82 0.30 34B 2.96 378.06 3.78 1.68 146.73 1.47 35B 2.54 282.18 2.82 0.43 348.51 3.49 37B 0.55 33.21 0.33 3.34 46.40 0.46
136 145 Table 5-6. Continued. Sample # Ct MARCKSCt GAPDH % of control MARCKS fold change Ct TNFCt GAPDH % of control TNF-fold change 40B 2.26 233.21 2.33 3.02 57.96 0.58 41B 3.09 413.13 4.13 1.16 209.39 2.09 42B 3.80 677.21 6.77 0.90 876.16 8.76 43B 3.30 480.19 4.80 2.57 79.02 0.79 44B 2.61 296.82 2.97 0.24 554.12 5.54 48B 0.30 39.41 0.39 5.10 13.71 0.14 49B 1.20 111.62 1.12 4.67 18.43 0.18 54B 4.60 1176.64 11.77 0.18 532.29 5.32 55B 1.64 151.85 1.52 2.76 68.98 0.69 57B 1.51 138.86 1.39 4.00 29.24 0.29
136 146 Table 5-7. Linear regression for correlations between RA pa tient RT-PCR data for various parameters and TNF. Y=A+B*X Cell type Sample Parameter Value Error t-Value Prob>|t| A 6.80520 3.11253 2.18639 0.03537 IL-17A B 0.03533 0.11688 0.30227 0.76419 A 61.14649 22.49072 2.71874 0.00884 IL-1 B 13.54019 0.92178 14.68922 <0.0001 A 2.92789 1.40129 2.08942 0.04149 MARCKS B 0.76921 0.05743 13.39353 <0.0001 A -117.58328 27.89971 4.21450 <0.0001 Monocytic Cells MCP-1 B 13.29173 1.14346 11.62409 <0.0001 A 0.45705 0.50370 0.90739 0.37098 IL-1 B 1.21444 0.20365 5.96327 <0.0001 A 0.65038 0.38486 1.68990 0.10077 MARCKS B 0.83305 0.15560 5.35363 <0.0001 A 9.0570 3.50184 2.58635 0.01445 Granulocytic Cells MCP-1 B 10.23483 1.41585 7.22876 <0.0001
136 147 Table 5-8. R, R-square, adj. R-square a nd standard deviation for corr elations between RA patie nt RT-PCR data for various parameters and TNF. Cell type Sample R R-Square (COD)Adj. R-Square Root-MSE (SD) N IL-17A -0.0503 0.00253 -0.02518 16.28726 38 IL-1 0.896 0.80281 0.79909 139.92565 55 MARCKS 0.8786 0.77193 0.76763 8.71813 55 Monocytic Cells MCP-1 0.8475 0.71826 0.71295 173.57764 55 IL-1 0.7255 0.52635 0.51155 2.17161 34 MARCKS 0.68737 0.47248 0.456 1.65926 34 Granulocytic Cells MCP-1 0.78753 0.6202 0.60833 15.09761 34
136 148 Table 5-9. ANOVA table for correlations between RA patie nt RT-PCR data for various parameters and TNF. Cell type Sample Item Degrees of Freedom Sum of Squares Mean Square F Statistic Prob>F Model 1 2.42E+01 2.42E+01 0.09136 0.76419 Error 36 9.55E+03 2.65E+02 IL-17A Total 37 9.57E+03 Model 1 4.22E+06 4.22E+06 215.77324 <0.0001 Error 53 1.04E+06 1.96E+04 IL-1 Total 54 5.26E+06 Model 1 1.36E+04 1.36E+04 179.38671 <0.0001 Error 53 4.03E+03 7.60E+01 MARCKS Total 54 1.77E+04 Model 1 4.07E+06 4.07E+06 135.11952 <0.0001 Error 53 1.60E+06 3.01E+04 Monocytic Cells MCP-1 Total 54 5.67E+06 Model 1 1.68E+02 1.68E+02 35.56062 <0.0001 Error 32 1.51E+02 4.72E+00 IL-1 Total 33 3.19E+02 Model 1 7.89E+01 7.89E+01 28.66131 <0.0001 Error 32 8.81E+01 2.75E+00 MARCKS Total 33 1.67E+02 Model 1 1.19E+04 1.19E+04 52.25498 <0.0001 Error 32 7.29E+03 2.28E+02 Granulocytic Cells MCP-1 Total 33 1.92E+04
136 149 Table 5-10. MARCKS and TNF pr otein levels in patient samples as determined by ELISA. Group Sample # MARCKS (ng/100,000 cells) TNF (pg/mL) Diagnosis Prognosis Therapy Group 1: Healthy controls 5 5 0.11 2.81 Healthy 11 11 0.10 587.35 Healthy 12 12 0.21 21.42 Healthy 13 13 0.70 3.76 Healthy 14 14 0.24 3.76 Healthy 15 15 0.23 3.87 Healthy 16 16 0.32 1.77 Healthy 50 50 0.09 4.05 Healthy 51 51 0.03 23.28 Healthy 52 52 0.29 40.65 Healthy 53 53 0.27 2.01 Healthy Group 2: RA+MTX 4 4 0.18 RA on Methotrexate 7 7 0.13 2.73 RA on Methotrexate 22 22 0.15 0.19 RA on Methotrexate 27 27 0.02 2.94 RA on Methotrexate 29 29 0.14 3.60 RA on Methotrexate 39 39 0.27 15.35 RA on Methotrexate 40 40 0.41 1.57 RA on Methotrexate 48 48 0.32 RA on prednisone and MTX Group 3: RA+TNF inhibitor 19 19 0.03 4.99 RA on Humira--has active RA 20 20 0.12 0.62 RA on Humira--5 months 21 21 0.06 RA on Humira--4 months 36 36 0.06 0.07 RA on Humira 38 38 0.51 0.77 RA on Humira--RA not active 46 46 0.25 RA on Humira 47 47 0.47 28.62 RA on Enbrel 54 54 0.50 23.80 RA on MTX and Enbrel
136 150 Table 5-10. Continued Group Sample # MARCKS (ng/100,000 cells) TNF (pg/mL) Diagnosis Prognosis Therapy Group 4: RA--no therapy 10 10 0.47 RA start Methotrexate 17 17 0.02 3.15 RA no therapy 30 30 0.15 1.83 RA starting Humira 31 31 0.15 5.74 RA Starting Humira 33 33 0.03 1.85 RA Starting Methotrexate AND Humira Group 5: Untreated early synovitis 18 18 0.12 24.27 polyarthritis, early synovitis developed RA no therapy 43 43 0.06 5.53 early synovitis developed RA no therapy 57 57 0.03 6.80 early synovitis, breast cancer developed RA no therapy Group 6: Early synovitis on MTX or prednisone 1 1 0.46 5.56 early synovitis MCTD+RA on prednisone 26 26 0.18 1.32 early synovitis on Methotrexate 34 34 0.11 early synovitis developed RA on prednisone 45 45 0.20 11.62 unknown developed RA on Methotrexate Group 7: JRA 9 9 0.07 0.74 JRA starting TNF inhibitor 49 49 0.04 6.61 JRA no therapy 55 55 0.02 7.60 JRA on Enbrel Group 8: MCTD 6 6 0.33 0.54 MCTD no therapy 8 8 0.27 0.05 MCTD, polymyocitis no therapy 24 24 0.05 MCTD Group 9: Other 25 25 0.14 3.21 Psoriatic arthritis Starting Methotrexate 28 28 0.90 1.32 SLE+RA on prednisone and MTX 32 32 0.22 Wegner's 37 37 0.10 5.88 Blau's syndrome on Humira 44 44 0.04 0.62 Psoriatic arthritis on Methotrexate
151 Figure 5-1. Histopaque double-gr adient for leukocyte isolation from whole blood. Centrifuge 700xg 30 minutes Blood Histopaque-1119 Histopaque-1077 Plasma AMononuclear Cells/Platelets BGranulocytes CErythrocytes
152 Figure 5-2. Correlation be tween mRNA levels of TNFand other proteins in mononuclear cells. A) IL-1 B) MARCKS, C) MCP-1, and D) IL-17A. The red line represents the best fit to the data; blue lines, upper and lower prediction limits; and green lines, upper and lower 95% correlation limits. A B
153 Figure 5-2. Continued. D
154 Figure 5-3. Correlation be tween mRNA levels of TNFand other proteins in granulocytes. A) IL-1 B) MARCKS and C) MCP-1. The red line represents the best fit to the data; blue lines, upper and lower prediction limits; and green lines, upper and lower 95% correlation limits. A B
155 Figure 5-3. Continued. C
156 Group1 Healthy Controls Group2 RA+ MTX Group3 RA+ TNF inhibitor Group4 RA no therapy Group5 Untreated Early Synovitis Group6 Early Synovitis+ MTX or pred Group7 JRA Group8 MCTD Group9 Other0 10 20 30 40 Serum TNF (pg/mL) Figure 5-4. TNF serum prot ein levels of different patient groups as determ ined by quantitative sandw ich ELISA. Horizontal lin es indicate the mean protein levels in each group
157 Group1 Healthy Controls Group2 RA+ MTX Group3 RA+ TNF inhibitor Group4 RA no therapy Group5 Untreated Early Synovitis Group6 Early Synovitis+ MTX or pred Group7 JRA Group8 MCTD Group9 Other0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 ng MARCKS/100,000 cells Figure 5-5. MARCKS cellular prot ein levels of different patient groups as determined by quant itative sandwich ELISA. Horizont al lines indicate the mean protein levels in each group
158 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS It had been widely accepted that using the phosphorylation site dom ain of MARCKS, an unstructured protein, in biochem ical assays can act as a surrogate for using the entire molecule. The rationale was that in an unfolded protein, al l parts of the protein should be easily accessible all the time. Whereas this may be true for some proteins, it certainly isnt for MARCKS. The unusual charge distribution on the protein makes ioni c interactions be tween the acidic ends of the protein and its central basic effect or domain very plausible, and th at is what we have shown in this study. A question then arises as to how thes e proteins can be activat ed inside the cell, and there are a few possibilities, which are worth investigating. Cells commonly use posttranslational modifications for altering function and interact ions of molecules: our EDC neutralization assays, wh ere only a few charges were modified yet caused an increase in activity of MARCKS, suggest that additi on of a phosphate group here or there could greatly activate the protein. Specific proteolysis of inhibiting domains or binding by other proteins may also free the effector domain and activate the protein in question. In addition, local changes in ionic strength may disrupt ionic interactions; th is may be achieved by changes in calcium or other ions whose concentrations are constantly changing in our cells. Many of these mechanisms can be used simultaneously to regulate these natively unfolded proteins. Identifying more intrinsically disordered proteins that are inactivated by intramolecular interactions would be an important next step in validating our observati ons with MARCKS. We will first start by looking at other proteins that contain the MARCK S phosphorylation site domain (e.g., adducin, diacylglycerol kinase ) to see whether they demonstrate similar properties as those observed for MARCKS.
159 Mapping the sites of intramolecular intera ctions for the PSD on MARCKS and other proteins may prove to be useful in the develo pment of biological therapies. The binding sites identified by mass spectrometry will first need to be tested and validated by biochemical assays. The actin cytoskeleton is believed to play an importa nt role in the pathogenesis of RA . If an inhibitor of MARCKS can be thus identified, it may be used in the treatment of autoimmune diseases such as RA in place of tumor necr osis factor inhibitors, because MARCKS is a downstream effector of TNF in immune cells. This may be helpful because inhibiting MARCKS may potentially have fewer side effects than inhibiting TNF, which plays many important roles in immunity. Inhibiting MARCKS using a competi tive inhibitor corresponding to the N-terminal domain of the protein has already been shown to block mucus hypersecretion in a mouse model of asthma,  with potential uses in cystic fibrosis and chronic bronchitis.
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180 BIOGRAPHICAL SKETCH Im an M. A. Al-Naggar was born on March 31s t, 1981 to an Egyptian fam ily living in Kuwait. She was Ashraf Al-Naggars and Wafa a Badawys first-born child, and eventually became the eldest of five. Iman started school at the age of three when she joined Lcole Franaise, a French pre-K in Kuwait. Iman stay ed in a French education system until the Ninth grade, when she received a French Brevet des co llges. She then joined the American School of Kuwait, where she stayed until graduation in J une of 1998 with a High School Diploma. Iman had always known she loved Biology most, and tw o teachers in particular are to thank for it: Monsieur Jean Rocques in 6th grade and Miss Lisa Myhre in high school. In high school, Iman was an alto on the Honor Choir and performed so los in many school concerts. After graduating high school, Iman joined the Faculty of Science at Kuwait University where she majored in Molecular Biology and minored in Biochemistry As an undergraduate student at Kuwait University, Iman joined the lab of Dr. Esmaeil Al-Saleh, where she did research on oil-degrading bacteria and really enjoyed the experience. Sh e graduated in June of 2002 with honors. At Kuwait University, Iman also met her husband, Mr. Ahmad M. Mahmoud, and they married in January of 2002. In August 2002, they moved toge ther to Gainesville, Florida where they both joined graduate programs at the University of Florida (She, the Interdisciplinary Program in Biomedical Sciences at the College of Medi cine, He, the Department of Mechanical and Aerospace Engineering at the College of Engin eering) and became avid Gator fans. Iman has always loved Science, Biology in part icular. Iman has always said that if she could go back in time and take only one thing with her from the future, it would be her Biology 101 book. She would just repeat a ll the brilliant experiments desc ribed and so be the discoverer of great things; more specifically Iman would want to be the di scoverer of Penicillin, which she believes to have been an extremely useful discover y. In fact, if Iman had to choose one person to
181 be, she would be Louis Pasteur! Iman carries he r experiments each and every day in the lab with his famous quote Chance favors the prepared mind in mind. During her years in graduate school, Iman made many friends and met special people. A few months before graduation, Iman and her husband were blesse d with a beautiful baby girl, whom they named Salma Ahmad Mahmoud. Salm a was born on September 12, 2007 at Shands Hospital at the University of Florida.