EXPRESSION AND PARTIAL CHARACTERIZATION OF TWO NEW PROTEINS THAT INTERACT WITH DEATH ASSOCIATED PROTEIN-3, A MITOCHONDRIAL RIBOSOME PROTEIN AND POSITIVE MEDIATOR OF APOPTOSIS By AMAR M. SINGH A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003
Copyright 2003 by Amar M. Singh
This thesis is dedicated to my late grandfather, Krishnanand Shookla, who taught me that the quest for knowledge through education leads to both success and happiness.
ACKNOWLEDGMENTS I would like to thank Dr. Thomas W. Oâ€™Brien for allowing me to work under his guidance, for without him none of this work would be possible. His stimulating conversations and witty sense of humor has given me not only a deeper appreciation for scientific research, but also a model of excellence for which to strive. I would next like to thank my committee members, Dr. Brian Cain and Dr. Nancy Denslow, for all of their patience and help in completing my research. I would like to give a much needed thanks to my fellow laboratory members, Aaron Hall and Ryan Norman, for all of their assistance, ideas, and encouragement. I would also like to give a special thanks to my parents, Mangal and Kaishmati, and sister, Devi, whose love, encouragement and support are never ending. Finally, I would like to thank my beautiful wife, Maureen, whose unconditional love is the reason I wake up every morning. iv
TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 The Mitochondrial Ribosome.......................................................................................2 DAP3 and its Interacting Proteins................................................................................4 2 MATERIALS AND METHODS.................................................................................9 Cloning of DIF1 and DIF2 into pET21b......................................................................9 Protein Expression and Purification of DIF1 and DIF2.............................................12 Immunization, Purification and Characterization of Chicken Antibodies..................14 Isolation and Sub-fractionation of Bovine Mitochondria...........................................16 Localization of DIF1 and DIF2..................................................................................18 3 RESULTS...................................................................................................................20 In Silico Analysis of DIF1 and DIF2..........................................................................20 Cloning and Expression of DIF1 and DIF2................................................................22 Antibody Preparation and Characterization for DIF1 and DIF2................................23 Mitochondrial Sub-fractionation and Localization of DIF1 and DIF2.......................24 4 DISCUSSION AND CONCLUSION........................................................................41 APPENDIX A DIF1 and DIF2 PRIMER SEQUENCES....................................................................45 B DNA AND PROTEIN SEQUENCES........................................................................46 DIF1 DNA Sequence..................................................................................................46 DIF1 Protein Sequence...............................................................................................47 v
DIF2 DNA Sequence..................................................................................................48 DIF2 Protein Sequence...............................................................................................48 C SECONDARY STRUCTURE ANALYSIS...............................................................49 Secondary Structure of DIF1 from Network Protein Sequence Analysis Website....49 Secondary Structure of DIF2 from Network Protein Sequence Analysis Website....54 D EXPRESSION PROFILES OF DIF1 AND DIF2......................................................57 Ludwig Transcript (LT) Viewer Results For DIF1....................................................57 Digital Northern Results for DIF1..............................................................................57 SAGE Anatomical Viewer Results for DIF1..............................................................59 Ludwig Transcript (LT) Viewer Results For DIF2....................................................61 Digital Northern Results for DIF2..............................................................................61 SAGE Anatomical Viewer Results for DIF2..............................................................63 LIST OF REFERENCES...................................................................................................65 BIOGRAPHICAL SKETCH.............................................................................................68 vi
LIST OF TABLES Table page 1. Molecular weights and isoelectric points of small subunit proteins...............................7 2. Molecular weights and isoelectric points of large subunit proteins................................8 vii
LIST OF FIGURES Figure page 1. A diagram of the apoptotic cascade................................................................................6 2. Alignment between DIF2 and Kv4.2............................................................................26 3. Alignment between DIF2 and B12...............................................................................27 4. Alignment between human DAP3 sequence and bovine DAP3 sequence...................28 5. Alignment between human and mouse DIF1 sequences..............................................29 6. Alignment between human and mouse DIF2................................................................30 7. A PCR of a part of DIF1 purchased from ATCC.........................................................31 8. A PCR used to amplify DIF2 purchased from ATCC..................................................32 9. DIF1 protein is over-expressed in E.coli......................................................................33 10. DIF1 protein purification............................................................................................34 11. DIF2 protein is over-expressed in E.coli....................................................................35 12. DIF2 protein purification............................................................................................36 13. Characterization of DIF1 chicken antibodies.............................................................37 14. Characterization of DIF2 chicken antibodies.............................................................38 15. A schematic diagram of the mitochondrial sub-fractionation experiment..................39 16. A series of western blots of each of the mitochondrial subfractions..........................40 viii
Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EXPRESSION AND PARTIAL CHARACTERIZATION OF TWO NEW PROTEINS THAT INTERACT WITH DEATH ASSOCIATED PROTEIN-3, A MITOCHONDRIAL RIBOSOME PROTEIN AND POSITIVE MEDIATOR OF APOPTOSIS By Amar M. Singh August 2002 Chair: Thomas W. Oâ€™Brien Major Department: Molecular Genetics and Microbiology Mitochondria are known for their roles in oxidative phosphorylation and the process of apoptosis. Mitochondrial ribosomes are responsible for the translation of 13 proteins which function in oxidative phosphorylation and the generation of ATP. One of the proteins on the mitochondrial ribosome, death associated protein-3 (DAP3), has also been found to be a mediator of programmed cell death. The screening of DAP3 in the yeast-two-hybrid system found 3 proteins with which it interacts. Two of these proteins were new and uncharacterized, while the third was a mitochondrial ribosome protein. The purpose of this present study was to further characterize the two interacting proteins, known as DAP3 interacting factors-1 and 2 (DIF1 and DIF2). Since neither of these proteins contained a mitochondrial targeting signal, the sub-cellular localization of ix
DIF1 and DIF2 was necessary. First, the proteins had to be expressed for use as antigens for the preparation of antibodies. Once the antibodies were made, mitochondria were isolated and sub-fractionated, and then screened for the presence of DIF1 or DIF2. Both DIF1 and DIF2 appeared in the matrix compartment of mitochondria. These findings, and the data obtained from in silico analysis of DIF1 and DIF2, have important implications. These proteins may be involved in the docking of mitochondrial ribosomes to the inner-membrane to initiate translation. Alternatively, these proteins may work in concert with DAP3 in its proapoptotic role. x
CHAPTER 1 INTRODUCTION Mitochondria have been termed the â€œpowerhouse of the cellâ€ for their role in oxidative phosphorylation and ATP synthesis. The ATP generated inside of mitochondria is responsible for providing energy for all cell functions in eukaryotes. More recently however, a new role of mitochondria have been observed. The release of cytochrome C from the intermembrane space of mitochondria into the cytoplasm of all metazoans allows for the formation of the apoptosome and subsequent progression of programmed cell death or apoptosis. In many cases, apoptosis begins with extracellular signaling molecules binding to death receptors such as Fas or TNFreceptor. Intracellular molecules such as FADD and TRADD bind to the cytoplasmic tails of these receptors to form the Death Inducing Signaling Complex (Chen and Wang, 2002). Further intracellular signaling takes place involving caspase-8 cleavage of Bid. Bid then interacts with other Bcl-2 members, which allow cytochrome C release through a still controversial mechanism (Kroemer and Reed, 2000). This mechanism is thought to involve membrane depolarization and protein complexes at membrane contact sites (Crompton, 2001). Once the apoptosome is formed from cytochrome C and APAF1, this 700 kDa complex will cleave and activate the effector molecule, caspase-3. Caspase-3 will cleave and interact with many other caspases and proteins that cause DNA fragmentation, chromatin condensation, phosphatidylserine externalization, blebbing, and the final formation of apoptotic bodies that are characteristic of apoptosis (see figure 1 by Joza et al., 2002). These apoptotic 1
2 bodies are then engulfed through phagocytosis by macrophages and are the final step of the cellâ€™s demise (Reed and Kroemer, 2000). The Mitochondrial Ribosome The isolation of mitochondrial ribosomes by Oâ€™Brien in 1967 led to considerable controversy, since these ribosomes were very different from both bacterial and cytoplasmic ribosomes (Oâ€™Brien and Kalf, 1967a; and Oâ€™Brien and Kalf, 1967b). Many believed that if mitochondria did have ribosomes they would be identical to bacteria, since mitochondria were derived from the invasion of a -proteobacteria into a proto-typical eukaryotic cell. Although mitochondrial ribosomes do share some features in common with bacterial ribosomes, many important differences were later discovered. For example, mitoribosomes have a lower sedimentation coefficient of 55S compared to the bacterial 70S. The small and large subunits of mitoribosomes are 28S and 39S, respectively, compared to the small bacterial ribosome subunit of 30S and large subunit of 50S (Oâ€™Brien, 2002). These alterations in sedimentation coefficients are the result of a lower rRNA content, but higher protein content. However, even with the lower sedimentation rate, the mitochondrial ribosomes are actually larger in size than their bacterial counterparts (Oâ€™Brien, 2002). The diminished rRNA content can be best noted in the lack of a 5S rRNA strand, which is found in all other ribosomes. Also, the 12S rRNA of the mitoribosome small subunit is shorter than the bacterial small subunit 16S rRNA. In addition, the 18S rRNA of the mitoribosome large subunit is shorter than the 23S bacterial large subunit rRNA (Anderson et al., 1981). The mitochondrial ribosome consists of 79 proteins, while the bacterial ribosome has only approximately 50 proteins. Only 16 proteins of the 31 in the small subunit of mitoribosomes, and 28 proteins of the
3 48 in the large subunit of mitoribosomes have sequence similarity to bacterial ribosome proteins (Koc et al., 2001a; Suzuki et al., 2001; and Oâ€™Brien, 2002; see tables 1 and 2). All of these mitoribosome proteins are translated in and imported from the cytoplasm into the mitochondria, while mitoribosomal RNA is strictly derived from the mitochondrial DNA. Therefore, two separate genomesâ€”the nucleus and mitochondriaâ€”are responsible for the assembly of the mitochondrial ribosome (Oâ€™Brien, 2002; and Oâ€™Brien et al., 1990). These mitoribosome proteins may play more than just a structural role as is commonly believed of all proteins found in other ribosomes. Most interestingly, mitochondrial ribosome protein S29 (MRP-S29) has been found to bind GTP and GDP with high affinity, unlike any other ribosome protein in nature (Denslow et al., 1991). This protein, earlier named MRP-S5, by Matthews, et al in 1982, has now been named death associated protein-3 (DAP3) as it has been implicated as a positive mediator of apoptosis (Kissil et al., 1995; and Kissil and Kimchi, 1997). Mitochondrial ribosomes are responsible for translating approximately 13 mRNA encoded by the mitochondrial genome. The transcribed mitochondrial mRNA does not have a Shine-Dalgarno sequence, nor a guanosine cap, and therefore translation begins at the 5â€™ end (Oâ€™Brien, 2002). These proteins translated by the mitoribosome include cytochrome C oxidase I and III (COX I and III) of the electron transport chain. Once these proteins are synthesized on mitochondrial ribosomes, they are exported into the mitochondrial inner-membrane and assembled with other COX subunits that are encoded by the nucleus and translated by cytoplasmic ribosomes (Saint-Georges et al., 2001). The Oxa1 and Mba1 proteins facilitate the export and assembly of the COX complex (Hell et al., 2001 and Preuss et al., 2001). Mitochondrial ribosomes are anchored to the matrix side of the inner-membrane, most likely by the small subunit, when undergoing active
4 translation (Denslow et al., 1989; and Oâ€™Brien, personal communication). How the ribosome is assembled onto the inner-membrane and the proteins that anchor them there are yet to be determined. In addition, since inner-membrane depolarization is a key event prior to cytochrome C release during apoptosis, the finding of a mitochondrial ribosome protein, DAP3, being involved in apoptosis may implicate the mitochondrial ribosome in cell death (Koc et al., 2001b). Alternatively, DAP3 may be a bifunctional protein being involved in both mitochondrial biogenesis and programmed cell death. DAP3 and its Interacting Proteins Death Associated Protein-3 (DAP3) has been found to be a pro-apoptotic mediator for Interferon-gamma, TNF-alpha, and Fas-induced cell death through antisense RNA knock-out experiments (Kissil et al., 1999). In addition, when the high affinity nucleotide binding P-loop motif for GTP and GDP is mutated, this protein loses its ability to induce apoptosis when overexpressed in HeLa cells (Kissil et al., 1999). DAP3 has also been suggested to interact with the glucocorticoid receptor and with the TNF related apoptosis inducing ligand (TRAIL) receptors (Hulkko et al., 2000; Hulkko and Zilliacus, 2002; and Miyazaki and Reed, 2001). The interactions of DAP3 with these receptors are questionable since this would place DAP3 in both the nucleus and the cytoplasm. However, all work to date has localized DAP3 strictly in the mitochondria and on the mitochondrial ribosome (Denslow et al., 1991 and Morgan et al., 2001). More recently, the interaction of DAP3 with the TRAIL receptor has been shown to be a false positive interaction occurring only during disruption of mitochondrial membranes (Berger et al., 2002). There are a few proteins that are released from mitochondria during apoptosis, such as cytochrome C and second mitochondrial activator of caspases (Smac); however,
5 DAP3 does not appear to be one of them, at least during Fas-induced cell death (Berger et al., 2000). Screening of Death Associated Protein-3 (DAP3) as a bait protein in the Yeast-2-Hybrid system against a cDNA library from human brain found 3 proteins with which it interacts (Song, 2000). One of the proteins, termed Interacting Protein-3 (IP-3), has now been found to be another mitochondrial ribosome protein, MRP-S35 (Koc et al., 2001). The other two interacting proteins (IP-1 and IP-2) appeared to be new and as of yet uncharacterized. The goal of the present study is to provide a partial characterization of IP-1 and IP-2, now renamed DAP3 interacting factors-1 and -2 (DIF1 and DIF2), respectively.
6 Figure 1. A diagram of the apoptotic cascade. It begins with the activation of the death receptors and formation of the DISC, leading to cytochrome C release and effector caspase activation (Joza et al., 2002).
7 Table 1. Molecular weights and isoelectric points of small subunit proteins (HGNC nomenclature) from human mitochondrial ribosomes, compared to E. coli ribosomal proteins (Oâ€™Brien, 2002)
8 Table 2. Molecular weights and isoelectric points of large subunit proteins (HGNC nomenclature) from human mitochondrial ribosomes, compared to E. coli ribosomal proteins (Oâ€™Brien, 2002)
CHAPTER 2 MATERIALS AND METHODS Cloning of DIF1 and DIF2 into pET21b Vector clones containing a partial sequence for DAP3 interacting factor-1 (DIF1), accession: BC008883, and most of the sequence for DAP3 interacting factor-2 (DIF2), accessions: NM_153331 or AKO27572, were purchased from American Type Culture Collection (ATCC). Primers were designed and purchased against the DIF1 and DIF2 vector sequences from GenoMechanix, LLC. (See Appendix A for primer sequences). A Sal I restriction endonuclease site flanked the forward primer, and a Not I restriction endonuclease site flanked the reverse primer for directional cloning. A polymerase chain reaction (PCR) Supermix (Invitrogen) was used to amplify the DIF1 and DIF2 DNA sequences. The reaction mixture consisted of 45L of the PCR supermix (22 mM Tris-HCl pH 8.4, 55 mM KCl, 1.65 mM MgCl 2 , 220 M dGTP, 220 M dCTP, 220 M dATP, 220 M dTTP, 22 Units Taq Polymerase/mL, stabilizers), 2 L each of the forward and reverse primers at a 1.5 M final concentration, and 1 L of the vector DNA at 10-50 ng, for a total reaction volume of 50 L. The PCR reaction proceeded as follows: an initial denaturing step for 2 min at 94C, a denaturing step of 30 seconds for all subsequent cycles, an annealing step at 60C for 30 seconds, and an elongation step at 72C for 1 minute and 30 seconds. This reaction was repeated for a total of 25 cycles in an Eppendorf themocycler. The PCR reactions were purified using the Qiagen PCR purification kit according to the manufacturerâ€™s guidelines. Briefly, the PCR reactions were resuspended in a 5X 9
10 volume of Buffer PB (proprietary formula) and passed through a Qiagen QIAquick Column. The columns were then centrifuged at 13,000 rpm in a standard tabletop microcentrifuge for 1 minute. The column was washed with 750 L of the ethanol containing-Buffer PE (proprietary formula) and eluted with 50 L Buffer EB (10 mM Tris-HCl, pH 8.5) by centifugation. The amplified DNA from the PCR reactions was digested using Sal I and Not I restriction enzymes (New England Biolabs). Approximately 2-3 g of DNA in a volume of 30 L was added to a microcentrifuge tube, containing 4 L Sal 1 Buffer (150 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 , 1 mM dithiothreitol pH 7.9 at 25C) and BSA (100 g/mL). Deionized water and 1 L each from both of the restriction enzymes were added to bring the final volume to 40 L. The digestion reactions proceeded overnight at 37C. The following day the digestion reactions were inactivated at 65C for 20 minutes. In addition, a pET21b expression vector, purchased from Novagen, was digested in a similar manner. The digested reactions were run on 1% agarose gels stained with Ethidium Bromide at 120V for 1 hour. The DNA bands were excised from the agarose under UV light. Using the Qiagen gel extraction kit, the DNA sequences for DIF1, DIF2, and pET21b were purified away from the agarose. The excised agarose was weighed, suspended in a 3X volume of Buffer QG (proprietary formula), and brought into solution by heating in a water bath at 50C for 10 minutes. Isopropanol at a 1X volume was added to the mixture and spun through a Qiagen QIAquick Column in a tabletop microcentrifuge at 13,000 rpm for 1 minute. The column was washed with Buffer PE and eluted with Buffer EB.
11 To estimate the concentration of digested and purified DNA, absorbance readings were measured using a spectrophotometer at 260/280 nm. The absorbance readings at 260 nm for pET21b, DIF1, and DIF2 were 0.1160, 0.0177, 0.0629, respectively. Using the conversion factor of 1 absorbance unit at 260nm equals 50 g DNA/mL and since the dilution factor was 50, the estimated DNA concentrations were 0.29 g/L, .044 g/L, and 0.157 g/L, respectively. The DIF1 and DIF2 inserts were ligated into the pET21b vector using a DNA ligase kit from Roche. A 3 to 1 insert to vector molar ratio was used for the ligation. The conversion factor of 1 mole x base-pair equals 650 g DNA, and the base-pair length of the DNA were used to estimate the molar concentration of the insert or vector per uL. The pET21b, DIF1, and DIF2 concentrations were 8.26 x 10 -14 mols/L, 3.64 x 10 -13 mols/L, and 1.13 x 10 -13 mols/L, respectively. The ligation reaction was set-up as described in the Roche kit instruction manual. The appropriate concentration of DNA to give an insert to vector ratio of 3 to 1 was added to 10 L of 2X buffer (proprietary formula), 4 L of 5X buffer (proprietary formula), and deionized water to bring the volume to 19 L. The final reaction volume was brought to 20 L by addition of 1 L of T4 DNA Ligase. The reaction proceeded for 30 minutes at room temperature and then left overnight at 4C. XL-10 gold E.coli purchased from Stratagene was used for the transformation of the ligation reactions. Approximately 1-10 ng of DNA was added to 40 L of the E.coli and kept on ice for 30 minutes. The cells were heat-shocked at 42C for 20 seconds and followed by a 2-minute incubation on ice. Then 950 L of LB broth was added to the cells and shaken at 37C for 1 hr at 225 rpm. Next 100 L of the reaction was plated
12 onto petri dishes containing LB agar with 100 g/mL ampicillin. The plates were incubated at 37C overnight. The following day, individual colonies were picked from the plates and grown in LB media containing ampicillin for 15 hours. Plasmids were extracted from the bacterial cultures using the Qiagen Miniprep kit as described in the manufacturerâ€™s protocol. Briefly, the cultures are pelleted and resuspended in 250 L Buffer P1 (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 g/mL Rnase A). 250 L Buffer P2 (200 mM NaOH, 1% SDS w/v) and then 350 L Buffer N3 (proprietary formula) are added which is followed by a 10-minute spin at 13,000 rpm in a tabletop microcentrifuge. The supernatant is collected and spun through a Qiagen minicolumn. The minicolumn is then washed with Buffer PE and eluted with Buffer EB. To determine if the insert was successfully ligated into the vector, the obtained plasmids were digested with Sal I and Not I restriction endonucleases and analyzed by electrophoresis on agarose gels as previously described. In additon, the obtained plasmid was subjected to PCR as described above to see if the insert was present and at an appropriate size. Protein Expression and Purification of DIF1 and DIF2 BL21 (DE3) E.coli cells were purchased from Stratagene and transformed using the DIF1 or DIF2-pET21b vector constructs. This transformation also used a heat shock protocol as previously described. To express DIF1 and DIF2, a single isolated colony was picked from a LB-ampicilin plate and grown in 2 mL of LB media supplemented with ampicillin for approximately 4 hours shaking at 225 rpm at 37C until the absorbance at 600 nm was between 0.4 and 1.0. Then, 500 L of the culture was used to innoculate 500 mL of LB media with ampicillin. This large culture was again shaken for
13 3-4 hours at 37C until the absorbance at 600 nm was close to 1.0. IPTG was added to a final concentration of 1 mM to induce the protein expression and the culture was grown for another 3 hours. The culture was then pelleted at 6500 x g at r max for 12 minutes and stored at C. To lyse the E.coli pellets, lysis buffer consiting of 50 mM Tris-HCl and 100 mM NaCl was used to resupend the pellets. Lysozyme was then added to a final concentration of 100 g/mL and shaken for 30 minutes at 4C. The resuspened pellets were then subjected to sonication for eight 1-minute intervals with 1-minute rests at 4C. The solutions were centrifuged in a Beckman JA-20 rotor at 17,700 x g at r max for 10 minutes, and the supernatant was kept for analysis. The pellets were resuspended in lysis buffer containing 0.5% Triton X-100 and again centrifuged. The supernatant was kept for analysis. The final pellets were resuspended in lysis buffer containing 8 M urea. The first supernatant, the triton supernatant, and the urea resuspension were analyzed by SDS-PAGE on 10% acrylamide gels and visualized by Coomassie staining. To purifiy the DIF1 and DIF2 proteins, Novagenâ€™s His-Bind Kit was used according to the manufacturerâ€™s batch protocol. The resin is washed with deionized water and charged with 50 mM NiSO 4 . The resin is collected by centrifugation at 500-1000 rpm in a tabletop microcentrifuge for 1 minute. The resin is washed with binding buffer solution (0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole, pH 7.9) supplemented in 8 M urea and collected by centrifugation. The DIF1 or DIF2 proteins are then incubated with the resin while shaking gently for 5-10 minutes. The resin is collected by centrifugation and washed twice with 3 resin volumes in binding buffer with 8 M urea. The resin is again collected by centrifugation and the supernatant is kept for analysis. The resin is then washed twice with 3 resin volumes of wash buffer (0.5 M NaCl, 20 mM
14 Tris-HCl, 60 mM imidazole, pH 7.9) with 8 M urea and collected by centrifugation. Again, the supernatant was kept for analysis. Next, the resin is resuspended with 3 resin volumes of elute buffer (0.5 M NaCl, 20 mM Tris-HCl, 1 M imidazole, pH 7.9) with 8 M urea for a few minutes and collected by centrifugation. The supernatant is collected and should have the protein of interest. Finally, the resin is stripped of any remaining protein using strip buffer (0.5 M NaCl, 20 mM Tris-HCl, 100 mM EDTA, pH 7.9), and the process can be repeated. Each supernatant is subsequently analyzed by SDS-PAGE on 10% acrylamide gels and visualized using Coomassie staining. The elute buffer supernatant was dialyzed using FisherBrand dialysis tubing overnight into phosphate-buffered saline with 2-3 buffer changes. The protein solution was then lyophilized overnight. Immunization, Purification and Characterization of Chicken Antibodies Approximately, 100-200 g of the lyophilized DIF1 or DIF2 proteins were resuspended in water, mixed with MDL-TPL adjuvant (Sigma) in a 50:50 ratio, and injected subcutaneously under the breast area of chickens. The chickens were boosted with a similar injection 3-4 weeks following the first immunization. Eggs were collected before the first injection and following each immunization. The eggs were cracked and the yolk was cut away from the surrounding albumen. The yolks were suspended in a 1X volume of phosphate buffered saline (approximately 10 mL/yolk) for 30 min at room temperature, and then centrifuged at 12,000 x g at r max for 20 minutes. The supernatant was collected and treated with a 2X concentration of chloroform, shaken every 30 minutes for 2 hours. The mixtures were centrifuged at 5000 x g at r max for 10 minutes and the supernatant containing IgY was removed. The IgY was then supplemented with
15 bovine serum albumin to a 1% final concentration, and stored at C. (Aulisio and Shelokov, 1967). To purify the antibodies specific to DIF1 or DIF2 from the total egg isolate of IgY, affinity column chromatography was used. DIF1 or DIF2 was conjugated to CNBractivated sepharose 4B from Amersham Biosciences. DIF1 or DIF2 was first dialyzed into the sepharose binding buffer, consisting of 0.2 M NaHCO 3 , 0.5 M NaCl at pH 9.0, and 8 M urea overnight with 2 buffer changes. One gram of resin is activated by the addition of 5-10 mL of 1 mM HCl in a beaker for 15 minutes. The resin is then washed with 500 mL of 1 mM HCl by vaccum filtration. The resin was scraped off the filter and added to 10 mL of binding buffer without urea into a 50 mL conical tube. DIF1 or DIF2 at 1-2 mg protein in 10 mL of binding buffer with urea was added to the conical tube. The tube was then shaken gently at room temperature for 2 hours. Blocking solution (0.2 M glycine, 0.5 M NaCl, pH 8.0) was added at an equal volume (20 mL), and shaken gently for 1 hour at room temperature. The resin was again collected by vacuum filtration and washed with 250 mL of binding buffer, 250 mL of wash buffer (0.25 M NaOAc-, pH 4.5 with acetic acid), 250 mL of binding buffer, and 250 mL of wash buffer. Finally the resin was washed with the buffer used for storage of the resin, Tris-buffered saline + Tween-20 (TBST), which consisted of 25 mM Tris-HCl, 125 mM NaCl, and 0.15% Tween-20, pH 8.0. The resin was placed into a column and stored in TBST. The antibodies purified from the chicken eggs were passed over the column containing the DIF1 or DIF2 conjugated to CNBractivated sepharose. The resin was washed with 20-30 mL of TBST and eluted with 3 mL of elute buffer (100 mM glycine, pH 2.3). The eluate was neutralized immediately upon release from the column as it dripped into a tube containing 150 L of 1 M Tris-HCl, pH 8.0, which was determined
16 prior to bring 3 mL of elute buffer to a pH between 7.0 and 8.0. Thirty miligrams of BSA (1% final concentration) was also added into the tube prior to elution to serve as a blocking agent. After elution, the protein-bound resin was neutralized by the passage of 10 mL of TBST through the column. To characterize the purified antibodies, western blots at different dilutions of the antibodies were performed. DIF1 or DIF2, at 1-2 g, were loaded onto a 10% acrylamide containing SDS-PAGE gels next to a marker and run for 70 V in the stacking gel, and 180 V in the separating gel. The proteins were transferred onto nitrocellulose by electroblotting overnight at 30 volts at 4C. The nitrocellulose membrane was then blocked in 3% BSA in TBST for 30 minutes. The antibodies were diluted 1/50, 1/100, and 1/200 in the TBST with 3% BSA and shaken for 1 hour at room temperature. The membrane was then washed 3 times with TBST at 10-minute intervals. The membrane was incubated with secondary rabbit anti-chicken antibodies conjugated to alkaline phosphatase (purchased from Sigma) at a 1/30,000 dilution in TBST with 3% BSA for 1 hour at room temperature. The membrane was washed as described above and developed by the use of BCIP/NBT tablets (purchased from Sigma) disolved in 10 mL of deionized water. The appropriate antibody concentration was empirically determined by visualization of the blot. Isolation and Sub-fractionation of Bovine Mitochondria Mitochondria from bovine liver were isolated as previously described (Oâ€™Brien and Denslow, 1996). Briefly, fresh liver was sliced and ground using a meat grinder. The ground meat was mixed with isolation (ISO) media (1.36 M Sucrose, 80 mM Tris-HCl, 4 mM EDTA, pH 7.5) and pulsed for 2-5 seconds, 6 to 8 times in Hamilton Beach kitchen blender. The liver mince was then passed through a screen and homogenized using a
17 Tekmar. The solution was centrifuged gently in a Beckman JA-10 rotor at 1590 x g at r max to remove the nuclei. The mitochondria-containing supernatant was removed and washed several times by centrifugation at 17,700 x g at r max , and the mitochondrial pellets were resupended to homogenicity. To calculate the concentration of mitochondria, the absorbance was measured at 550 nm and then compared to a standard curve (0.5 A 550 = 184 g/mL). To sub-fractionate mitochondria, a freshly prepared 1 gram sample in 24 mL ISO was treated with 13.4 mL digitonin from a 6 mg/mL stock solution in ISO. The final concentration was 0.08 grams digitonin/1 gram mitochondria and the mitochondrial concentration was 25 mg/mL. The digitonin-ISO mixture was brought into solution by heating in a microwave for 10-second intervals. The solution was cooled before addition to the mitochondria. The solution mixture was incubated for 15 minutes at 4 C and then centrifuged in a Beckman JA-20 rotor at 17,400 x g at r max for 10 minutes. This first supernatant contained outer-membrane, intermembrane space, and matrix proteins. The pellet containing partially disrupted mitoplasts was then resuspended and homogenized in mitofrac buffer (20 mM HEPES, 40 mM KCl, 10 mM MgCl 2 , 1 mM DTT, at pH 7.5). The resuspended mitoplasts were sonicated 8 times for 1-minute intervals with 1-minute rests to release matrix proteins. This solution was centrifuged in the JA-20 rotor at 500 x g at r max for 45 minutes. This second supernatant contained inner-membrane and matrix proteins. The two supernatants were then centrifuged for 120,000 x g at r max for 30 minutes. The first supernatant contained intermembrane space and matrix proteins, while its pellet contained outer-membrane proteins. The second supernatant contained matrix proteins, while its pellet contained inner-membrane proteins. The two pellets were solubilized by treatment with ISO containing 1.6% Triton X-100. These solutions were
18 then centrifuged at 40,000 x g at r max for 20 minutes. The outer-membrane and inner-membrane proteins were subsequently collected from the supernatants. Each fractionâ€”outer-membrane, intermembrane space and matrix, inner-membrane, or matrixâ€”at 0.04% was run on 10% SDS-PAGE gels and electroblotted onto nitrocellulose. Antibodies against cytochrome C (LabVision), cytochrome C oxidase IV (Molecular Probes), and HSP60 (StressGen Biotechnologies) were used in western blots at the appropriate concentrations as suggested by the manufacturer. Western blots were conducted as previously described except with the substitution of an anti-mouse conjugated to alkaline phosphatase purchased from Sigma for the secondary antibody. Once again, the blots were developed by use of BCIP/NBT tablets purchased from Sigma. Localization of DIF1 and DIF2 To prepare immuno-affinity columns, 3 mL of affinity-purified antibodies isolated from 4 egg yolks (which may be used at a 1/200 dilution on western blots) were conjugated to CNBractivated sepharose 4B from Amersham Biosciences. This procedure is the same as previously described for the coupling of DIF1 and DIF2. Each of the mitochondrial subfractions containing outer-membrane, intermembrane space and matrix, inner-membrane, or matrix, prepared as described above, were passed over the column containing the antibody-linked sepharose. The sepharose resin was washed with 20 mL mitofrac wash buffer (100 mM KCl, 20 mM MgCl2, 25 mM Tris and 0.15% Tween-20, pH 7.5) and then eluted with 3 mL elute buffer (100 mM glycine, pH 2.3). The eluate was neutralized by 150 L of 1 M Tris-HCl, pH 8.0. Each eluate was concentrated with a Chemicon Microconcentrator according to the manufacturerâ€™s instructions. The sample is centrifuged in the concentrator at 7000 rpm for 4-5 hours in a
19 Beckman JA-20 rotor and the protein is collected onto a membrane filter while the solution is spun through. The protein samples were then eluted off the concentrator membrane with 80 L of concentrator elution buffer (50 mm Tris-HCl, 2% SDS w/v, pH 6.8). Each sample was run on 10% SDS-PAGE gels and electroblotted onto nitrocellulose membranes. These membranes were used in western blot analysis with antibodies against DIF1 or DIF2, as described above.
CHAPTER 3 RESULTS In Silico Analysis of DIF1 and DIF2 The DNA sequences for DAP3 interacting factors-1 and 2 were identified from screening DAP3 against a cDNA library in the yeast-two hybrid system. These DNA sequences did not encode the entire protein and in many cases the sequences differed in length (Song, 2000). The smallest DNA sequences of DIF1 and DIF2, identified in the screen, thus represent a putative binding domain for DAP3 (Appendix B). To determine the full-length DNA and protein sequences for DIF1 and DIF2, BLAST analysis was used on the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST/). To investigate the more detailed characteristics of these proteins several other in silico analyses were conducted. Using the full-length proteins, further BLAST analyses were done to see if any similar proteins had been characterized. BLAST analysis of DIF1 did not find any proteins with significant similarity. However, searching with DIF2 found two proteins that were similar to the DIF2 amino-terminus. DIF2 had significant homology with a potassium channel tetramerization domain protein, Kv 4.2 (accession NP_036413), having 44.8% identity over a 67 amino acid overlap with an E-value of 8.3 x 10 -8 (Sansom, 1998). In addition, DIF2 had homology with a TNF-alpha induced protein, B12 (accession: Q13829), having 38.3% identity over a 133 amino acid overlap with an E-value of 7.8 x 10 -14 (Sarma et al., 1992; and Wolf et al., 1992; see Figures 2 and 3). BLAST analysis was also conducted on EST databases for different species, including mice and cows. Generally speaking mitochondrial ribosome proteins are 20
21 approximately 70-90% similar between species (Oâ€™Brien, 2002; see Figure 4). Interestingly, DIF1 is also within this range, showing a 73.5% similarity between mouse and human sequences (Figure 5). Surprisingly, DIF2 has a 99.2% similarity between mouse and human sequences, suggesting that this protein is strongly evolutionally conserved (Figure 6). To analyze the secondary structure of DIF1 and DIF2, the sequences were entered into the Network Protein Sequence Analysis website (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html). For both DIF1 and DIF2, the sequences are composed mostly of -helices, random coils and a few short extended strands, suggesting that both proteins are mostly globular in structure (Appendix C). To determine the expression profiles of DIF1 and DIF2; that is, their abundance in multiple tissue types, the SAGEâ€”Serial Analysis of Gene Expressionâ€”anatomic viewer was used at the Cancer Genome Anatomy Project website (http://cgap.nci.nih.gov/SAGE/AnatomicViewer). The accession numbers were entered into the website, and a 10 base-pair SAGE tag is identified, based upon the furthest downstream sequence after a Nla III restriction site. The tag is then screened against several cDNA library databases of multiple tissue types. The number of hits in each library determines the proteinâ€™s relative abundance. Both DIF1 and DIF2 seem to be ubiquitously expressed, but at very low levels (Appendix D). Their expression profiles are thus at an equivalent stoichiometric expression level as DAP3 and also, like DAP3, these proteins are found in most cell types. DAP3 is a mitochondrial ribosome protein and contains a standard amino-terminal import signal (Morgan et al., 2000 and Koc et al., 2000). To determine if DIF1 and DIF2 also contain such a signal, their respective sequences were searched on MitoProt and
22 TargetP (http://mips.gsf.de/cgi-bin/proj/medgen/mitofilter and http://www.cbs.dtu.dk/services/TargetP/). Neither of the interacting proteins contained such an import signal. If these proteins were indeed mitochondrial, they would most likely contain internal import signals, which have not been well characterized (Oâ€™Brien, personal communication). Cloning and Expression of DIF1 and DIF2 Antibodies against DIF1 and DIF2 were needed for future experiments to localize the proteins. Therefore, the DIF1 and DIF2 proteins were expressed for use as antigens to develop the antibodies. First, the DNA sequences for DIF1 and DIF2, purchased from ATCC, were used for the creation of an expression vector. To express DIF1 and DIF2, the pET vector system from Novagen was used, since it allows the expression of a large amount of protein in E.coli. Using standard molecular biology cloning techniques, the DIF1 and DIF2 DNA sequences were amplified using PCR (Figures 7 and 8), digested, and ligated into the pET21b vector. This vector contains a C-Terminal hexahistidine tag, which has a high affinity for nickel and allows for quick and easy purification on nickel-bound resin. As with all pET vectors, protein expression is under control of the Lac promotor and is induced by the addition of IPTG to the media. IPTG binds to and inhibits the Lac repressor allowing the protein to be expressed. Once the expression vectors were constructed, they were transformed into BL21-DE3 E.coli. The expressed proteins were induced with IPTG and harvested in E.coli as described in the Material and Methods. Each crude fraction containing lysis buffer, lysis buffer with Triton X-100, or lysis buffer with urea, was then analyzed by electrophoresis on SDS-PAGE gels and stained with Coomassie Blue. High protein expression was found mostly in the urea containing samples, suggesting the proteins were highly
23 expressed and formed inclusion granules (see arrows on Figures 9 and 11). The DIF1 and DIF2 proteins were then isolated using Novagenâ€™s His-Bind Kit. Resin charged with nickel were bound by the proteins, washed, eluted with high concentrations of imidazole, and subsequently stripped of the nickel. To assess the purity of the isolated protein, each fraction (wash, elute, and strip) was run on SDS-PAGE gels and stained with Coomassie Blue (Figures 10 and 12). The level of purity was empirically determined to be 80-90%, and the protein concentration was determined from the gel to be approximately 10 mg protein per liter of culture grown as described in the Materials and Methods. The isolated protein was then dialyzed against PBS and lyophilized. Antibody Preparation and Characterization for DIF1 and DIF2 Chickens were used to create antibodies against DIF1 and DIF2. The lyophilized proteins were resuspended in water and mixed with adjuvant in a 50:50 ratio. Approximately 200 g of protein were injected into the chickens and were again boosted after a 3-4 week interval. Eggs were collected daily from the chickens. Antibodies were isolated from the chicken egg yolks using chloroform to remove lipids as described by Aulisio et al in 1967. Since the antibodies isolated from the yolks would be the full repertoire of the chicken, it was necessary to further purify the DIF1 and DIF2 antibodies from the total antibody isolate for experimental use. Affinity chromatography was used to purify the antibodies by binding the DIF1 or DIF2 proteins to CNBractivated sepharose. The protein-coupled resins were then placed into a column. The total antibody isolates from several chicken eggs were pooled and passed over the resin, washed, and eluted. To characterize the antibodies, DIF1 or DIF2 proteins were run on SDS-PAGE gels and blotted onto nitrocellulose. The prepared antibodies were then diluted at various concentrations with TBST and used in western blotting. For both DIF1
24 and DIF2 antibodies, the appropriate dilution was empirically determined to be at 1/200 (Figures 13 and 14). Also, antibodies against the contaminating E.coli proteins from the DIF1 and DIF2 protein purification were found in the chickens. However, these proteins were not likely to be conserved with mammalian proteins, and thus the contaminating antibodies would not be detrimental to future experiments. Mitochondrial Sub-fractionation and Localization of DIF1 and DIF2 As mentioned earlier, neither DIF1 nor DIF2 have a standard mitochondrial import signals. Thus, for a meaningful interaction to exist between these newly identified proteins and DAP3, the sub-cellular location of these proteins must be determined. In addition, since mitochondrial ribosome proteins are at a rather low concentration in the cell, the interacting factors of a mitochondrial ribosome protein are also likely to be at a low concentration. Therefore, the preparation of at least a gram of mitochondria was thought to be necessary to purify DIF1 and DIF2 from the mitochondrial subfractions. Fresh mitochondria were prepared and purified from bovine liver as previously described (Oâ€™Brien and Denslow, 1996). A 1-gram sample was then sub-fractionated into the mitochondrial compartments using a combination of various strategies as depicted in Figure 15 (Schnaitman et al., 1967 and Liu et al., 2000). The basic procedure was to partially disrupt the mitochondrial outer and inner-membranes using digitonin. After centrifugation all intermembrane space soluble proteins and outer-membrane proteins were in the supernatant, while partially disrupted mitoplasts were pelleted. Sonication of the pellets released mitochondrial matrix proteins. Finally, after further centrifugation, the supernatants contained the intermembrane space and matrix soluble proteins. In addition, the pellets containing outer and inner-membrane proteins were resuspended in Triton X-100.
25 To determine the successful separation of each mitochondrial sub-fraction, an equal percentage of each fraction was loaded onto SDS-PAGE gels and then western blots for various markers were performed. Antibodies against cytochrome C (Cyt. C), an intermembrane space marker; cytochrome C oxidase IV (COX IV), an inner-membrane marker; and Heat Shock Protein-60 (HSP6), a matrix marker, were used in the experiment (Figure 16). The antibody against cytochrome C identified a 46-kDa protein in fraction 2. This protein represents apocytochrome C, which is associated with the outer-side of the inner-membrane, and was found to be in the intermembrane space during mitochondrial fractionation experiments. In addition HSP60 was also found in fraction 2. This fraction thus represents proteins from the intermembrane space and matrix. COX4 was only detected in fraction 3, suggesting that this fraction contains inner-membrane proteins. COX4 was not found in the outer membrane fraction, suggesting that only weak disruption of the inner-membrane had occurred. At higher concentrations of digitonin, such as 1.2 mg per 10 mg of mitochondria, COX4 was detected in the fraction 1, suggesting that more inner-membrane mitochondrial disruption had occurred (data not shown). Finally, in fraction 4, the only mitochondrial marker detected was HSP60, suggesting that this fraction contained matrix proteins (Figure 16). To isolate DIF1 or DIF2 from each fraction, antibodies against these proteins were conjugated to sepharose. Each mitochondrial fraction was then incubated with the sepharose, washed, and eluted. Each fraction was then concentrated to an equal volume and western blots were performed using the DIF1 or DIF2 antibodies (Figure 16). Both DIF1 and DIF2 seemed to co-purify with HSP60, suggesting mitochondrial matrix localization. However, this fractionation scheme cannot rule out the possibility that one or both of these proteins may be associated with the inner-membrane.
26 10 20 30 40 50 60 DIF2 VTLNVGGHLYTTSLTTLTRYPDSMLGAMFGGDFPTARDPQGNYFIDRDGPLFRYVLNFLR ..:::.: . : :: ::::..::. :: . : .::.::: .::..::: : Kv4.2 IVLNVSGTRFQTWQDTLERYPDTLLGSSER-DFFYHPETQ-QYFFDRDPDIFRHILNFYR 50 60 70 80 90 100 70 DIF2 TSELTLP :..: : Kv4.2 TGKLHYP Figure 2. Alignment between DIF2 and Kv4.2, a potassium channel tetramerization domain protein. Alignment shows 44.8% identity over a 67 amino acid overlap with an E-value of 8.3e-08.
27 10 20 30 40 50 60 DIF2 VTLNVGGHLYTTSLTTLTRYPDSMLGAMFGGDFPTARDPQGNYFIDRDGPLFRYVLNFLR : ::::: :: :.. .:::. :.:: :::.: . . : .: .::: : : .::.:: TNF-AL VQLNVGGSLYYTTVRALTRH-DTMLKAMFSGRMEVLTDKEGWILIDRCGKHFGTILNYLR 30 40 50 60 70 80 70 80 90 100 110 DIF2 TSELTLPLDFKEFDLLRKEADFYQIEPLIQ-C---LNDPKPLY-PMDTFEEVVELSSTRK . .::: . .:. : :: .: :. :.. : :.: : : :. .. .. :. .. TNF-AL DDTITLPQNRQEIKELMAEAKYYLIQGLVNMCQSALQDKKDSYQPVCNIPIITSLKEEER 90 100 110 120 130 140 120 130 DIF2 LSKYSN-PVAVII : . :. ::. .. TNF-AL LIESSTKPVVKLL 150 160 Figure 3. Alignment between DIF2 and B12, a TNF-alpha induced protein. Alignment shows 38.3% identity over a 133 amino acid overlap with an E-value of 7.8e-14.
28 10 20 30 40 50 60 hDAP3 MLKGITRLISRIHKLDPGRFLHMGTQARQSIAAHLDNQVPVESPRAISRTNENDPAKHGD ::::.:::.::.:::::::: :.:::: : .::::::::.: :::::: ::::::::. bDAP3 MLKGMTRLVSRVHKLDPGRFSHLGTQAPQCPVAHLDNQVPTERTRAISRTLENDPAKHGE 10 20 30 40 50 60 70 80 90 100 110 120 hDAP3 QHEGQHYNISPQDLETVFPHGLPPRFVMQVKTFSEACLMVRKPALELLHYLKNTSFAYPA :: ::::::: :.:.::::::::::::::::::.::::::::::::::::::::.::.:: bDAP3 QHVGQHYNISIQELKTVFPHGLPPRFVMQVKTFNEACLMVRKPALELLHYLKNTNFAHPA 70 80 90 100 110 120 130 140 150 160 170 180 hDAP3 IRYLLYGEKGTGKTLSLCHVIHFCAKQDWLILHIPDAHLWVKNCRDLLQSSYNKQRFDQP .::.:::::::::::::::.::::::::::::::::::::::::::::::.::::::::: bDAP3 VRYVLYGEKGTGKTLSLCHIIHFCAKQDWLILHIPDAHLWVKNCRDLLQSTYNKQRFDQP 130 140 150 160 170 180 190 200 210 220 230 240 hDAP3 LEASTWLKNFKTTNERFLNQIKVQEKYVWNKRESTEKGSPLGEVVEQGITRVRNATDAVG :::: :::::::.:::::.:::::.::. ::::::::::::.::::::: :::::::::: bDAP3 LEASIWLKNFKTANERFLSQIKVQDKYIRNKRESTEKGSPLAEVVEQGIMRVRNATDAVG 190 200 210 220 230 240 250 260 270 280 290 300 hDAP3 IVLKELKRQSSLGMFHLLVAVDGINALWGRTTLKREDKSPIAPEELALVHNLRKMMKNDW :::::::::::::.:.:::::::.:::::::::::::::::.::::::..:::::.:::: bDAP3 IVLKELKRQSSLGVFRLLVAVDGVNALWGRTTLKREDKSPITPEELALIYNLRKMVKNDW 250 260 270 280 290 300 310 320 330 340 350 360 hDAP3 HGGAIVSALSQTGSLFKPRKAYLPQELLGKEGFDALDPFIPILVSNYNPKEFESCIQYYL .::::: ..:::::::::::::::::::::::::.::::::::::::::::::.:::::: bDAP3 RGGAIVLTVSQTGSLFKPRKAYLPQELLGKEGFDTLDPFIPILVSNYNPKEFEGCIQYYL 310 320 330 340 350 360 370 380 390 hDAP3 ENNWLQHEKAPTEEGKKELLFLSNANPSLLERHCAYL :::::::::: ::::::::::::: ::.:::: :::: bDAP3 ENNWLQHEKAHTEEGKKELLFLSNRNPGLLERLCAYL 370 380 390 Figure 4. Alignment between human DAP3 sequence and bovine DAP3 sequence. Alignment shows 87.4% identity over a 397 amino acid overlap.
29 Human -----------------------------------------------------------M Mouse DSLDRSGSQACEDKEEAAPPYPVIHVDPDACVLPDAALCAFTVLDDMLVTLAQGPTQWKM * Human QLFEQPCPGEDPRPGGQIGEVELSSYTPPAGVPGKPAAPHFLPVLCSVSPSGSRVPHDLL Mouse QLFERPCAGEEPLPRGQIGEVELSTCTPPGGVPEKPAAPRFLPVLCCVFPPDSRAPHGHP ****:**.**:* * *********: ***.*** *****:******.* *..**.**. Human GGSGGFTLEDALFGLLFGADATLLQSPVVLCGLPDGQLCCVILKALVTSRSAPGDPNALV Mouse QGCGCFTLEEALFGLLFGVDATLLQSPVILCGLPDGQLCCVVLKALVTSGLAPGDPKVLV *.* ****:********.*********:************:******* *****:.** Human KILHHLEEPVIFIGALKTEPQAAEAAENFLPDEDVHCDCLVAFGHHGRMLAIKASWDESG Mouse KILHHLEEPVIFIGALRAEPHEEEAAGELLPGQHEHSDCLVALGHQGRTLAIKASWSESG ****************::**: *** ::**.:. *.*****:**:** *******.*** Human KLVPELREYCLPGPVLCAACGGGGRVYHSTPSDLCVVDLSRGSTPLGPEQPEEGPGGLPP Mouse NLVPELREYCLPGPVLCAACDRDGHVYHSTPSDLCVVDLTRRDSPWNPEKPDGAIGGLPS :*******************. .*:**************:* .:* .**:*: . ****. Human MLCPASLNICSVVSLSASPRTHEGGTKLLALSAKGRLMTCSLDLDSEMPGPARMTTESAG Mouse VLCPASLNICSALALCVTARAPTGSTELLALSSKGRLITCSLDLNSEAPVPAKMAMANAG :**********.::*..:.*: *.*:*****:****:******:** * **:*: .** Human QKIKELLSGIGNISERVSFLKKAVDQRNKALTSLNEAMNVSCALLSSGTGPRPISCTTST Mouse QKIKELLLDIGDVSERVSFLKKAVDQRNKAITSLNEAMNVSCALLSHPEGDRPIACTITT ******* .**::*****************:*************** * ***:** :* Human TWSRLQTQDVLMATCVLENSSSFSLDQGWTLCIQVLTSSCALDLDSACSAITYTIPVDQL Mouse SWSRLELRDMLMATCTLENSSSFSLDQGWTLCIQVLTSSSALDLDGTGSAFTYTIPVDRL :****: :*:*****.***********************.*****.: **:*******:* Human GPGARREVTLPLGPGENGGLDLPVTVSCTLFYSLREVVGGALAPSD-SEDPFLDECPSDV Mouse GPGSRREVTLPLGPSESGVLDLPVTMSCWLFYSLREVVGAALAPSDPLEAPYLEQFP-LS ***:**********.*.* ******:** **********.****** * *:*:: * Human LPEQEGVCLPLSRHTVDMLQCLCFPGLAPPHTRAPSPLGPTRDPVATFLETCREPGSQPA Mouse LPKQEGVCLPLCKRTVDMLQCLRFAGAATHPAQAPCMPGPACEPVETFLKTCQAPGSEPT **:********.::******** *.* *. ::**. **: :** ***:**: ***:*: Human GPASLRAEYLPPSVASIKVSAELLRAALKDGHSGVPLCCATLQWLLAENAAVDVVRARAL Mouse GAASLRAKYLPPSTASIRVSAGLLRAALEDSHSGFHLCSATLRWLLAENAAVDVVRAQTL *.*****:*****.***:*** ******:*.***. **.***:**************::* Human SSIQGVAPDGANVHLIVREVAMTDLCPAGPIQAVEIQVESSSLADICRAHHAVVGRMQTM Mouse SSIQGIAPDGTDVNLTVHEVAVTDLSPAGPIQAVEIQVESSSLANMCRAHHAIIRRIQTM *****:****::*:* *:***:***.******************::******:: *:*** Human VTEQAAQGSSAPDLRVQYLRQIHANHETLLREVQTLRDRLCTEDEASSCATAQRLLQVYR Mouse VTEQAALGSSPPDLRMQYLQQIHANHQELLREVQALRDQLCTEDELSSCSTAQKLLHIYK ****** ***.****:***:******: ******:***:****** ***:***:**::*: Human QLRHPSLILL Mouse QLRNPSLVLL ***:***:** Figure 5. Alignment between human and mouse DIF1 sequences. Alignment shows 73.5% identity over 730 amino acid overlap.
30 10 20 30 40 50 60 human MDNGDWGYMMTDPVTLNVGGHLYTTSLTTLTRYPDSMLGAMFGGDFPTARDPQGNYFIDR ::::::::::.::::::::::::::::::::::::::::::::::::::::::::::::: mouse MDNGDWGYMMSDPVTLNVGGHLYTTSLTTLTRYPDSMLGAMFGGDFPTARDPQGNYFIDR 80 90 100 110 120 130 70 80 90 100 110 120 human DGPLFRYVLNFLRTSELTLPLDFKEFDLLRKEADFYQIEPLIQCLNDPKPLYPMDTFEEV ::::::::::::::::::::::::::::::::::::::::::::::::.::::::::::: mouse DGPLFRYVLNFLRTSELTLPLDFKEFDLLRKEADFYQIEPLIQCLNDPRPLYPMDTFEEV 140 150 160 170 180 190 130 140 150 160 170 180 human VELSSTRKLSKYSNPVAVIITQLTITTKVHSLLEGISNYFTKWNKHMMDTRDCQVSFTFG :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: mouse VELSSTRKLSKYSNPVAVIITQLTITTKVHSLLEGISNYFTKWNKHMMDTRDCQVSFTFG 200 210 220 230 240 250 190 200 210 220 230 human PCDYHQEVSLRVHLMEYITKQGFTIRNTRVHHMSERANENTVEHNWTFCRLARKTDD ::::::::::::::::::::::::::::::::::::::::::::::::::::::::: mouse PCDYHQEVSLRVHLMEYITKQGFTIRNTRVHHMSERANENTVEHNWTFCRLARKTDD 260 270 280 290 300 310 Figure 6. Alignment between human and mouse DIF2. Alignment shows 99.2% identity over a 237 amino acid overlap.
31 DIF1 DNA Figure 7. A PCR of a part of DIF1 purchased from ATCC. The amplified DNA is 649 base pairs in length and corresponds to most of the putative DAP3 interaction domain.
32 DIF2 DNA Figure 8. A PCR used to amplify DIF2. The amplified DNA corresponds to 663 base pairs and encodes most of the full sequence purchased from ATCC.
33 LANES: 1 2 3 4 5 UnpurifiedDIF1453121.5 MW (kDa) Figure 9. DIF1 protein is over-expressed in E.coli. DIF1 was highly expressed in the urea resuspension fraction. Lane 1, marker; Lane 2, lysis buffer supernatant; Lane 3, lysis buffer + Triton X-100 supernatant; Lane 4, urea resuspension; Lane 5, uninduced supernatant control.
34 LANES: 1 2 3 4 5Purified DIF1453121.5MW (kDa) Figure 10. DIF1 protein purification. DIF1 was purified using affinity chromatography on nickel-bound resin and was eluted using imidazole. Lane 1, marker; Lane 2, elution sample; Lane 3, flow-thru sample; Lane 4, wash sample; Lane 5, resin strip sample.
35 LANES: 1 2 3 4 Unpurified DIF2MW (kDa)372520 Figure 11. DIF2 protein is over-expressed in E.coli. DIF2 was highly expressed in the urea resuspension fraction. Lane 1, marker; Lane 2, lysis buffer supernatant; Lane 3, lysis buffer + Triton X-100 supernatant; Lane 4, urea resuspension.
36 LANES: 1 2 3 4 5 MW ( kDa ) 37 25 Purified DIF2 20 Figure 12. DIF2 protein purification. DIF2 was purified using affinity chromatography on nickel-bound resin and was eluted using imidazole. Lane 1, marker; Lane 2, elution sample; Lane 3, wash sample; Lane 4, resin strip sample; Lane 5, flow-thru sample.
37 Dilutions: 1 /50 1/100 1/200 L N : 1 2 3 4 567 DIF1 Figure 13. Characterization of DIF1 chicken antibodies. Approximately, 1 ug of DIF1 protein was loaded in lanes 2, 4, and 6; and 100 ng of DIF1 protein was loaded in lanes 3, 5, and 7. Lane 1 is the marker; Lanes 2 and 3 is a western blot with DIF1 antibodies at a 1/50 dilution; Lanes 4 and 5 is a western blot at 1/100 dilution; Lanes 6 and 7 is a western blot at a 1/200 dilution.
38 LN: 1 2 3Dilutions: 1/50 1/100 1/200 DIF2 Figure 14. Characterization of DIF2 chicken antibodies. Approximately, 1 ug of DIF2 protein was loaded into each lane. Lanes 1 is a western blot with DIF2 antibodies at a 1/50 dilution; Lane 2 is a western blot at 1/100 dilution; Lane 3 is a western blot at a 1/200 dilution.
39 Mitochondria freshly Isolated from Bovine liver (1 g) Treated w/ digitonin (.8 mg dig/ 10 mg mit protein)shaken 15â€™, 4 C, centrifuged 17,400 x g 10 minSupernatantPellet Centrifuge at 120K xg 30 minSupernatantâ€œIntermembrane Space + Matrixâ€Pelletsolubilize in 1.6% Triton X-100centrifuged 40K xg 20 min Supernatantâ€œOuter Membraneâ€Junk Pellet Resuspended in mitofrac buffersonicated 18--10s burstscentrifuged 500 x g 45 minSupernatantJunk pellet Supernatantâ€œMatrixâ€Pelletsolubilize in 1.6% Triton X-100centrifuged 40K xg 20 min Supernatantâ€œInner membraneâ€Junk Pellet Centrifuge at 120K x g 30 minFraction 2Fraction 1Fraction 4Fraction 3 Figure 15. A schematic diagram of the mitochondrial sub-fractionation experiment. See text for details.
40 OM IMS+Ma IM Ma-COX4-HSP60-DIF1-DIF2-Cyt. C45 15757525 MW(kDa)Fractions: 1 2 3 4 Figure 16. A series of western blots of each of the mitochondrial subfractions. cytochrome C (Cyt. C) is present in fraction 2, cytochrome C oxidase IV (COX IV) is present in fraction 3, and Heat Shock Protein 60 (HSP60) is in fractions 2 and 4. These blots suggest that fraction 2 is intermembrane space and matrix proteins, fraction 3 is inner-membrane proteins, and fraction 4 is matrix proteins. Both DIF1 and DIF2 are found in fractions 2 and 4, co-localizing with HSP60, suggesting the matrix as their sub-cellular location.
CHAPTER 4 DISCUSSION AND CONCLUSION Death associated protein-3 (DAP3) was independently identified as both a small-subunit mitochondrial ribosome protein and as a positive mediator of programmed cell death (Matthews el al., 1982 and Kissil et al., 1995). DAP3 has also been shown to have a high affinity GTP/GDP binding site of unknown function (Denslow et al., 1991). However, the mutation of this site has been reported to render its proapoptotic ability inactive (Kissil et al., 1999). Whether DAP3 is truly a bifunctional protein, or if its function in the mitochondrial ribosome and in the apoptotic cascade overlaps, is still debatable. The screening of DAP3 against a cDNA library using the yeast-two-hybrid system identified two new proteins with which DAP3 interacts (Song, 2000). These two new proteins have now been renamed DAP3 interacting factors-1 and 2 (DIF1 and DIF2). The purpose of this present research investigation sought to further characterize these two proteins. Since DAP3 is a mitochondrial protein and contains a traditional mitochondrial targeting import sequence, the determination of the subcellular location of DIF1 and DIF2 was of the utmost importance. For a true, meaningful interaction to exist between DAP3 and the interacting factors, these proteins would most likely be localized to the mitochondria. More specifically, these proteins should be located either in the matrix or on the matrix-side of the inner-membrane, as this is where DAP3 can be found. In addition, since DAP3 is on the mitochondrial ribosome and interacts with one or more 41
42 proteins from the ribosome, the interactions with DIF1 or DIF2 may not occur at the same time due to steric hindrance. Also, the interactions of DIF1 or DIF2 with DAP3 may be transient and required only for ribosome and inner-membrane assembly. Neither DIF1 nor DIF2 have a mitochondrial targeting sequence; therefore, to determine if they were mitochondrial proteins, antibodies had to be made against them. To develop DIF1 and DIF2 as antigens for the production of antibodies, the proteins were expressed in E.coli using the pET vector system. Next, the proteins were purified by virtue of their hexahistidine tags on nickel-coated resin. Once ample quantities of the proteins were made, chickens were immunized, and antibodies were extracted from the chickensâ€™ eggs. The antibodies were then affinity purified over antigen-conjugated resin and characterized by use of western blotting. Mitochondria were next isolated from bovine liver and fractionated by use of digitonin, sonication, or the detergent Triton X-100, and centrifugation steps. Each fraction containing outer-membrane, intermembrane space and matrix, inner-membrane, or matrix was then analyzed by western blots with antibodies against specific compartmental marker proteins. The antibodies used for these blots were cytochrome C for the intermembrane space compartment, cytochrome C oxidase IV (COX4) for the inner-membrane compartment, and Heat Shock Protein-60 (HSP60) for the matrix compartment. Once the validity of each compartment was established, each compartmental fraction was passed over DIF1 or DIF2 antibody conjugated resin, washed, and eluted. The eluates were concentrated and then used for western blots with the DIF1 or DIF2 antibodies. Both DIF1 and DIF2 were shown to co-purify with HSP60 suggesting matrix localization, as was hypothesized. Although the discovery that DIF1 and DIF2 being mitochondrial matrix proteins was not a surprise, it does provide evidence for their interactions with DAP3 to be
43 physiologically relevant. Many new and interesting questions may now be raised regarding the function of these new interacting proteins; such as, are they a part of the mitochondrial translation system, the apoptotic cascade, or like DAP3, both? The mechanism of the mitochondrial ribosome docking to the inner-membrane, to initiate translation, has remained elusive. The possibility of these proteins being involved in such a mechanism is speculative, but worth investigating. DIF2 may be a two-domain protein, having the C-terminal portion binding to DAP3, while the N-terminal half, resembling a potassium channel tetramerization domain, may be another protein-protein interaction domain. This second potential protein-protein interaction domain may be involved in anchoring or â€œhanding-offâ€ to other protein complexes on the inner-membrane. In addition, since DAP3 is present in most cell types, and DIF1 and DIF2 also appear to be present in diverse tissue types, as seen from the digital northern blot analysis (Appendix D), they may function in concert with DAP3 in its proapoptotic role. DAP3, being a positive mediator of apoptosis through the TNF-alpha induced pathway, and DIF2, having some similarity to a TNF-alpha induced protein, may not merely be a coincidence. Also, mitochondrial membrane depolarization is known to be a key step in apoptosis. While, the mitochondrial ribosomeâ€”already known as having a protein, DAP3, that mediates apoptosisâ€”is bound to the inner-membrane during translation. These phenomena may then suggest the involvement of the DAP3 interacting proteins in the apoptotic cascade. There are many future experiments that may be done to further characterize the DIF1 and DIF2 proteins. A northern blot containing multiple tissue samples would help to validate the proteinsâ€™ expression profiles obtained in silico. Also, DIF1 and especially
44 DIF2 are strongly conserved between mammalian species. Thus, these proteins may play an important role in mitochondrial functions. The generation of DIF1 and DIF2 knock-outs in cell culture and mice may be a valuable tool in determining the function of these proteins. Finally, over-expression of the DAP3 interacting proteins in cell-culture may help to determine if these proteins do indeed play a role in programmed cell death. In conclusion, the identification of DIF1 and DIF2 as mitochondrial matrix proteins has generated important implications for mitochondrial translation and mitochondrial-mediated apoptosis. This discovery has led to many new and exciting hypotheses, which will undoubtedly open many future areas of research investigation.
APPENDIX A DIF1 AND DIF2 PRIMER SEQUENCES DIF1 Forward Primer 5--CCTCgtcgacGTGGATCTGTCTCGGGGA DIF1 Reverse Primer 5--TCACgcggccgcGAGCACCGGGGCCGAGCT DIF2 Forward Primer 5--GGAGgtcgacGCTATATGATGACTGACC DIF2 Reverse Primer 5--CTTCgcggccgcCCTACAGAAAGTCCAGTT The restriction endonuclease sites (Sal I for the forward primer and Not I for the reverse primers) are in lower-case. The uppercase letters allow for binding to the template DNA, purchased from ATCC. Extra bases 5 to the restriction sites allow for direct digestion after the PCR reaction. 45
APPENDIX B DNA AND PROTEIN SEQUENCES DIF1 DNA Sequence >Accession BCOO8883 ATGCAGCTGT TTGAGCAGCC CTGTCCTGGG GAGGACCCCC GGCCAGGAGG CCAGATCGGT GAGGTGGAGC TGTCCTCCTA CACGCCCCCA GCCGGGGTCC CAGGAAAGCC TGCAGCCCCC CACTTCCTTC CAGTGCTGTG CTCTGTGTCA CCATCAGGCT CCAGGGTCCC GCACGACCTC CTCGGGGGCT CCGGGGGCTT CACGCTGGAG GACGCCCTCT TCGGGCTCCT CTTTGGAGCT GATGCCACCC TCCTGCAGTC ACCTGTGGTC CTCTGTGGTC TCCCTGATGG CCAGCTCTGC TGTGTGATCC TGAAGGCCCT GGTCACCTCC AGGTCAGCCC CTGGTGACCC AAATGCCCTT GTCAAGATCC TCCATCACCT GGAGGAGCCC GTCATCTTCA TAGGGGCCTT GAAGACAGAG CCACAGGCTG CAGAAGCTGC AGAGAATTTT CTGCCTGACG AGGATGTGCA CTGTGACTGC CTGGTGGCCT TTGGTCACCA CGGCCGGATG CTGGCCATCA AGGCCAGCTG GGATGAGTCC GGGAAGCTGG TGCCCGAGCT GCGGGAGTAC TGCCTCCCAG GCCCTGTGCT CTGCGCTGCC TGTGGCGGGG GTGGCCGCGT GTACCACAGC ACCCCTTCTG ACCTCTGTGT GGTGGATCTG TCTCGGGGAA GCACCCCGCT GGGCCCTGAG CAGCCCGAAG AAGGCCCGGG AGGCCTGCCC CCCATGCTGT GCCCAGCCAG CCTGAACATC TGCAGTGTCG TCTCGCTGTC CGCGTCTCCC AGGACGCATG AAGGTGGCAC CAAGCTCCTG GCCCTGTCCG CCAAAGGCCG CCTGATGACC TGCAGCCTGG ACCTGGACTC TGAGATGCCT GGCCCAGCCA GGATGACCAC AGAGAGTGCA GGTCAGAAAA TAAAGGAGCT GCTGTCTGGA ATTGGCAACA TCTCTGAGAG AGTGTCTTTT CTAAAGAAGG CGGTTGACCA GCGGAACAAG GCACTGACAA GCCTCAACGA GGCCATGAAC GTGAGCTGTG CACTGCTGTC AAGCGGCACG GGCCCCAGAC CCATCTCCTG CACCACCAGC ACCACCTGGA GCCGCCTGCA GACACAGGAT GTGCTCATGG CCACCTGCGT GCTAGAGAAC AGCAGCAGCT TCAGCCTGGA CCAGGGGTGG ACCCTGTGCA TCCAGGTGCT CACCAGCTCC TGTGCTCTCG ACCTGGACTC GGCCTGCTCC GCCATCACCT ACACCATCCC CGTGGACCAG CTCGGCCCCG GTGCTCGGCG GGAGGTGACG CTACCCCTGG GCCCTGGTGA GAACGGCGGG CTCGACCTGC CCGTGACCGT GTCCTGCACG CTGTTCTACA GTCTCAGGGA GGTGGTGGGC GGGGCCCTTG CCCCCTCAGA CTCTGAGGAC CCCTTTCTGG ATGAGTGCCC CTCCGACGTC CTGCCCGAGC AAGAGGGTGT TTGCCTGCCC CTGAGCAGGC ACACAGTGGA CATGCTGCAG TGTCTGTGCT TCCCTGGCCT GGCCCCGCCA CACACACGGG CCCCCTCCCC ACTCGGCCCC ACCCGAGACC CTGTGGCCAC TTTTCTGGAA ACTTGTCGGG AGCCTGGCAG CCAGCCAGCA GGACCCGCCT CCCTGCGGGC CGAGTACCTG CCCCCATCTG TGGCTTCCAT CAAGGTGTCG GCGGAGCTGC TCAGAGCTGC CTTGAAGGAC GGCCACTCAG GCGTGCCCCT GTGCTGTGCC ACCCTGCAGT GGCTCCTTGC TGAGAATGCT GCTGTGGACG TCGTGAGGGC CCGAGCACTA TCTTCCATCC AGGGAGTGGC CCCTGATGGC GCCAACGTTC ACCTCATCGT CCGAGAGGTG GCCATGACCG ACCTGTGCCC AGCAGGGCCC ATCCAGGCCG TGGAGATTCA AGTGGAAAGC TCCTCTCTGG CCGACATTTG CAGGGCGCAC CATGCCGTTG TCGGGCGCAT GCAGACGATG GTGACAGAGC AGGCCGCCCA GGGCTCCAGC GCTCCTGATC TCCGTGTGCA GTACCTCCGC CAGATCCACG CCAACCACGA GACACTGCTG CGGGAGGTGC AGACCCTGCG CGACCGGCTC TGCACGGAGG ATGAGGCCAG CTCCTGTGCC ACCGCCCAGA GGCTGCTACA GGTGTACCGG CAGCTGCGCC ACCCCAGCCT CATCCTGCTG TGA 46
47 DIF1 Protein Sequence >Hypothetical protein FLJ22175 (DIF1) 76,970 Da, pI 5.07. Bold sequence represents putative DAP3 interaction domain MQLFEQPCPG EDPRPGGQIG EVELSSYTPP AGVPGKPAAP HFLPVLCSVS PSGSRVPHDL LGGSGGFTLE DALFGLLFGA DATLLQSPVV LCGLPDGQLC CVILKALVTS RSAPGDPNAL VKILHHLEEP VIFIGALKTE PQAAEAAENF LPDEDVHCDC LVAFGHHGRM LAIKASWDES GKLVPELREY CLPGPVLCAA CGGGGRVYHS TPSDLCVVDL SRGSTPLGPE QPEEGPGGLP PMLCPASLNI CSVVSLSASP RTHEGGTKLL ALSAKGRLMT CSLDLDSEMP GPARMTTESA GQKIKELLSG IGNISERVSF LKKAVDQRNK ALTSLNEAMN VSCALLSSGT GPRPISCTTS TTWSRLQTQD VLMATCVLEN SSSFSLDQGW TLCIQVLTSS CALDLDSACS AITYTIPVDQ LGPGARREVT LPLGPGENGG LDLPVTVSCT LFYSLREVVG GALAP SDSED PFLDECPSDV LPEQEGVCLP LSRHTVDMLQ CLCFPGLAPP HTRAPSPLGP TRDPVATFLE TCREPGSQPA GPASLRAEYL PPSVASIKVS AELLRAALKD GHSGVPLCCA TLQWLLAENA AVDVVRARAL SSIQGVAPDG ANVHLIVREV AMTDLCPAGP IQAVEIQVES SSLADICRAH HAVVGRMQTM VTEQAAQGSS APDLRVQYLR QIHANHETLL REVQTLRDRL CTEDEASSCA TAQRLLQVYR QLRHPSLILL
48 DIF2 DNA Sequence >Accession NM_153331 ATGGATAATG GAGACTGGGG CTATATGATG ACTGACCCAG TCACATTAAA TGTAGGTGGA CACTTGTATA CAACGTCTCT CACCACATTG ACGCGTTACC CGGATTCCAT GCTTGGAGCT ATGTTTGGGG GGGACTTCCC CACAGCTCGA GACCCTCAAG GCAATTACTT TATTGATCGA GATGGACCTC TTTTCCGATA TGTCCTCAAC TTCTTAAGAA CTTCAGAATT GACCTTACCG TTGGATTTTA AGGAATTTGA TCTGCTTCGG AAAGAAGCAG ATTTTTACCA GATTGAGCCC TTGATTCAGT GTCTCAATGA TCCTAAGCCT TTGTATCCCA TGGATACTTT TGAAGAAGTT GTGGAGCTGT CTAGTACTCG GAAGCTTTCT AAGTACTCCA ACCCAGTGGC TGTCATCATA ACGCAACTAA CCATCACCAC TAAGGTCCAT TCCTTACTAG AAGGCATCTC AAATTATTTT ACCAAGTGGA ATAAGCACAT GATGGACACC AGAGACTGCC AGGTTTCCTT TACTTTTGGA CCCTGTGATT ATCACCAGGA AGTTTCTCTT AGGGTCCACC TGATGGAATA CATTACAAAA CAAGGTTTCA CGATCCGCAA CACCCGGGTG CATCACATGA GTGAGCGGGC CAATGAAAAC ACAGTGGAGC ACAACTGGAC TTTCTGTAGG CTAGCCCGGA AGACAGACGA CTGA DIF2 Protein Sequence >DIF2; 27,611 Da, pI 4.23. Bold sequence represents putative DAP3 interaction domain, underlined sequence represents portion similar to Kv4.2 tetramerization domain MDNGDWGYMM TDPVTLNVG G HLYTTSLTTL TRYPDSMLGA MFGGDFPTA R DPQGNYFIDR DGPLFRYVLN FLRTSELTL P LDFKEFDLLR KEADFYQIEP LIQCLNDPKP LYPMDTFEEV VELSSTRKLS KYSNPVAVII TQLTITTKVH SLLEGISNYF TKWNKHMMDT RDCQVSFTFG PCDYHQEVSL RVHLMEYITK QGFTIRNTRV HHMSERANEN TVEHNWTFCR LARKTDD
APPENDIX C SECONDARY STRUCTURE ANALYSIS Secondary Structure of DIF1 from Network Protein Sequence Analysis Website 49
51 Sequence length : 730 DPM : Alpha helix (Hh) : 222 is 30.41% 3 helix (Gg) : 0 is 0.00% 10 Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 212 is 29.04% Beta turn (Tt) : 73 is 10.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 223 is 30.55% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% DSC : Alpha helix (Hh) : 100 is 13.70% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 203 is 27.81% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 427 is 58.49% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00%
52 GOR4 : Alpha helix (Hh) : 195 is 26.71% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 147 is 20.14% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 388 is 53.15% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% HNNC : Alpha helix (Hh) : 240 is 32.88% 3 helix (Gg) : 0 is 0.00% 10 Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 107 is 14.66% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 383 is 52.47% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% PHD : Alpha helix (Hh) : 227 is 31.10% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 161 is 22.05% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 342 is 46.85% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% Predator : Alpha helix (Hh) : 220 is 30.14% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 105 is 14.38% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 405 is 55.48% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% SIMPA96 : Alpha helix (Hh) : 219 is 30.00% 3 helix (Gg) : 0 is 0.00% 10 Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 129 is 17.67% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 382 is 52.33% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% SOPM : Alpha helix (Hh) : 250 is 34.25%
53 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 145 is 19.86% Beta turn (Tt) : 57 is 7.81% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 278 is 38.08% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% Sec.Cons. : Alpha helix (Hh) : 199 is 27.26% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 127 is 17.40% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 369 is 50.55% Ambigous states (?) : 35 is 4.79% Other states : 0 is 0.00%
54 Secondary Structure of DIF2 from Network Protein Sequence Analysis Website Sequence length : 237 DPM : Alpha helix (Hh) : 77 is 32.49% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 66 is 27.85% Beta turn (Tt) : 17 is 7.17% Bend region (Ss) : 0 is 0.00%
55 Random coil (Cc) : 77 is 32.49% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% DSC : Alpha helix (Hh) : 16 is 6.75% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 71 is 29.96% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 150 is 63.29% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% GOR4 : Alpha helix (Hh) : 55 is 23.21% 3 helix (Gg) : 0 is 0.00% 10 Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 68 is 28.69% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 114 is 48.10% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% HNNC : Alpha helix (Hh) : 84 is 35.44% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 34 is 14.35% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 119 is 50.21% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% PHD : Alpha helix (Hh) : 71 is 29.96% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 58 is 24.47% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 108 is 45.57% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% Predator : Alpha helix (Hh) : 44 is 18.57% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 19 is 8.02% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 174 is 73.42% Ambigous states (?) : 0 is 0.00%
56 Other states : 0 is 0.00% SIMPA96 : Alpha helix (Hh) : 85 is 35.86% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 31 is 13.08% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 121 is 51.05% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% SOPM : Alpha helix (Hh) : 96 is 40.51% 3 10 helix (Gg) : 0 is 0.00% Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 39 is 16.46% Beta turn (Tt) : 22 is 9.28% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 80 is 33.76% Ambigous states (?) : 0 is 0.00% Other states : 0 is 0.00% Sec.Cons. : Alpha helix (Hh) : 71 is 29.96% 3 helix (Gg) : 0 is 0.00% 10 Pi helix (Ii) : 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% Extended strand (Ee) : 36 is 15.19% Beta turn (Tt) : 0 is 0.00% Bend region (Ss) : 0 is 0.00% Random coil (Cc) : 115 is 48.52% Ambigous states (?) : 15 is 6.33% Other states : 0 is 0.00%
APPENDIX D EXPRESSION PROFILES OF DIF1 AND DIF2 Ludwig Transcript (LT) Viewer Results For DIF1 Tag Tag Sequence Tag Position BP Number Tag Freq CAGGGAGCTC 1 3477 69 AAAGTGCTTG 2 3046 4 GGTTAGAGGG 3 2966 1 CAGACGATGG 4 2676 2 Digital Northern Results for DIF1 Search query: CAGGGAGCTC Color Code Tags per 200,000 <2 <4 <8 <16 <32 <64 <128 <256 <512 >512 Library Total Tags in Library Tags per200,000 ColorCode SAGE_Brain_glioblastoma_B_pooled 56428 14 SAGE_Ovary_cystadenoma_CL_ML10-10 55144 14 SAGE_Colon_adenocarcinoma_CL_SW837 61148 13 SAGE_Breast_carcinoma_epithelium_AP_DCIS7 89184 11 SAGE_Pancreas_adenocarcinoma_B_91-16113 33582 11 SAGE_Brain_astrocytoma_gradeI_B_H1043 75922 10 SAGE_Brain_glioblastoma_CL_H54+EGFRvIII 56982 10 SAGE_Breast_carcinoma_CL_ZR75_1_tamoxifen 40052 9 SAGE_Brain_normal_thalamus_B_1 24015 8 SAGE_Colon_normal_B_NC2 48479 8 SAGE_Kidney_embryonic_CL_293+beta-catenin 23519 8 SAGE_Prostate_adenocarcinoma_CL_LNCaP 22344 8 SAGE_Colon_adenocarcinoma_B_Tu102 55700 7 SAGE_Ovary_adenocarcinoma_B_OVT-7 53514 7 57
58 SAGE_Brain_oligodendroglioma_B_1001 32442 6 SAGE_Breast_carcinoma_B_95-348 60343 6 SAGE_Breast_carcinoma_B_IDC-5 60451 6 SAGE_Ovary_carcinoma_CL_ES2-1 31159 6 SAGE_Pancreas_adenocarcinoma_B_96-6252 33213 6 SAGE_Pancreas_carcinoma_CL_ASPC 31224 6 SAGE_Pancreas_normal_CS_H126 32223 6 SAGE_Peritoneum_mesothelioma_B_12 32529 6 SAGE_Prostate_carcinoma_CL_A+ 30298 6 SAGE_Prostate_carcinoma_CL_LNCaP_no-DHT 62160 6 SAGE_Prostate_normal_B_2 64058 6 SAGE_White_Blood_Cells_Breast_carcinoma_AP_DCIS6 66176 6 SAGE_Brain_glioblastoma_CL_H54+LacZ 66908 5 SAGE_Brain_medulloblastoma_B_1273 38614 5 SAGE_Brain_ependymoma_B_R1023 122690 4 SAGE_Brain_medulloblastoma_B_98-05-P608 48451 4 SAGE_Brain_medulloblastoma_CL_mhh-1 47858 4 SAGE_Breast_carcinoma_B_DCIS-5 43098 4 SAGE_Breast_carcinoma_metastasis_B_2 49794 4 SAGE_Breast_carcinoma_myoepithelium_AP_DCIS6 81452 4 SAGE_Breast_normal_epithelium_AP_1 48729 4 SAGE_Colon_adenocarcinoma_B_Tu98 41371 4 SAGE_Kidney_embryonic_CL_293-control 41955 4 SAGE_Kidney_normal_B_1 40993 4 SAGE_Prostate_carcinoma_CL_PC3_AS2 40768 4 SAGE_Brain_astrocyte_normal_CL_NHA5 51481 3 SAGE_Brain_astrocytoma_gradeIII_B_H1020 51573 3 SAGE_Brain_ependymoma_B_1150 62373 3 SAGE_Brain_glioblastoma_control_CL_H247 60428 3 SAGE_Brain_normal_cerebellum_B_1 50385 3 SAGE_Breast_carcinoma_B_DCIS-4 60605 3 SAGE_Breast_normal_B_hyperplasia1 61704 3
59 SAGE_Colon_adenocarcinoma_CL_Caco2 60682 3 SAGE_Colon_adenocarcinoma_CL_HCT116 55641 3 SAGE_Muscle_normal_B_young 53947 3 SAGE_Prostate_normal_MD_PR317 59277 3 SAGE_Stomach_adenocarcinoma_B_G234 64925 3 65314 3 SAGE_Vascular_normal_CS_VEGF+ 57316 3 SAGE_Vascular_normal_CS_control 51642 3 SAGE_Brain_astrocytoma_grade_II_B_H563 88568 2 SAGE_Brain_glioblastoma_B_H1110 68986 2 SAGE_Brain_normal_cortex_B_BB542 94233 2 SAGE_Breast_carcinoma_B_95-347 67070 2 SAGE_Breast_carcinoma_B_IDC-3 68891 2 SAGE_Breast_carcinoma_epithelium_AP_DCIS6 72857 2 SAGE_Breast_normal_stroma_AP_1 79152 2 SAGE_Muscle_normal_B_old 53889 0 Brain Retina No Data Lung No Data Heart No Data Breast Stomach SAGE_Vascular_Endothelium_Breast_carcinoma_AP_DCIS6 SAGE Anatomical Viewer Results for DIF1
60 Pancreas Liver No Data Kidney No Data Colon Peritoneum Spinal Cord No Data Ovary Placenta No Data Prostate Muscle No Data No Data Skin White Blood Cells
61 Ludwig Transcript (LT) Viewer Results For DIF2 Tag Tag Sequence Tag Position BP Number Tag Freq GAAATGTCTG 1 2269 49 TAGGGTGACC 2 2107 7 ATGCCTTTGA 3 2078 50 TGCCTGTTCA 4 1895 2 Digital Northern Results for DIF2 Search query: GAAATGTCTG Color Code Tags per 200,000 <2 <4 <8 <16 <32 <64 <128 <256 <512 >512 Library Total Tags in Library Tags per 200,000 ColorCode SAGE_Brain_medulloblastoma_B_97-05-P015 69971 17 SAGE_Brain_glioblastoma_B_GBM1062 59762 13 SAGE_Retina_Peripheral_normal_B_1 59661 13 SAGE_Pancreas_carcinoma_CL_ASPC 31224 12 SAGE_Brain_ependymoma_B_R1023 122690 11 SAGE_Brain_astrocytoma_grade_II_B_H388 106285 9 SAGE_Brain_ependymoma_B_R628 120431 8 SAGE_Brain_astrocyte_normal_CL_NHA5 51481 7 SAGE_Brain_astrocytoma_gradeIII_B_H1020 51573 7 SAGE_Brain_medulloblastoma_B_96-04-P019 52645 7 SAGE_Brain_oligodendroglioma_B_H988 27864 7 SAGE_Spinal_cord_normal_B_1 54785 7 SAGE_Brain_ependymoma_B_1150 62373 6 SAGE_Brain_glioblastoma_control_CL_H247 60428 6 SAGE_Brain_normal_cortex_B_pool6 62451 6 SAGE_Brain_oligodendroglioma_B_1001 32442 6 SAGE_Breast_normal_AP_myoepithelial1 57222 6
62 SAGE_Breast_normal_endothelium_AP_1 33115 6 SAGE_Ovary_carcinoma_CL_ES2-1 31159 6 SAGE_Pancreas_normal_CS_H126 32223 6 SAGE_Brain_astrocytoma_grade_II_B_H359 105764 5 SAGE_Brain_ependymoma_B_R353 73822 5 SAGE_Breast_normal_AP_Br_N 37121 5 SAGE_Retina_Peripheral_normal_B_2 105312 5 SAGE_Brain_medulloblastoma_B_98-05-P608 48451 4 SAGE_Brain_normal_cerebellum_B_BB542 40500 4 SAGE_Breast_carcinoma_B_DCIS-5 43098 4 SAGE_Kidney_embryonic_CL_293-control 41955 4 SAGE_Kidney_normal_B_1 40993 4 SAGE_Ovary_adenocarcinoma_B_OVT-6 41443 4 SAGE_Ovary_normal_CL_IOSE29EC-11 47159 4 SAGE_Ovary_normal_CS_HOSE_4 47728 4 SAGE_Prostate_carcinoma_CL_PC3_AS2 40768 4 SAGE_Brain_glioblastoma_CL_H392 55990 3 SAGE_Brain_normal_cerebellum_B_1 50385 3 SAGE_Breast_carcinoma_B_IDC-4 64095 3 SAGE_Breast_carcinoma_B_IDC-5 60451 3 SAGE_Colon_adenocarcinoma_CL_Caco2 60682 3 SAGE_Colon_adenocarcinoma_CL_SW837 61148 3 SAGE_Gastric_cancer_B_G189 63075 3 SAGE_Ovary_adenocarcinoma_B_OVT-7 53514 3 SAGE_Ovary_cystadenoma_CL_ML10-10 55144 3 SAGE_Prostate_normal_MD_PR317 59277 3 SAGE_Universal_reference_human_RNA_CL 51729 3 SAGE_Vascular_normal_CS_VEGF+ 57316 3 SAGE_Brain_astrocytoma_grade_II_B_H563 88568 2 SAGE_Brain_glioblastoma_CL_H54+LacZ 66908 2 SAGE_Brain_normal_cortex_B_BB542 94233 2 81452 2 SAGE_Breast_carcinoma_myoepithelium_AP_DCIS6
63 SAGE_Breast_normal_stroma_AP_1 79152 2 SAGE_Endothelial_hemangioma_B_146 75680 2 SAGE_Gastric_carcinoma_B_xenograft_X101 69749 2 SAGE_Placenta_first_trimester_normal_B_1 89265 2 SAGE_Placenta_normal_B_1 118083 1 SAGE_Retina_normal_B_HMAC2 102417 1 SAGE Anatomical Viewer Results for DIF2 Brain Retina No Data Lung No Data Heart No Data Breast Stomach Pancreas Liver No Data Kidney No Data Colon Peritoneum
64 Spinal Cord No Data Ovary Placenta No Data Prostate Muscle No Data No Data Skin White Blood Cells
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BIOGRAPHICAL SKETCH Amar Singh was born in Leicester, England, where he lived for nine years. He and his family then moved to New Jersey. Five years later they relocated to Cape Coral, Florida, where his parents still reside. Amar received his BS in microbiology with a minor in business at the University of Florida in 2000. In 2002, he married his beloved girlfriend of five years, Maureen Hodges. After receiving his MS in medical sciences, he will be continuing his education in the Interdisciplinary Program at the University of Florida in molecular cell biology. 68