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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2010-05-31.

Permanent Link: http://ufdc.ufl.edu/UFE0021861/00001

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Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2010-05-31.
Physical Description: Book
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

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Subjects / Keywords: Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Electronic Thesis or Dissertation

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Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Lyons, Thomas J.
Electronic Access: INACCESSIBLE UNTIL 2010-05-31

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021861:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021861/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2010-05-31.
Physical Description: Book
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Lyons, Thomas J.
Electronic Access: INACCESSIBLE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021861:00001


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1 HUMAN PROGESTIN ADIPOQ RECEPTORS, IDENTIFICATION OF PAQR3 AS A NEW POSSIBLE ADIPONECTIN RECEPTOR By IBON GARITAONANDIA 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

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2 2008 Ibon Garitaonandia

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3 To my loving family

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4 ACKNOWLEDGMENTS This project was funded by the University of Florida and the NIH Grant R21 DK07481201. I would also like to thank the Departm e nt of Chemistry for the financial support. I am grateful to my research advisor Dr. Tom Lyons and my doctoral dissertation committee, Dr. Gail Fanucci, Dr. Nicole Hore nstein, Dr. William Dolbier and Dr. David Silverman, for their continuous support a nd guidance throughout all these years. I thank former and current members of the Lyons group, Jessica, Brian, Lisa, Nancy, Lidia, for their friendship and guidance throughout these years. Special thanks go to Jessica, whose unconditional friendship, help and useful discussions over the years made the laboratory such a positive place for work. Finally I thank my parents, Pedro and Mari a, my sister Alaitz and my grandmother Herminia for being my number one support system.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT...................................................................................................................................12 CHAP TER 1 INTRODUCTION..................................................................................................................14 Type 2 Diabetes................................................................................................................ ......14 Adiponectin.................................................................................................................... .........14 Adiponectin Receptors.......................................................................................................... ..19 PAQR Receptors................................................................................................................. ....23 Human Class I PAQRs....................................................................................................23 Human Class II PAQRs...................................................................................................24 Human Class III PAQRs................................................................................................. 25 Ceramidase Activity........................................................................................................ 25 2 EXPRESSION AND CHARACTERIZATION OF THE HUMAN CL ASS I PAQR PROTEINS IN ESCHERICHIA COLI ...................................................................................38 Introduction................................................................................................................... ..........38 Materials and Methods...........................................................................................................38 Cloning of Human PAQRs..............................................................................................38 Growth Conditions..........................................................................................................39 Detection of Expression.................................................................................................. 40 Coomassie staining................................................................................................... 40 His-tag in-gel staining.............................................................................................. 40 Western blot.............................................................................................................40 Purification......................................................................................................................41 Ceramidase Assay........................................................................................................... 42 Results and Discussion......................................................................................................... ..43 Expression of PAQRs in Escherich ia coli .......................................................................43 Ceramidase Assay........................................................................................................... 44 3 EXPRESSION AND CHARACTERIZATION OF THE HUMAN CL ASS I PAQR PROTEINS IN SACCHAROMYCES CEREVISAE ................................................................51 Introduction................................................................................................................... ..........51 Materials and Methods...........................................................................................................52

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6 Yeast Strains and Media.................................................................................................. 52 Cloning of Human PAQR...............................................................................................52 Growth Conditions..........................................................................................................52 Membrane Protein Isolation............................................................................................53 Western Blot....................................................................................................................53 -galactosidase Reporter Assays .....................................................................................54 GFP Fluorescence............................................................................................................55 Adiponectin Binding.......................................................................................................55 Results and Discussion......................................................................................................... ..56 Repression of FET3 by PAQRs ....................................................................................... 56 Thaumatin as a Ligand for Izh2p..................................................................................... 57 Human Adiponectin Receptors........................................................................................ 58 Inhibitors and Activators of Human Adiponectin Receptors.......................................... 59 PAQR3 and PAQR4........................................................................................................ 60 Involvement of PKA and AMPK....................................................................................62 Ceramidase Activity........................................................................................................ 63 Fluorescence Studies.......................................................................................................63 Adiponectin Binding.......................................................................................................66 4 EXPRESSION AND CHARACTERIZATION OF THE HUMAN CL ASS I PAQR PROTEINS IN SF9 INSECT CELLS....................................................................................94 Introduction................................................................................................................... ..........94 Materials and Methods...........................................................................................................95 Cloning of Human Class I PAQR Proteins..................................................................... 95 Growth of Sf9 Insect Cells ...............................................................................................95 Transfection of Sf9 Insect Cells ....................................................................................... 96 Cell Extract Preparati on and Western Blot..................................................................... 96 Adiponectin Binding.......................................................................................................96 Results and Discussion......................................................................................................... ..97 Expression of PAQRs in Sf9 cells ...................................................................................97 Adiponectin binding........................................................................................................ 97 5 CONCLUSIONS.................................................................................................................. 100 APPENDIX A YEAST STRAINS................................................................................................................ 103 B MEDIA COMPOSITION..................................................................................................... 104 C PRIMERS USED FOR CLONING......................................................................................105 LIST OF REFERENCES.............................................................................................................109 BIOGRAPHICAL SKETCH.......................................................................................................118

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7 LIST OF TABLES Table page 1-1 Human PAQR family.........................................................................................................27 2-1 Accession numbers for the cDNA of the human PAQR genes from Open Biosystem s.........................................................................................................................47 2-2 Number of rare codon of human PAQR genes in Escherich ia coli ...................................50 3-1 Number of rare codons of human PAQR genes in Saccharomyces cerevisiae ................67 3-2 EC50 values for PAQR1, PAQR2 and PAQR3 with adiponectin in Saccharomyces cerevisiae...........................................................................................................................80 4-1 Number of rare codons of the human PAQR genes in Sf9 ...............................................98 6-1 Strains used in this study................................................................................................. .103

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8 LIST OF FIGURES Figure page 1-1 Phylogenetic tree of human class I PAQR proteins. .......................................................... 28 1-2 Hydropathy plots fo r the class I PAQRs ............................................................................ 29 1-3 Multiple sequence alignment of human class I PAQRs, Izh1p and Izh3p......................... 30 1-4 Third conserved motif of class I PAQR proteins ............................................................... 31 1-5 Hydropathy plots fo r the class II PAQRs .......................................................................... 32 1-6 Hydropathy plot fo r the class III PA QRs........................................................................... 33 1-7 Reaction catalyze by alkaline ceramidases........................................................................ 33 1-8 Multiple sequence alignment of PAQR and alkaline ceramidase sequences from different organism s............................................................................................................34 1-9 The three highly conserved motifs f ound in PAQRs and alkaline ceram idases................ 35 1-10 Third conserved motif found in PAQRs and alkaline ceram idases................................... 36 1-11 Hypothetical mechanism of action of PAQRs...................................................................37 2-1 Structure of C6 NBD ceramide.......................................................................................... 47 2-2 Structure of NBD hexanoic acid........................................................................................ 48 2-3 Western blot of the purificati on of PAQR3 using His Bind Colum n................................ 48 2-4 TLC analysis of reaction of C6 NB D ceram ide with cells expressing PAQR1.................49 2-5 TLC analysis of reaction of SCDase with C6 NBD ceramide........................................... 49 3-1 Repression of FET3 by overexpression of Izh1-4p in S. cerevisiae ..................................68 3-2 Loss of repression of FET3 by Izhp expression in 0.05% galactose ................................. 69 3-3 Repression of FET3 by Izh2p when 1 M thaumatin is added. ......................................... 69 3-4 Repression of FET3 in cells carrying vector control at 10 M thaum atin......................... 70 3-5 Repression of FET3 in cells lack ing all four IZH genes ( IZH1-4 ) at 100 M thaumatin...................................................................................................................... ......70 3-6 Repression of FET3 by overexpression of PAQR1 ........................................................... 71

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9 3-7 Repression of FET3 by PAQR2 upon additi on of adiponectin..........................................71 3-8 Dose response assay with adiponectin...............................................................................72 3-9 Expression of PAQR1 and PAQR2 in Saccharo myces cerevisiae ....................................72 3-10 Repression of FET3 by PAQR1 dependent on the concentration of galactose. ................ 73 3-11 Repression of FET3 by PAQR1 upon additi on of adiponectin..........................................73 3-12 Dose response assay of PAQR1 with adiponectin............................................................. 74 3-13 Activation of PAQR1 by C1q............................................................................................74 3-14 Dose response assay of PAQR1 to C1q............................................................................. 75 3-15 Inhibition of PAQR1 by TNF.........................................................................................75 3-16 Repression of FET3 by overexpression of PAQR3 and PAQR4. ......................................76 3-17 Expression of human class I PAQRs in Saccharo myces cerevisae ...................................76 3-18 Repression of FET3 by PAQR3 dependent on the concentration of galactose. ................ 77 3-19 Repression of FET3 by PAQR4 dependent on the concentration of galactose. ................ 77 3-20 Loss of repression of FET3 by PAQR3 and PAQR4 at 0.05% galactose..........................78 3-21 Activation of PAQR3 by adiponectin. PAQR4 di d not respond to adiponectin................ 78 3-22 Dose response assay of PAQR3 with adiponectin............................................................. 79 3-23 Dose response assay of PAQR4 with adiponectin............................................................. 79 3-24 Dose response with adiponectin of hum an class I PAQRs in S. cerevisae ........................80 3-25 Repression of FET3 by PAQR7 and PAQR2 by a dd ition of progesterone and adiponectin respectively..................................................................................................... 81 3-26 Repression of FET3 by PAQR5, PAQR8 and PAQR1 by addition of progesterone and adiponectin respectively .............................................................................................. 81 3-27 No repression of FET3 by PAQR3 and PAQR4 by addition of progesterone ...................82 3-28 Dose response of PAQR4 with progesterone.....................................................................82 3-29 Loss of repression of FET3 in tpk2 strain by overexpressi on of hum an class I PAQRs.......................................................................................................................... .....83

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10 3-30 Loss of repression of FET3 in tpk2 strain by activation with adiponectin of hum an class I PAQRs.................................................................................................................. ..83 3-31 Loss of repression of FET3 in snf4 strain by overexpressi on of hum an class I PAQRs.......................................................................................................................... .....84 3-32 Loss of repression of FET3 in snf4 strain by activation with adiponectin of hum an class I PAQRs.................................................................................................................. ..84 3-33 Loss of repression of FET3 by PAQR1, PAQR2 and Ypc1p by addition of Derythro -MAPP....................................................................................................................85 3-34 Dose response assay of PAQR1 with Derythro -MAPP ................................................... 86 3-35 Dose response assay of PAQR2 with Derythro -MAPP ................................................... 86 3-36 Repression of FET3 by N-term inal GFP tagged human class I PAQRs............................ 87 3-37 Repression of FET3 by activation w ith adiponectin of the N-terminal GFP tagged human class I PAQRs........................................................................................................87 3-38 Western blot of the expression of N-term inal GFP tagged human class I PAQRs in S. cerevisiae ...........................................................................................................................88 3-39 Western blot of the soluble fraction of N-term inal GFP tagged PAQR2 sample probed with anti-GFP antibodies....................................................................................... 88 3-40 GFP fluorescence of human class I PAQRs in S. cerevisiae .............................................89 3-41 GFP fluorescence of empty vector pGREG575 in S. cerevisiae .......................................89 3-42 Repression of FET3 by C-term inal GFP tagged human class I PAQRs............................ 90 3-43 Repression of FET3 by activation w ith adiponectin of the C-terminal GFP tagged human class I PAQRs........................................................................................................90 3-44 Western blot for the expression of C-term inal GFP tagged human class I PAQRs in S. cerevisiae .......................................................................................................................91 3-45 Western blot of the solu ble fraction of C-term inal G FP tagged human class I PAQRs.... 91 3-46 Fluorescence of C-terminal GFP tagged human class I PAQRs in S. cerevisiae ..............92 3-47 Binding of whole cells to FAM-labeled adiponectin ......................................................... 93 3-48 Effect of adiponectin on Izhps........................................................................................... 93 4-1 Western blot of expre ssion of Hum an Class I PAQRs in Sf9 insect cells.........................98

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11 4-2 Binding of PAQR1 to FAM-Adiponectin in Sf9 cells ....................................................... 99 4-3 Binding of PAQR2, PAQR3 and PAQR4 to FAM-Adiponectin in Sf9 cells.................... 99

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12 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 HUMAN PROGESTIN ADIPOQ RECEPTORS, IDENTIFICATION OF PAQR3 AS A NEW POSSIBLE ADIPONECTIN RECEPTOR By Ibon Garitaonandia May 2008 Chair: Thomas J. Lyons Major: Chemistry Type 2 diabetes is a health problem of wo rldwide proportions with increasing incidence every year. It is linked to obes ity and it is caused by the failure of the body to respond normally to insulin. A recently discovered antidiabetic agent is a hormone secreted by the adipocytes known as adiponectin. Adiponectin plays an import ant role in the etiology of obesity induced Type 2 diabetes through the re gulation of glucose uptake and fatty acid oxidation. The receptors for adiponectin are two integral membrane proteins known as PAQR1 and PAQR2. PAQR1 and PAQR2 bind to adiponectin, transmit its intracellular signal and belong to a larger family of proteins known as the human P rogestin A dipoQ R eceptor or human PAQR family. This family is distantly related to another family of proteins known as the alkali ne ceramidases. We believe that the mechanism by which the PAQRs transmit th eir signal is the hydrol ysis of ceramide. The original goal of this pr oject was to heterologously express the human PAQRs in Escherichia coli and test these proteins for ceramidas e activity. Unfortunately we could not detect expression of these protei ns and the ceramidase activity c ould not be properly tested. So the yeast Saccharomyces cerevisiae was used instead as expression system. In this organism, the expression levels were not very high, only detect ed by western blot, and instead of purification,

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13 in vivo studies were performed. Through these studies it was found that PAQR3, another member of the human PAQR family, responds to adiponectin in Saccharomyces cerevisiae and could be a new adiponectin receptor. To further corroborat e this finding, binding to fluorescently labeled adiponectin was studied. Inconclu sive results were obtained in S. cerevisiae so the Sf9 insect cells were used instead for studying binding to adiponectin. Unfortunately, only expression and binding was seen for PAQR1, and binding of PA QR3 to adiponectin could not be proven. Although further corroboration for PAQR3 is n eeded, this finding could have great future implications in the fight against Type 2 diabetes and cardiovascular diseases.

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14 CHAPTER 1 INTRODUCTION Type 2 Diabetes Type 2 diabetes is the m ost common metabolic disease in the world. The incidence of type 2 diabetes is rapidly rising, and there are already more than 171 million diabetic individuals. It is expected that by the year 2030, the numb er will increase to 366 million (Kahn et al. 2006). In the United States, it is the leadi ng cause of blindness, end-stage renal disease and nontraumatic loss of limb (Hogan et al. 2003). Type 2 diabetes is caused by the failure of the body to respond normally to the action of insulin. This results fr om impairment in both insulin sensitivity and insulin secretion (Elbers et al ., 2007). Type 2 diabetes is a disorder that depends on both genetic and non-genetic (environmental a nd lifestyle) factors (Ianucci et al ., 2007). Adiponectin A m ajor player in the development of Type 2 diabetes is adipose tissue, which is composed of many different type s of cells, with adipocytes being the most predominant ones. The adipocytes store lipids in the form of trigly recides and cholesterol es ters in lipid droplets. These droplets are specialized organelles that ac count for more than 95% of the mass of the adipocyte and changes in the amount of lipid stored can affect the size of the cell, ranging from 25 to 250 M (Desruisseaux et al ., 2007). The immense size of the liquid droplet within the adipocyte is the reason why these ce lls were originally viewed as lipid storage cells. But now it is well known that the adipose tissue ac ts as an endocrine organ that plays a central role in overall energy homeostasis. It does so by the production and release of hormonal factors known as adipokines. There are several of these adipokines including adiponectin (Scherer et al ., 1995), leptin (Zhang et al ., 1994), resistin (Steppan et al ., 2001), tumor necrosis factor alpha (TNF) (Hotamisligil, 1999), plasminogen activator inhibitor type-1 (PAI-1) (Shimomura et al ., 1996),

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15 adipsin (Cook et al ., 1987), serum amyloid A3 (Lin et al ., 2001), omentin (Yang et al ., 2006), visfatin (Fukuhara et al ., 2005) and RBP4 (Yang et al ., 2005), among others. They are involved in the regulation of energy homeostasis through effects on both central and peripheral tissues (Kim et al ., 2007). Out of all of the adipokines, adiponectin is the only one produced exclusively by the adipocyte (Desruisseaux et al ., 2007). All other known adipoki nes can be synthesized by other tissues in addition to the adipose tissue. Adiponectin, also known as ACRP30, apM1, adipoQ and GBP28 was independently discovered by four different groups The first ones to discover it were Scherer and colleagues in 1995 and they named it Adipocyte complement-re lated protein of 30 kDa (Acrp30) (Scherer et al ., 1995). They identified the cDNA of adiponectin through a screening syst em that would allow for the detection of genes up-regulated in adipocyte differentiation (Scherer et al ., 1995). Then in 1996, it was idependently identified by Spiegelman and colleagues and named AdipoQ (Hu et al ., 1996). Subsequently, it was identified by Ma tsuzawa and colleagues and named adipose most abundant gene transcript 1 (apM1) (Maeda et al ., 1996) and by Tomita and colleagues and named gelatin-binding protein of 28 kDa (GBP28) (Nakano et al ., 1996). Adiponectin is composed of four different dom ains: an amino-terminal signal sequence, a variable region, a collegenous domain, and a car boxy-terminal globular domain (Ekmekci and Ekmekci, 2006). In its most basic form, adipone ctin is a homotrimer of three 30 KDa subunits (Trujillo and Scherer, 2005). The trimers asso ciate through the formation of disulfide bonds within the collagenous domains of each monomer to form higher order structures. The higher order structures include low molecular weight hexamers of 180 KDa and high molecular weight (HMW) multimers of 16 to 18 mers of more than 400 kDa (Trujillo and Scherer, 2005). Adiponectin with all its di fferent complexes accounts for 0.01% of total serum protein,

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16 circulating at 3-30 g/mL (Arita et al ., 1999). Almost all adiponectin found in plasma exists as full length adiponectin in the form of low molecular weight trimer-dimer or HMW complexes, and only a small amount is found in the globular form (Pajvani et al ., 2003). Females have higher circulating levels in serum of the HMW complex than males (Waki et al ., 2003; Combs et al ., 2003). It has been reported that the levels of the HMW complex, but not the total amount of adiponectin, correlate with an improveme nt in insulin sensitivity (Pajvani et al ., 2004). Indeed, a good predictable marker for Type 2 diabetes and metabolic syndrome is m onitoring the levels of HMW adiponectin (Pajvani et al ., 2004). Studies have shown that bacterially pr oduced, full-length or globular domain of adiponectin administered to mice resulted in decr eased circulating levels of glucose, free fatty acids and triglycerides (Fruebis et al ., 2001; Yamauchi et al ., 2001). The increase in glucose uptake and lipid oxidation in muscle, have been attributed to the activation of AMPK in vitro (Fruebis et al ., 2001; Yamauchi et al ., 2002). This decrease in circul ating free fatty acids due to increased muscle lipid oxidation is thought to improve insulin signaling in muscle and whole body insulin sensitivity (Yamauchi et al ., 2001; Yamauchi et al ., 2002). The effects of bacterially produced adiponectin were only show n on muscle and had no effect on the liver, because the multimerization of ad iponectin into HMW multimers is essential for its activity on the liver. The bacterially produced adiponectin is secreted as lower ordered forms, because in this system there are no post-translational modi fications in the collagenous domain that would allow for the proper folding and orientation of the disulphide bonds within these domains (Shapiro and Scherer, 1998). On the other hand, adiponectin produced by mammalian cells form both low and high molecular weight multimers. Mice administered with this adiponectin showed a decrease in hepatic glucose production (Berg et al ., 2001).

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17 In skeletal muscle, adiponectin increases the expression of mol ecules involved in the transport of fatty acids, their combustion and in energy dissipation (Kadowaki and Yamauchi, 2005). All these changes lead to a decrease concentration of triglycerides in the skeletal muscle (Yamauchi et al ., 2001b). It has been reported that an in crease in the trig lyceride content can interfere with the activation of the insulin-stimulated phosphatid ylinositol (PI) 3kinase and the subsequent translocation of th e glucose transporter 4, thus leading to a decrease in glucose uptake and an increase in insulin resistance (Shulman et al ., 2000). So a decrease in the triglyceride content in skeletal muscle by adiponectin tr eatment contributes to an improved insulin signal transduction (Kadowaki and Yamauchi, 2005). Adiponectin also leads to the activation of the peroxisome proliferators-activated receptor alpha (PPAR ) (Yamauchi et al ., 2001a). PPAR is a ligand activated transcriptional factor receptor that is involved in the regulati on of lipid and glucose metabolism (Bordet et al ., 2006). The activation of PPAR by adiponectin increases fatty acid combustion and energy consumption, which leads to a decrease in the co ncentration of triglyce rides in the liver and skeletal muscle and consequently an in crease in insulin sensitivity (Yamauchi et al ., 2001). Activation of PPAR upon treatment with ad iponectin was observed in vivo (Yamauchi et al ., 2001) and in vitro in C2C12 myocytes (Yamauchi et al ., 2003). Adiponectin is also known to stimulate -oxidation and glucose uptake through the phosphorylation and activation of the AMP-activ ated protein kinase (AMPK) (Yamauchi et al ., 2002). The downstream effects of AMPK ar e phosphorylation and inhibition of acetyl coenzyme-A carboxylase (ACC), fatty-acid combus tion, glucose uptake, l actate production in myocytes, and a reduction in proteins involved in gluconeogenesis (Yamauchi et al ., 2002). The activation of AMPK by adiponectin is also seen w ith other adipokines, such as leptin (Minokoshi

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18 et al ., 2002). This activation might be the comm on mechanism by which all these insulin sensitizing hormones stimulate glucose uptak e and fatty acid combustion (Kadowaki and Yamauchi, 2005). Apart from its insulin sensitizing action, adipone ctin also has antiatherosclerotic actions. Adiponectin inhibits atherosc lerosis and plaque formati on by inhibiting expression of inflammatory cytokines and adhesion molecules, including intracellular adhesion molecule-1 (Matsuzawa et al ., 2004), vascular cellular a dhesion molecule-1 (Funahashi et al ., 1999) and Eselectin (Arita et al ., 2002). Adiponectin also inhibits the activation of nuclear factorB through the inhibition of I B phosphorylation (Ouchi et al ., 2000). The adhesion of monocytes to endothelial cells might be inhi bited by adiponectin through the s uppression of the nuclear factorB (Ouchi et al ., 1999). Adiponectin also inhibits athero sclerosis by suppression of cholesterol uptake by inhibiting the expression of scavenge r receptors. Scavenger receptors are surface receptors that bind and internaliz e modified lipoproteins and are involved in the development of atherosclerotic lesions (van Berkel et al ., 2005). There are several different kinds of scavenger receptors and adiponectin has been shown to suppress the expression of the scavenger receptor class A-1, which is involved in foam cell formation (Ouchi et al ., 2001). Adiponectin also inhibits the expression of the platelet-derived growth factor the heparin-binding epidermal growth factor (EGF)-like growth factor, the ba sic fibroblast growth f actor, and the epidermal growth factor (EGF) (Arita et al ., 2002). The mechanism of inhibi tion is thought to involve the mitogen-activated protein kinase (MAPK) si gnaling pathway (Kadowaki and Yamauchi, 2005). Adiponectin has also been reported to have antiangiogenesis and antitumor activity via induction of caspase mediated endot helial cell apoptosis (Brakenhielm et al ., 2004). The activation induced by adiponectin of a cascade of caspases (caspase -8, -9, and -3), leads to cell

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19 death and greatly inhibits prim ary tumor growth (Brakenhielm et al ., 2004). Impaired tumor growth is associated with decreased neovasculari zation, which leads to an increase in tumor cell apoptosis (Brakenhielm et al ., 2004). The induction of endothelia l apoptosis by adiponectin as a mechanism for antiangiogenesis raises the pos sibility of adiponectin having therapeutic implications in the treatment of angi ogenesis dependent diseases (Brakenhielm et al ., 2004). Furthermore, adiponectin has been reported to alleviate alcoholic and nonalcoholic fatty liver diseases (Xu et al ., 2003) and liver fibrosis (Kamada et al ., 2003) in mice. This clearly shows the multiple physiological and pathophysiologi cal roles of adiponecti n. Future studies are needed to really comprehend the broad physiology of adiponectin. Genome wide scans have revealed three chro mosomal regions linked to type 2 diabetes (3q, 15q, and 20q) (Mori et al ., 2002). Interestingly, the adipon ectin gene is located in the chromosomal region 3q27, linking adiponectin not only functionally but also genetically to insulin resistance and Type 2 diabetes (Mori et al ., 2002). Several single nucleotide polymorphisms (SNP) have been detected in the adiponectin gene that lead to decrease plasma adiponectin levels, increase in insulin resi stance and Type 2 diabetes. Among these were SNP276 in intron 2 (Hara et al ., 2002), SNP45 in exon 2 (Stumvoll et al ., 2002), and SNP-11377 and SNP-11391 in the promoter region (Vasseur et al ., 2002). Additional mutations in the adiponectin gene that generate the Gly84Arg and Gly90Ser mutants, allow adiponectin to assemble into trimers and hexamers but not HMW species (Waki et al ., 2003). These mutants were clinically associated with Type 2 di abetes, supporting the idea that the HMW multimers have a more potent effect against diabetes than the trimers and hexamers (Waki et al ., 2003). Adiponectin Receptors In 2003, Kadowaki and colleagues cloned the cDNA for the adiponectin receptors from the cDNA library fro m human skeletal muscle by screening for globular adiponectin binding

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20 (Yamauchi et al ., 2003). The cDNA analyzed encoded a seven transmembrane domain protein designated as human AdipoR1. Afte r the identification of the first receptor, a second adiponectin receptor was discovered by comparing homologous gene sequences in the human and mouse databases. Kadowaki and colleagues found a gene with 67% amino acid sequence identity to AdipoR1, and was termed AdipoR2 (Yamauchi et al ., 2003). AdipoR1 is ubiquitously expressed, but most a bundantly expressed in the skeletal muscle. AdipoR2 on the other hand is most abundantly expr essed in the liver. AdipoR1 has affinity for both full length and globular adi ponectin, whereas AdipoR2 has affi nity for full length but very low affinity for globular adiponectin (Yamauchi et al ., 2003). Studies done with RNA interference showed that suppression of AdipoR1 significantly reduces globular adiponectin binding, whereas suppression of AdipoR2 greatly reduces full-length adiponectin binding (Yamauchi et al ., 2003). The reason for this sp ecificity is that globular ad iponectin only exists as a trimer, whereas full length adiponectin can form HMW multimers. As it was previously mentioned, only the HMW multimers of adiponec tin are active on the liver and AdipoR2 is mostly expressed in the liver. AdipoR1 on the ot her hand, is mostly expressed in the muscle where globular adiponectin does have an effect. AdipoR1 and AdipoR2 are integral membrane proteins with the N te rminus internal, and the C terminus external, which is opposite to the topology of all other G protein-coupled receptors (GPCRs) (Yamauchi et al ., 2003). The adiponectin receptors do not seem to be GPCRs, because overexpression of either Adip oR1 or AdipoR2 has little effect on cAMP, cGMP, and intracellular calcium levels (Yamauchi et al ., 2003). The adiponectin receptors activate a series of signaling molecules such as PPAR AMPK, and p38 MAPK leading to an increase in gluc ose uptake and fatty-aci d oxidation (Yamauchi et

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21 al ., 2003). Suppression of AMPK or PPAR partially reduces the fatty acid oxidation stimulated by adiponectin. Furthermore, suppression of AMPK or p38 MAPK partially reduces the glucose uptake (Yamauchi et al ., 2003). The reduction in plasma glucose levels and proteins involved in gluconeogenesis in the liver is decreased in a dominant negative AMPK (Yamauchi et al ., 2003). These data clearly indicates th at AdipoR1 and AdipoR2 serve as receptors for adiponectin and mediate the adiponectin induced bi ological functions through AMPK, PPAR and p38 MAPK. In 2004, Lodish and colleagues reported anot her adiponectin bindi ng protein, known as Tcadherin (Hug et al ., 2004). This is a glycosylphosphatidylin ositol-anchored extracellular protein that is expressed in endothelial and smooth muscle cells (Hug et al ., 2004). T-cadherin is capable of binding adiponectin but is believed not to have an effect on adiponectin signaling, because it lacks an intracellular domain (Hug et al ., 2004). This protein is thought to be only an adiponectin binding protein and not a true receptor because it is not expressed in the liver, which is the most important target organ of adiponectin (Nawrocki et al ., 2006; Combs et al ., 2001). In 2006, Mao and colleagues revealed that the N-terminal cytoplasmic domain of AdipoR1 interacts with APPL1 (adaptor protein containing pleckstri n homology domain, phosphotyrosinebinding domain, and leucine zipper motif 1) (Mao et al ., 2006). It was also found that the Cterminal extracellular domain of Adi poR1 interacts with adiponectin (Mao et al ., 2006). The interaction of APPL1 with AdipoR1 was s timulated by adiponectin binding, and played important roles in adiponectin signaling such as lipid oxida tion and glucose uptake (Mao et al ., 2006). This finding helped to corrob orate that the adiponectin recep tors directly interact with adiponectin and mediate its downstream effects. Expression levels of AdipoR1 and AdipoR2 are decreased in obesity linked insulin resistance and Type 2 diabetes (Tsuchida et al ., 2004). Possible treatments for insulin resistance

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22 and Type 2 diabetes would be increasing AdipoR expression or the use of agonists of these receptors (Yamauchi et al ., 2003; Tsuchida et al ., 2004). It has been repo rted that adiponectin receptor expression levels may be regulated by agonists of the nuclear receptors PPAR PPAR and liver X receptor (Chinetti et al ., 2004). Another study showed that activation of PPAR and PPAR enhanced the action of ad iponectin by increasing the concentration of adiponectin receptors, which lead to an amelioration of th e obesity linked insulin resistance (Tsuchida et al ., 2005). A recent study done by Linden and colleagues has shown that AdipoR1 and AdipoR2 may have opposing effects on energy metabolism (Brujsell et al ., 2007). AdipoR1 and AdipoR2 knockout mice were generated and the mice were st udied in terms of body weight, body fat, food intake, glucose tolerance, energy expenditu re, spontaneous locomotor activity, receptor downstream signaling, and plasma biochemistry. AdipoR1 knockout mice showed increased adiposity associated with decreased glucose tolerance, spontaneous locomotor activity, and energy expenditure, whereas AdipoR2 knockout mice were lean and resistant to high-fat diet induced obesity associated with improved gluc ose tolerance and higher spontaneous locomotor activity. So, possible therapeutic approaches for the treatment of obesity, Type 2 diabetes, and cardiovascular diseases would be the activation of Adi poR1 or inactivation of AdipoR2 (Brujsell et al ., 2007). The yeast homolog of AdipoR1, Izh2p (Implicated in zinc homeostasis) is a receptor for osmotin (Narasimhan et al ., 2005). Osmotin is a plant defense pr otein that induces apoptosis in yeast. It is a member of the pathogenesis related-5 (PR-5) family of proteins which are ubiquitous in fruits and vegetables, extremely stab le and active even when digested or inhaled by humans. Interestingly, osmotin was shown to activate AMPK via adiponectin receptors in

PAGE 23

23 mammalian C2C12 myocytes (Narasimhan et al ., 2005). Osmotin and the globular domain of adiponectin are structura lly similar (Narasimhan et al ., 2005). Examining the structural similarities of osmotin and adiponectin can give insights into the design of potential adiponectin receptor agonists (Narasimhan et al ., 2005). These new agonists that mimic or enhance the action of adiponectin could be new therapeutic strategi es for the treatment of insulin resistance, the metabolic syndrome, and Type 2 diabetes. PAQR Receptors The adiponectin recep tors belong to a la rger family of proteins known as P rogestin A dipoQ R eceptor family (PAQR). This family is pres ent in eukaryotes and prokaryotes (Tang et al ., 2005). AdipoR1 and AdipoR2, also known as PAQR1 and PAQR2 respectively, belong to the human PAQR family. There are 11 members of the human PAQR family divided into 3 different classes (Table 1-1). Human Class I PAQRs The hum an class I PAQRs are composed of PAQR1, PAQR2, PAQR3 and PAQR4. Figure 1-1 shows a phylogenetic tree of the class I PAQR proteins fro m different organisms. PAQR1 and PAQR2 are more closely re lated and clustered in the sa me clade, whereas PAQR3 and PAQR4 form their own separate clades. These proteins are characterized by the presence of seven transmembrane domains. Figure 1-2 sh ows the hydropathy plots for the class I PAQR proteins from different organisms. All four proteins are predic ted to have seven transmembrane domains and in bold is shown the average hydropat hy value for all the sequences from different organisms. A multiple sequence alignment was generated with the human class I PAQRs and two of the PAQRs from Saccharomyces cerevisiae, Izh1p and Izh3p. Three highly conserved motifs were found (Figure 1-3). The first motif is found at the beginning of transmembrane domain 1 (TM1) and consists of Ex2Nx3H. The second motif is found at the end of TM2 and at

PAGE 24

24 the beginning of TM3 and consists of Sx3HxnD, which resembles the well known catalytic triad. And finally the last conserved motif is found at the end of TM6 and at the beginning of TM7 and consists of PEx3PGxnHx3H. Figure 1-4 shows in more detail the last conserved motif. PAQR1 and PAQR2 sequences are characterized by an Hx2FH in the last conserved motif, whereas PAQR3 have an Hx2WH and PAQR4 an Hx2MH. These proteins are belie ved to be localized to the plasma membrane with a cytoplasmic N-termi nus and an extracellular C-terminus, but this has only been confirmed for PAQR1 and PAQR2 (Yamauchi et al ., 2003). Nothing has been published so far about PAQR3 and PAQR4 and th e localization, topology and function of these proteins remains unknown. We have recently disc overed that PAQR3 responds to adiponectin and might be a new adiponectin receptor. We still do not know what the function for PAQR4 might be. These results will be discussed in later chapters. Human Class II PAQRs The hum an class II PAQRs are compos ed by PAQR5, PAQR6, PAQR7, PAQR8 and PAQR9. The human PAQR5, PAQR7 and PAQR8 genes encode membrane Progestin Receptors, also known as mPR mPR and, mPR respectively. These are non-genomic or posttranscriptional hormone receptors that rapidl y respond to progesterone without the need for transcription, as opposed to the nuclear progesterone receptors (Zhu et al ., 2003) which require transcription for signal transduc tion. The hydropathy plots for the cl ass II proteins are shown in Figure 1-5. These proteins are predicted to ha ve 8 transmembrane domains, but it has been suggested that they are GP CRs and only have 7 (Thomas et al ., 2007). We have heterologously expressed these proteins in Saccharomyces cerevisiae and proven that they signal without the need of heterotrimeric G proteins, discarding th e possibility of them being GPCRs. This work was performed by Jessica Smith. There is noth ing published about PAQR6 and PAQR9 and the function of these proteins is unknown. But again we have shown in our laboratory that PAQR6

PAGE 25

25 and PAQR9 respond to progesterone and might be new membrane pr ogestin receptors. The work for PAQR6 was done by Brian Kupchak and PAQR9 by Jessica Smith. Human Class III PAQRs The hum an class III PAQRs are composed of PAQR10 and PAQR11, also known as MMD2 and MMD1 respectively. Thei r genes are up regulated during monocyte to macrophage differentiation (Rehli et al ., 1995). Hydropathy plots were also generated and these proteins are predicted to have seven transmembrane domains (F igure 1-6). They are not as closely related as the class I and class II are. Ceramidase Activity Searches perfor med by Dr. Lyons of the NCBI database using PSI-BLAST, revealed that the PAQRs display distant similarity with a family of proteins known as alkaline ceramidases. Alkaline ceramidases catalyze the hydrolysis of ceramide (Figure 1-7). Both families consist of membrane proteins with at least 7 transmembr ane domains. A multiple sequence alignment was generated with the PAQR and alkaline ceramidas es sequences from different organisms and three highly conserved motifs were found (Figure 18). The first motif, motif A, consists of Ex23Nx3H found at the N-terminus of transmembrane domain 1 (TM1) (Figure 1-9). Motif B, consists of Sx3HxnD at the end of TM2. And the third motif, motif C, consists of Hx3H found in the loop region between TM6 and TM7. The third c onserved motif is shown in more detail in Figure 1-10. Alkaline ceramidase s are characterized by a HAxWH motif, which is also found in the PAQR10 and PAQR11 sequences. We have hypothesized that, even though these receptors recognize different ligands, they might transmit the same signal. This signal could be the hydrolysis of ceramide (Figure 1-11). The sphingoid base generated could be the activating molecule of a downstream signaling cascade. Sphingolipids have been shown to improve the response of cultured cells to insulin

PAGE 26

26 (Chavez et al ., 2005). Ceramides on the other hand, redu ce the response of cultured cells to insulin (JeBailey et al ., 2007), and accumulate in muscle cells of obese individuals with type 2 diabetes (Straczkowski et al ., 2007). Also, disruption of sphi ngolipid metabolism has been shown to alleviate the effects of adiponectin in cell culture (Kase et al ., 2007). So a role for the adiponectin receptors and the PAQRs in general, as alkaline ceramidases would be consistent with what is known about adiponectin and insulin signaling.

PAGE 27

27 Table 1-1. Human PAQR family. There are 11 me mbers of this family divided into three different classes, Class I, Class II and Class III. PAQR Class Other Name Function PAQR1 I AdipoR1 Adiponectin Receptor PAQR2 I AdipoR2 Adiponectin Receptor PAQR3 I LOC152559 Unknown PAQR4 I FlJ30002 Unknown PAQR5 II mPR Progesterone Receptor PAQR6 II FLJ22672 Unknown PAQR7 II mPR Progesterone Receptor PAQR8 II mPR Progesterone Receptor PAQR9 II LOC344838 Progesterone Receptor PAQR10 III MMD2 Unknown PAQR11 III MMD1 Unknown

PAGE 28

28 Figure 1-1. Phylogenetic tree of human class I PAQR proteins.

PAGE 29

29 Figure 1-2. Hydropathy plots for th e class I PAQRs. Panel A show s the hydropathy plot for the AdipoR1 and AdipoR2 sequences from diffe rent vertebrate species. Panel B shows the hydropathy plot for PAQR3 and Panel C for PAQR4. In bold is shown the average hydropathy value for all the sequences. All four proteins are predicted to have 7 transmembrane domains. Plots generated by Dr. Tom Lyons.

PAGE 30

30 Figure 1-3. Multiple sequence alignment of huma n class I PAQRs, Izh1p and Izh3p. The highly conserved motifs are shown in grey and the light grey rectangles demark the transmembrane domains. Shaded in black are the conserved amino acids in the AdipoR1 and AdipoR2 sequences that ar e also present in other sequences.

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31 Figure 1-4. Third conserved motif of class I P AQR proteins. PAQR1 and PAQR2 sequences are characterized by an Hx2FH, whereas PAQR3 have an Hx2WH and PAQR4, an Hx2MH

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32 Figure 1-5. Hydropathy plots for the class II PAQRs. In Panel A is shown the hydropathy plot for the PAQR5 and PAQR6 sequences from different vertebrate species. Panel B shows the hydropathy plot for PAQR7 a nd PAQR8 and Panel C for PAQR9. In bold is shown the average hydropathy value fo r all the sequences. All 5 proteins are predicted to have 8 transmembrane doma ins. The third transmembrane domain has been highlighted in grey because it has previously been ignored by other investigators, but it is cl early conserved throughout th e species. Plots generated by Dr. Tom Lyons.

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33 Figure 1-6. Hydropathy plot for the class III PAQRs. In bold is shown the average hydropathy value for all the vertebrate sequences. P AQR10 and PAQR11 are predicted to have 7 transmembrane domains. Plot generated by Dr. Tom Lyons. Figure 1-7. Reaction catalyze by alkaline ceramidas es. The hydrolysis of ceramide generates a sphingoid base and a fatty acid molecule.CH3OH OH NH O CH3 CH3OH OH NH2 +OH O CH3

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34 Figure 1-8. Multiple sequence alignment of PAQR and alkaline cera midase sequences from different organisms. The three highly conserved motifs are highlighted in grey and are found at TM1, TM2, TM3, TM6 and TM7.

PAGE 35

35 Figure 1-9. The three highly conserved motifs found in PAQRs and alkaline ceramidases.

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36 Figure 1-10. Third conserved motif found in PAQRs and alkaline ceramidases.

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37 Figure 1-11. Hypothetical mechanism of action of PAQRs. Upon binding of their ligands, the PAQRs might catalyze the hydrolysis of cer amide to generate a sphingosine base. The sphingosine base could be the activator of a dow nstream signaling cascade.

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38 CHAPTER 2 EXPRESSION AND CHARACTERIZATION OF THE HUMAN CLASS I PAQR PROTEINS IN ESCHERICHIA COLI Introduction The expression of the hum an class I PAQRs was first attempted in Escherichia coli because it is a commonly used organism for th e expression of large amounts of protein. The heterologous expression of other human membrane proteins has been previously reported in E. coli (Fawzi et al ., 2001; Baneres et al ., 2003; Ishigaki et al ., 2005). Once we obtained high levels of expression, the proteins could be purified and later characterized. To do this, ceramid ase assays would be performed in vitro with purified protein. The caveat to this approach, is th at it cannot be determined if th e proteins properly folded after purification. So to solve this problem, ceram idase assay would be first performed with cell extracts expressing the human PAQRs. If increa sed ceramidase activity is observed compared to cells expressing the empty vector it would be a good indication that these proteins are acting as alkaline ceramidases. Then this assay could be used for determining if the proteins folded properly. In case expression of the human class I PAQRs is not detected, expression of the other members of the human PAQR family was also attempted. This way, the ceramidase activity could still be tested. If the human PAQRs happen to have ceramidase activ ity, it would prove the unifying theory that even though these receptors recognize different ligands, they transmit their intracellular signal through the same mech anism, the hydrolysis of ceramide. Materials and Methods Cloning of Human PAQRs The hum an PAQR genes were cloned into the pKM260 and pKM263 expression plasmids (Melcher, 2000). In both plasmids, the genes are under the control of the T7 promoter but in

PAGE 39

39 pKM263 the translated proteins contain N-termin als 6 x His (His) and gl utathione S-transferase (GST) tags, whereas in pKM260, only an N-termin al His tag. These tags were intended for detection and purificatio n purposes. The GST tag has been pr eviously used for expression and purification of GPCRs in E.coli (Kiefer et al ., 1996). pKM263 also has a tobacco etch virus (TEV) cleavage site that allows the removal of the tags after purification. The PAQR genes were cloned from their cDNAs [Open Biosystems, Tabl e 2-1] by the use of polymerase chain reaction (PCR) with Pfu Turbo DNA polymerase [Takara].T he primers used are listed in Appendix C. These PCR fragments were digested with the appr opriate restriction enzymes and ligated into the plasmids with T4 DNA polymerase [New England Biolabs]. The plasmids were then transformed into E. coli TOP10 cells via electroporation and were purified with the use of the Wizard Plus Minipreps [Promega]. Finally the plasmids were sent for sequencing to the DNA Sequencing Core Facility [University of Florida]. Growth Conditions The plasm ids containing the PAQR genes were transformed into BL21(DE3) [Novagen], C41(DE3) and C43(DE3) [Gifts of Dr. Bowie, UCLA] cells. The last tw o strains have been shown to facilitate overexpression of membrane proteins (Miroux et al ., 1996). Successful transformants were grown in Luria Bertani (LB) medium containing 100 g/mL ampicillin, shaken at 250 rpm, at different te mperatures (37, 30, 25, and 20C). After OD600 reached 0.6-1, different concentrations of isopropyl-D-thiogalactoside (IPTG) (0.25, 0.5, 0.75 and 1 mM) were used to induce expression. The cultures we re grown for several more hours and the cells were harvested for later analysis.

PAGE 40

40 Detection of Expression Coomassie staining The pellets were resuspended in 2X Laemm li Sa mple Buffer and incubated at 37C for 30 min. The samples were not boiled to prevent ag gregation of the membrane proteins. After incubation, the samples were treated with RQ1 RNase-Free DNase [Promega] for 5 min at room temperature to sheer the released DNA and reduce the viscosity of the samples. The samples were loaded on a 10% SDS-PAGE and run at 100 V. The gels were finally stained overnight in Coomassie Blue Stain. His-tag in-gel staining After electrophoresis, the gel was fixed for 1 hour with fixi ng solution (50% ethanol, 10% acetic acid, 40% ultra p ure wate r). The gel was then washed tw ice with ultra pure water and incubated for 1 hour with InVision His-tag In-gel Stain [Invitrogen #LC6030]. This stain is based on a fluorescent dye conjugate d to a nickel: nitrilotriacetic acid (NTA) complex that binds to His-tagged fusion proteins allowing their det ection from a mixture of other proteins. It was then washed twice with 20 mM phosphate buffer pH 7.8. The dye is then excited at 302 nm and the bands are visualized with a UV transillum inator equipped with a CCD camera [Bio-Rad]. Western blot After electrophoresis the prot eins were transferred onto a PVDF m embrane [Bio-Rad] at 100V for 1 hour at 4C. The membrane had previ ously been pre-wetted in 100% methanol and equilibrated in transfer buffer before blotting. The membrane was then blocked overnight at 4C with shaking in PBST1 buffer (20 mM NaH2PO4, 150 mM NaCl, 0.1% Tween 20, pH 7.4). The membrane was then incubated for 1 hour at room temperature with His-Detection Reagent, from the Universal His Western Blot Kit 2.0 [Clont ech #635642], at a ratio of 1:1000 in PBST2 buffer (20 mM NaH2PO4, 500 mM NaCl, 0.1% Tween 20, 3mM imid azole, pH 7.4). This His-Detection

PAGE 41

41 Reagent is based on a BD TALONTM [Clontech] resin that is c onjugated to biotin through a carrier molecule and binds to the His tagged fu sion proteins. The membrane was then washed twice with PBST2 buffer and four more times with PBST3 buffer (20 mM NaH2PO4, 500 mM NaCl, 0.1% Tween 20, pH 7.4). Then it was incu bated for 30 min. with Streptavidin-HRP [Clontech] at a ratio of 1:8000 in PBST3 buffer. This is a streptavidin molecule, conjugated to horseradish peroxidase (HRP), which recognizes and binds to biotin. The membrane was then washed six times with PBST3 buffer to re move any unbound Streptavidin-HRP. Then it was incubated for 1 min. with Detection solution [Cl ontech], which contains peroxide, luminol and enhancer. The HRP reacts with peroxide generating O2. Then O2 reacts with luminol, generating unstable peroxide that decomposes to a molecule with electrons in the excited state. As the electrons go from the excited state to the ground st ate, the excess energy is liberated in the form of a photon, visible as blue light. After incuba tion, the excess solution was decanted and the membrane was wrapped with a sheet protector. The protected membrane was placed in a film cassette with x-ray film [Thermo Scientific ] and the film was exposed and developed. Purification Transformed cells were grown in 1L of LB me dium containing 100 g/ml of ampicillin. The culture was grown at 30C with shaking at 250 rpm until an OD600 = 0.6-1.0 was reached. The cells were then induced with 1mM IPTG and grown for 8 more hours. They were then harvested by centrifuging at 4000 rpm for 15 min at 4C and resuspended in 30 ml of cold lysis buffer (20 mM Tris-HCl, 5 mM imidazole, 0.5 M NaCl, 2 % Triton X-100, at pH 7.9) supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF) and bacterial protease inhibitor cocktail [American Bioanalytical]. Th e cells were then lyse d by several rounds of sonication, with cooling on ice between each round, until the samp le was no longer viscous. The

PAGE 42

42 resulting suspension was centrifuged at 15,000 rpm fo r 30 min at 4C to remove the cell debris and the supernatant was saved. The supernatant was combined with pre-equilibrated His-MagTM Agarose Beads [Novagen #71002] and incubated for 5 min. at 4C. The bead s were then collected with a magnet and washed 3 times with washing buffer supplemented w ith protease inhibitors. The protein was then eluted by resuspending the beads for 5 min. with elution buffer (20 mM Tris-HCl, 1 M imidazole, 0.5 M NaCl, pH 7.9) supplemented also with protease inhibitors. The eluted protein was collected and analyzed by SDS-PAGE. The supernatant was also applied to a Hi s-Bind Column [Novagen] that had been previously packed with a nickel-charged resi n [Novagen]. The column was washed first with binding buffer (0.5 M NaCl, 20 mM Tris-HCl, 10 mM imidazole, pH 7.9) and then twice with washing buffer (20 mM Tris-HCl, 60 mM imidazo le, 0.5 M NaCl, pH 7.9). The protein then was eluted with elution buffer (20 mM Tris-HCl 1 M imidazole, 0.5 M NaCl, pH 7.9) and all fractions were collected and analyzed by SDSPAGE. Again, all buffers contained protease inhibitors to avoid protein degradation. Ceramidase Assay Alkaline ceram idase activity was measur ed following a published protocol (He et al ., 1999). Briefly, 5 L of cell extract was mixed with 5 L of reaction buffer (25 mM Tris-HCL buffer pH 8.0, 0.25% (w/v) Triton X-100, 5 mM CaCl2 and 0.2 mM N-[6-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino]hexanoyl]-Derythro -Sphingosine (C6 NBD Ceramide) [Avanti Polar Lipids]) and incubated at 37 C for 12 hours. C6 NBD ceramide is a ceramide molecule with an N-acyl group attached to an NBD fluorophore (Figure 2-1). Once the reaction was completed, 5 L of the assay mixture was added to 95 L of ethanol and centrif uged at 10,000 rpm for 5 min at 4 C. Then, 20 L of the supernatant was applied to a Thin Layer Chromatography (TLC)

PAGE 43

43 plate [Fisher] along with the sta ndards C6 NBD Ceramide and NBD hexanoic acid [Avanti Polar Lipids] (Figure 2-2). The TLC plate was then dried and developed with a mixture of chloroform/methanol/25% ammonia (90:20:0.5, v/v) The bands were visualized with a handheld UV detector and a gel docum entation system with UV detec tion capability [Bio-Rad]. Results and Discussion Expression of PAQRs in Escherichia coli The focus of this work w as to express and purify the human class I PAQRs in Escherichia coli but in case their expression failed, all th e other human PAQRs were tried. The expression was first tried with pKM263. Unfortunately non-d etectable levels of e xpression were obtained with this plasmid. A possible probl em could have been that the bi g GST tag (26 kDa) might have prevented proper folding of these proteins into the membrane, leading to their later degradation. So a plasmid with just an N-terminal His-tag was tried, pKM260. But again non-detectable levels of expression were obtained. It is possible that a small N-termin al His tag could have affected folding of these proteins into the membrane. A di fferent tag or a C-terminal His tag could have had better results with expression. Different E. coli strains and different growth conditions were used for expression. First, cells were grown at 37C, which is the optimal growth temperature for E. coli Then the temperature was lowered to 30, 25 and 20C. Lo wering the temperature is known to help the folding of membrane proteins into the membra ne (Laage and Langosch, 2001). Concentrations of IPTG were also varied, from 1 mM, which is the usual con centration, to 0.75, 0.5 and 0.25 mM. Reducing the concentration of i nducer can facilitate the foldi ng into the membrane by slowing down the production of protein. But in all cases non-detectable levels of expression were obtained.

PAGE 44

44 Analysis of expression was first done with Coomassie Blue Staining of the SDS-PAGE. This method is valid whenever the expression le vels are high, as it is the case for soluble proteins. Membrane proteins usually have lower expression leve ls than soluble proteins and sometimes are not detected by Coomassie Blue St aining. This is what happened with the human PAQRs, their expression could not be de tected by Coomassie Blue Staining. A stain with a higher detection limit was used instead, the InVision His-tag In-gel Stain. With this stain, up to 30 ng of His-tagged fusi on protein can be detected. But there was a problem with this stain, it turned out to be not very specific to wards the His-tag fusion proteins and bound to many other proteins from E. coli, not allowing the clear detection of the PAQRs. So finally expression of these proteins was analyzed by western blot. The Universal His Western Blot Kit 2.0 was used for this purpose wh ich can detect as littl e as 0.5 ng of His-tagged fusion protein. Unfortunately, the expression levels of the PAQRs were not detected by western blot. In order to concentrate the proteins to see expression, purification of these proteins was attempted. Both agarose beads and a His-bind column [Novagen] were used for this purpose, but again expression of these proteins could not be detected. Figure 2-3 shows the western blot for the purification of N-terminal His tagged PAQR3 in Escherichia coli with a His-bind column. Lane 1 corresponds to the pellet collected from the centrifugati on at 15,000 rpm after sonication. Lanes 2-4 correspond to the three washes of the His-bind column, and lanes 5-6 to the eluted fractions. No protein bands were det ected in any of the fractions. Ceramidase Assay Even though we could not detect expression of these proteins, ceram idase assays were still performed with the crude cell extracts. The samples were analyzed by TLC along with the standards NBD hexanoic acid and C6 NBD Ceramide No ceramidase activity was detected for

PAGE 45

45 any of the PAQRs tested. Figure 2-4 shows the TLC of the ceramidase assay performed with crude cell extracts expressing PAQR1. The standa rds were loaded in the first two lanes, NBD hexanoic acid (lane 1) and C6 NBD ceramide (l ane 2). Lane 3 shows the reaction mixture of cells expressing PAQR1 with C6 NBD ceramide. Only unreacted C6 NBD ceramide was present in lane 3, and there was no NB D hexanoic acid, meaning that the cells expressing PAQR1 had not hydrolyzed C6 NBD ceramide. As a negative c ontrol, cells expressing the empty vector were incubated with C6 NBD ceramide and loaded in lane 4. Only unreacted C6 NBD ceramide was present in this lane. As a positive control for this assay, C6 NBD ceramide was incubated with sphingolipid ceramide N-deacylase (SCDase) from Pseudomonas sp. [Sigma] and the TLC was run (Figure 25). The standards were loaded in the first two lanes, C6 NBD ceramide (lane 1) and NBD hexanoic acid (lane 2). The reaction of SCDase with C6 NBD ceramide was loaded on lane 3, and both C6 NBD ceramide and NBD hexanoic acid we re present in this lane, but the band for NBD hexanoic acid was very faint. SCDase had partially hydrolyzed C6 NBD ceramide, but the reaction did not go to completion because SCDase has a preference for glycosylceramides (Ito et al ., 1995). Finally the negative control, where no SC Dase was added, was loaded in lane 4. There was no hydrolysis of ceramide because NBD hexanoic acid was not present in this lane 4. It cannot be concluded from these results that the PAQRs have no ceramidase activity. The assay conditions used might not have been the ap propriate for the PAQRs. A different substrate with longer fatty acid chain can be used, such as C12 NBD ceramide. Maybe pH = 8.0 is not the optimal pH for the PAQRs to hydrolyze ceramide, so different pHs could be tested. Instead of TLC, a different system with higher detection limit could be used, such as HPLC with a fluorescence detector. In case ceramida se activity is still not observed, 3H-labeled ceramide

PAGE 46

46 could be used as substrate. The reaction produc ts would also be sepa rated by TLC. Then the spots for the reaction products would be scrapped off the TLC plate and quantified using a scintillation counter. If ceramidase activity is still not detected after all these different conditions, a possible explanation would be that the expres sion levels of the human PAQRs in Escherichia coli are not high enough for the ceramidase activity to be detect ed. The low expression levels could be due to the fact that the human PAQR gene s contain too many rare codons for E. coli expression system to recognize (Table 2-2). To account for this an additional plasmid, pRARE plasmid (Novagen), was used to aid in the expression. This is a plas mid that increases rare tRNA levels by increasing the levels of the respective tRNA genes. In sufficient tRNA pools can lead to premature translation termination and amino acid misinc orporation. Unfortunately, expression of the PAQRs was still not observed when the cells were cotransformed with the pRARE plasmid. A possible explanation for this could be that the pRARE plasmid di d not provide all the necessary tRNAs to cover all the different rare codons found in the human PAQRs genes. So an alternative to this would be to codon-optimize the human P AQR genes, that is, change the rare codons for others more recognizable by the E. coli system. This option was not followed because it would have been very expensive or time consumi ng. Instead, a different or ganism was used for expression of these proteins, the yeast Saccharomyces cerevisiae

PAGE 47

47 Table 2-1. Accession numbers for the cDNA of th e human PAQR genes from Open Biosystems. PAQR gene Accession numbers for cDNA PAQR1 BC010743 PAQR2 BC051858 PAQR3 BC031256 PAQR4 BC033703 PAQR5 BC039234 PAQR6 BC058509 PAQR7 BC042298 PAQR8 BC030664 PAQR9 BC118666 PAQR10 BC067905 PAQR11 BC026324 Figure 2-1. Structure of C6 NBD ceramide. C6 NBD ceramide is a ceramide with an N-acyl group attached to an NBD fluorophore. CH3OH OH NH O NH N O N NO2

PAGE 48

48 Figure 2-2. Structure of NBD hexanoic acid. This is a hexanoic acid labeled with an NBD fluorophore Figure 2-3. Western blot of the purification of PAQR3 using His Bind Column. Lane1Pellet; Lane 2 First wash; Lane 3Second wash; Lane 4 Third wash; La ne 5First eluted fraction; Lane 6Second eluted fraction. No protein was detected in any of the fractions. OH O NH N O N NO2

PAGE 49

49 Figure 24. TLC analysis of reaction of C6 NBD ceramide with cells expressing PAQR1. Lane 1NBD hexanoic acid; Lane 2 C6 NBD Ceramide; Lane 3 Reaction of cells expressing PAQR1 with C6 NBD ceramide; La ne 4Reaction of cells expressing the empty vector with C6 NBD ceramide. Figure 2-5. TLC analysis of reaction of SCDase with C6 NBD ceramide. Lane 1C6 NBD ceramide; Lane 2NBD hexanoic acid; Lane 3Reaction of SCDase with C6 NBD ceramide; Lane 4Same as lane 3 but with no SCDase added.

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50 Table 2-2. Number of rare codon of human PAQR genes in Escherichia coli These were calculated with Gene Designer with a threshold of 5% (Villalobos et al ., 2006). PAQR gene Rare codons PAQR1 49 PAQR2 58 PAQR3 41 PAQR4 34 PAQR5 35 PAQR6 37 PAQR7 22 PAQR8 28 PAQR9 25 PAQR10 26 PAQR11 33

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51 CHAPTER 3 EXPRESSION AND CHARACTERIZATION OF THE HUMAN CLASS I PAQR PROTEINS IN SACCHAROMYCES CEREVISAE Introduction As m entioned in the previous chapter, we were unable to de tect expression of the human class I PAQRs in Escherichia coli So we switched to the eukaryote, Saccharomyces cerevisiae to express and characterize these proteins. This organism was chosen for three main reasons. First, the Human PAQR genes have significantly less rare codons in Saccharomyces cerevisiae than in E. coli (Table 3-1). Second, the signal transduction mechanism via PAQR receptors is similar in humans and yeast. And third, if we wanted to study a single PAQR receptor in mammalian cells, it would require the use of small interfering RNA (siRNA) or knockdown strains to eliminate effects of other receptors making the study more complicated. The use of siRNA is not necessary in Saccharomyces cerevisiae, because this organism does not possess adiponectin or membrane progestin receptors. So the study these proteins in yeast would be much easier because there would not be signal interference from other human PAQRs. S. cerevisiae do posses PAQR proteins, called Izh1-4p (Implicated in zinc homeostasis) (Lyons et al ., 2000), but they are involved in zi nc and iron homeostasis and have no role in adiponectin or progesterone signaling. Saccharomyces cerevisiae is a widely used model organism and the heterologous expression of ot her human membrane proteins, including GPCRs, has been previously reported (Wedekind et al ., 2006; Niebauer and Robinson, 2006; Minic et al ., 2005; Pausch et al ., 2004; Kong et al ., 2002; Lagane et al., 2000). Although expression levels are usually not as high as the ones obtained in E.coli human proteins that cannot be recombinantly expressed in E. coli have a better chance of being expressed in S. cerevisiae because of its closer resemblance to the mammalian system.

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52 Materials and Methods Yeast Strains and Media The strains used are listed in Appendix A and the com positions for the media are shown in Appendix B. Cloning of Human PAQR The hum an class I PAQR genes were cloned into the pGREG536, pGREG575 and pGREG600 expression plasmids (Jansen et al ., 2005). In all three plas mids, the genes are under the control of the galactose inducible GAL1 promoter, but the protei ns expressed have an Nterminal hemagglutinin (HA) tag in pGREG536, an N-terminal green fluorescence protein (GFP) tag in pGREG575, and a C-terminal GFP tag in pGREG600.The human PAQR genes were cloned from their cDNAs (Table 2-1) by PCR with Pfu Turbo DNA polymerase. The primers used are listed in Appendix C. These PCR fragments were insert ed into the Sal I site of pGREG536, pGREG575, and pGREG600 plasmi ds via homologous recombination (Jansen et al ., 2005). Transformations were performed by the standard lithium acetate protocol (Guthrie et al ., 1991) and the transformed colonies were selected for by plating on synthetic medium with the appropriate amino acids. The plasmids were isolated from yeast by using a modified standard protocol (Burke et al ., 2000) and they were transformed into E. coli TOP10 via electroporation. The plasmids were later purified from E. coli cultures and sent fo r sequencing to the DNA Sequencing Core Facility [U niversity of Florida]. Growth Conditions The parental yeast wild type haploid, BY 4742, was transformed with plasm ids containing the PAQR genes. Overnight cultures were grown in synthetic dextrose (SD) media supplemented with the appropriate amino acids. These cultures were used to re-inocu late 150 mL of low iron media (LIM) containing galactose and 1 M FeCl3 at OD600 = 0.2. Cells were grown at 30C

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53 until OD600 = 0.8-1.0 was reached and harvested by cen trifugation at 4C. The pellets were washed twice with ice-cold water and stored at -80C. Membrane Protein Isolation The pellets were thawed and dissolved in 600 L of m itochondrial isolation buffer (MIB) (0.6M Mannitol, 20mM HEPES pH 7.4, 1mM ED TA) supplemented with 1mM PMSF and 10 L/mL of protease inhibitor cocktail for fungal and yeast extracts [Sig ma]. The cells were disrupted by vortexing six times with acid washed glass beads for 1 minute at 4C, with cooling on ice in between. They were then centrifuged at 3,000 x g for 10 min and the supernatant was collected. To isolate the membrane proteins the supernatant was centrifuged at 100,000 x g for 90 min in an Optima TLX ultracentrifuge [B eckman Coulter]. After centrifugation, a yellow pellet was observed at the bottom of the tube which contained the membrane proteins. The supernatant was saved for late r analysis and the pellet wa s resuspended in MIB buffer supplemented with protease inhibitors. The protei n concentration of all samples was determined with the BCA Protein Assay Kit [Thermo Scientific]. Western Blot Equal am ounts of total protein were loaded on each well of a 10% SDS-PAGE and run at 100V. Proteins were then transfer red onto a nitrocellulose membra ne [BioRad] at 80V for 1 hour at 4C. The membrane was then blocked overnight at 4C with shaking in 15 mL of PBST (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4, 0.05% Tween-20) with 1% Bovine Serum Albumin (BSA). It was washed twice with PBST and incubated at room temperature for 1 hour with shaking with rabbit an ti-HA polyclonal antibody [Santa Cruz Biotechnology sc-805] at a ratio of 1:500 in 1% BSA in PBST. The membra ne was washed four times with PBST and incubated for 1 hour with goat anti-rabbit IgGHRP [Santa Cruz Biotechnology sc-2004] at a ratio of 1:8000 in 1% BSA in PBST. It was wa shed again four more times with PBST and

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54 incubated for 5 min. with SuperSignal West Pico Chemiluminescent Substrate [Thermo Scientific]. Finally the x-ray f ilm was exposed and developed. The membrane was then washed twice with PBST and incubated for 15 min at 37C with Restore Western Blot Stripping Buffer [Thermo Scientific] to strip the antibodies from the membrane. The membrane was then blocked for 1 hour at room temperature in 1% BSA in PBST and washed twice with PBST. Then, it was incubated for 1 hour with mouse anti-porin (yeast mitochondrial) monoclonal antibody (Invitrogen #A6449) at a ratio of 1:2000 in 1% BSA in PBST and washed four times with PBST. It was later incubated for 1 hour with goa t anti-mouse IgG-HRP (Bio-Rad #170-6515) and washed four times with PBST. Finally it was in cubated with the chemiluminescent substrate and the x-ray film was exposed and developed. -galactosidase Reporter Assays -galactos idase reporter assays were performed following a st andard protocol (Guarente, 1983). Briefly, after growing cells to mid-log phase in LIM, they were harvested and resuspended in Z-buffer (60mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH 7.0) at 4C. The cells were permeabilized by vortexing with chloroform and 0.1% sodium dodecyl sulfate (SDS). The cells were then aliquo ted into a 96-well plate [Costar] and incubated with o-nitrophenyl-D-galactopyranoside (ONPG) [Sigma]. The hydrolysis of ONPG (colorless) to o-nitrophenol (yellow) and galactose is followed by measuring the OD420. The OD600 was also measured to normalize for the nu mber of cells. The reaction was then stopped with Na2CO3, and the OD420 and OD600 were read in a SAFIRE microplate reader. The galactosidase activity was reported in Miller Units as follows: (OD420 x 1000) / (mL of culture x reaction time (min.) x OD600).

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55 GFP Fluorescence Overnight cultures were used to re-inoculate LIM cultures containing 2 % galactose at an OD600 = 0.2. Cells were grown overnight until an OD600 = 0.6-0.8 was reached and then loaded onto microscope slides [Fisher] and analyzed w ith a Zeiss Axiovert S100 Inverted Microscope with an oil immersed 63x objective. To observe green fluorescence prot ein (GFP) fluorescence, the cells were excited with a 488 nm Argon la ser and emission was detected at 520 nm. The expression of the GFP constructs was also ch ecked by western blot. The isolation of the membrane proteins and the western blot were performed as previously described, but this time rabbit anti-GFP antibody (Invitrogen #A11122) at a ratio of 1:3000 in 1% BSA in PBST and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnol ogy sc-2004) at a ratio of 1:5000 in 1% BSA in PBST were used as primary and secondary antibodies, respectively. As a loading control, the membrane was stripped from the anti-GFP antib odies and probed against porin the same way as before. -galactosidase reporter assays were also performed to check functional expression of the GFP constructs. Adiponectin Binding Both whole yeast cells and spheroplasts (yeast c ells lacking their cell wall) were used in this experiment. To prepare spheropl asts, 150 mL cultures were grown to OD600 = 1, harvested and washed twice with sterile wate r. Cells were resuspended at OD600 = 20 in 0.1 M Tris-SO4, pH 9.4, 10 mM DTT and 50 mM -mercaptoethanol and incubated at 30C for 30 min. with shaking at 250 rpm. During this incubation the outer mannoprotein layer was loosened. The cells were later harvested and washed first with 1.2 M sorbitol and then with spheroplasting buffer (1.2M sorbitol, 10 mM potassium phosphate pH 7.2, 0.5 x YPD). They were finally resuspended at OD600 = 50 in spheroplasting buffer with 10-25 Units/ OD600 of Zymolyase or Lyticase and incubated at 30C for 45 min. with shaking at 100 rpm. During this incubation, the (1 3)-

PAGE 56

56 glucan layer was lysed. Cells were then washed with 20 mL of PBS and the spheroplasts were recovered by centrifugation at 600 x g for 10 min. The supernatant containing the spheroplasts was transferred to a new tube and diluted to OD600 = 0.01 with PBS. Whole cells or spheroplasts we re then incubated at 4C with different concentrations of FAM-adiponectin [Phoenix Pharmaceuticals] for 1 hour. FAM is a mono-5-(ONDG) carbonyfluorescein dye with absorption at 495 nm and emission at 519 nm. They were incubated at 4C to avoid endocytosis of adiponectin. Samples were analyzed using a Flow Cytometry Instrument (FACS Vantage SE Turbosort). Results and Discussion Repression of FET3 by PAQRs It has been recently discovered th at ove rexpression of the PAQR proteins from S. cerevisiae (Izhps) causes repression of the FET3 gene (Kupchak et al ., 2007). This gene encodes the Fet3p protein, a cell surface multic opper ferroxidase that oxidizes Fe2+ to Fe3+ (De Silva et al ., 1995). Fet3p forms a complex with Ftr1p, an iron transporter, and imports Fe3+ during iron deficiency (Askwith et al ., 1994). The mechanism by which overexpression of the PAQRs causes repression of FET3 is not yet fully understood, but it is thought to involve the AMP dependent kinase (AMPK) and the cAMP-d ependent protein kinase (PKA) (Kupchak et al ., 2007). The -galactosidase reporter assay was used to monitor the repression of FET3 by PAQR overexpression. In this assay, yeas t cells were transfor med with two plasmids, one containing the PAQR gene under the control of the GAL1 promoter, and the other containing the lacZ gene under the control of the FET3 promoter. So, whenever th e PAQR proteins are being overexpressed, they would transmit a signal that would end up with the repression of the FET3

PAGE 57

57 promoter, and decreased transcription of the lacZ gene. The production of the lacZ gene product, -galactosidase, is followed by m easuring the hydrolysis of ONPG. Figure 3-1 shows the repression of FET3 by overexpression of the PAQRs from S. cerevisiae (Izh1-4p). This was first shown by Brian Kupchak. In low iron (1 M Fe3+), cells overexpressing the Izh proteins have decreased -galactosidase activity compared to cells carrying the empty expression vector. Lower -galactosidase activity means that the FET3 promoter is repressed when th e Izh proteins are overexpressed. Whereas, in high iron conditions (1 mM Fe3+), the FET3 promoter is repressed regardless of Izhp expression level. The reason for this is that the FET3 gene is expressed during iron deficien cy, to facilitate the transport of Fe3+ inside the cell, but during iron repletion, Aft1p, an iron responsive transcriptional activator, no longer binds to the FET3 promoter and the FET3 gene is not transcribed. These assays were performed in the presence of 2% galactose, wh ich means that the Izh proteins were being overexpressed. Thaumatin as a Ligand for Izh2p The PAQR genes are un der the c ontrol of a galactose inducible promoter, so the expression levels of these proteins can be regulated by ch anges in the concentration of galactose in the medium. When the concentration of galactose is 0.05%, cells expressing Iz hps no longer repress FET3 in low iron conditions (Figure 3-2). This wa s again first shown by Brian Kupchak. In this assay, the total sugar concentration was mainta ined at 2% by adding raffinose to the media because without a source of carbohydrates the yeast cells would not be able to survive. At 0.05% galactose, there is no longer repression of FET3 by the PAQRs, so the activation of these receptors by a ligand was st udied. Out of the four PAQRs from S. cerevisiae only the ligand for Izh2p is known. This ligand is osmotin, which is a protein from Nicotiana tabacum that belongs to the pathogenesis related-5 (PR-5) family of defens ins. Osmotin is a plant defense

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58 protein that induces apoptosis in S. cerevisiae (Narasimhan et al ., 2005). Unfortunately osmotin is not commercially available, a nd we could not use it in our a ssay. Instead we used thaumatin, another member of the PR-5 family which is commercially available. Thaumatin was first isolated from the fruits of the West African rain forest shrub Thaumatococcus daniellii and is currently used as an artificial sweetener. When thaumatin was added at a concentration of 1 M, Izh2p was activated causing the repression of FET3 (Figure 3-3). Thaumatin had no e ffect on the other Izhps at this concentration. This result indicates that thauma tin could be a newly unc haracterized ligand for Izh2p. At higher levels of thaumatin (10 M) repression of FET3 was seen in a strain carrying vector control because of the production of Izh2p from the genomic copy of the IZH2 gene (Figure 3-4). And at very high levels of thaumatin, 100 M, repression of FET3 was seen in cells lacking all four IZH genes (Figure 3-5). The Izh2p independent effect of thaumatin is consistent with the previous observation that an IZH2 deletion does not eliminate th e sensitivity of yeast to osmotin (Narasimhan et al ., 2005). Human Adiponectin Receptors Once the rep ression of FET3 by Izhps was observed, the human adiponectin receptors were studied to see if they would have the same effect. They did. When the human adiponectin receptor PAQR1 was heterologously overexpressed in S. cerevisiae it caused repression of FET3 (Figure 3-6). PAQR2 on the other hand, did not repress FET3 when overexpressed. It is possible that PAQR2 might require its li gand, adiponectin, to elicit FET3 repression. And that was actually the case. When adiponec tin was added at a concentration of 100 pM, cells expressing PAQR2 repressed FET3 (Figure 3-7). To confirm this result a dose response assay was done with adiponectin (Figure 3-8). As the concentrati on of adiponectin increa sed, the repression of FET3 by PAQR2 increased. Adiponectin on the other hand, had no effect on FET3 on cells expressing

PAGE 59

59 the empty vector, showing that adiponectin, per se, causes no repression of FET3 The difference in -galactosidase activity for PAQR1 and PAQR 2 could be due to expression levels. Using western blots with antibodies against the HA ta g we saw expression for PAQR1, but not PAQR2, in the yeast Saccharomyces cerevisiae (Figure 3-9). PAQR2 might be expressed, but we could not detect expression of PAQR2 with anti-HA antibodies. There is a possibility that the HA tag could be cleaved and PAQR2 can no longer be de tected using anti-HA antibodies. As a loading control, the blot was stripped and probed agains t porin, a protein expressed in the mitochondrial membrane. The same amount of porin was presen t in the PAQR1 and PAQR2 lanes (Figure 3-9), meaning that the same amount of total membrane protein had been loaded. At 2% galactose, overexpression of PAQR1 causes repression of FET3 but if the concentration of galactose is grad ually decreased, the repression of FET3 can be relieved (Figure 3-10). At a concentration of 0.05% galact ose, there is no longer repression of FET3 by PAQR1. To test if PAQR1 would be activated by its li gand, as in the case of PAQR2, adiponectin was added to the medium. Once a dded, there was repression of FET3 caused by activation of PAQR1 (Figure 3-11). To confirm this result, a dose response assay was done ag ain with adiponectin (Figure 3-12). And as before, when the concentration of adiponectin incr eased, the repression of FET3 by PAQR1 also increased. So repression of FET3 was seen by either overexpression or activation of the human adiponectin receptors. Inhibitors and Activators of Human Adiponectin Receptors This assay is really usefu l because it confirms that the hum an adiponectin receptors are functionally expressed in Saccharomyces cerevisiae and it also allows for the detection of inhibitors and activators of these receptors. This assay is currently in the process of being patented (A Colorimetric Assa y for Inhibitors and Activators of Adiponectin Receptors, UF#12636). This assay would be useful for the design of antidiabetic and an ti-atherogenic drugs. We

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60 have recently discovered with this assay th at the complement factor C1q, a homolog of adiponectin (Innamorati et al ., 2006), also activates PAQR 1 and causes repression of FET3 (Figure 3-13). The concentration required for acti vation with C1q (3 nM) is much higher than the one used with adiponectin (100 pM), meaning that C1q is not as pot ent an activator as adiponectin. A dose response assay was done with C 1q (Figure 3-14), and as the concentration of C1q increases, so does the repression of FET3 by PAQR1. On the other hand, tumor necrosis factor (TNF), a hormone with the opposite physiological effects of adiponectin (Navarro and Mora-Fernandez, 2006), has been shown to partially inhibit the repression of FET3 by PAQR1 (Figure 3-15). This was firs t shown by Brian Kupchak. TNFcould be a newly uncharacterized inhibitor of the human adiponectin receptors. Th e concentrations required for inhibition, 500 nM, are relatively high. Apart from identifying activators and inhibitors, this assay can also be used for structure-function studies. Mutants that no longer respond to adiponectin could give some insight into the amino acids needed for adiponectin signaling. This is curren tly being investigated in our laboratory. PAQR3 and PAQR4 After observing the effect of the human ad iponectin receptors (PAQR1 and PAQR2) on FET3 the other m embers of the human class I PAQR family (PAQR3 and PAQR4) were studied. When PAQR3 and PAQR4 were heterologous ly expressed in yeast, they also caused repression of FET3 (Figure 3-16). The expression levels of these proteins were analyzed by western blot. We saw expression for PAQR3, but not PAQR4, which could not be detected with anti-HA antibodies (Figure 3-17). Again as a load ing control, the membrane was stripped and probed against porin. Equal amounts of porin were found in all lanes, indicating that the same amount of total protein was present in all samp les. A possible explana tion for PAQR4 would be

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61 that the HA tag is cleaved, as in the case of PAQR2, and PAQR4 can no longer be detected with anti-HA antibodies. As with PAQR1, the repression of FET3 was relieved when the c oncentration of galactose was gradually decreased for PAQR3 (Figures 3-18) and PAQR4 (Figure 3-19). And at a concentration of 0.05% galactose, there is no longer repression of FET3 by PAQR3 and PAQR4 (Figure 3-20). Because of the close sequence homology of PAQR3 and PAQR4 to the adiponectin receptors, it was thought that these pr oteins could also be acting as adiponectin receptors. So whenever adiponectin was added to these receptors, we saw that PAQR3, but not PAQR4, was activated by adiponectin and repressed FET3 (Figure 3-21). This could mean that PAQR3 is a newly uncharacterized adiponectin r eceptor. To confirm this result a dose response assay was done with adiponectin, showing that, as the concentrati on of adiponectin increased, the repression of FET3 by PAQR3 also increased (Figure 3-22). PAQR4 on the other hand, was unaltered by the concentration of adiponectin and did not repress FET3 (Figure 3-23). Figure 324 shows a dose response with adiponectin for all four human class I PAQRs. PAQR3 requires higher concentrations of adiponectin than PAQR1 and PAQR2 to repress FET3 The half maximal effective concentration (EC50), which is the concen tration of adiponectin that induces a response halfway between the ba seline and maximum, was calculated for PAQR1, PAQR2 and PAQR3 (Table 3-2). The EC50 values are used as a measure of drug potency. This is the first time the EC50 for PAQR3 has been reported. The EC50 values for PAQR1 and PAQR2 had previously been reported in mammalian cells (Yamauchi et al ., 2003) and were 0.54 nM for PAQR1 and 6.23 nM for PAQR2. Thes e values are much higher than the ones obtained in Saccharomyces cerevisiae, 0.686 pM for PAQR1 and 2.377 pM for PAQR2. A possible explanation for these lower values in S. cerevisiae would be that the PAQR receptors are

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62 expressed in S. cerevisiae without the presence of the human proteins that could affect binding to adiponectin. To further validate the finding that PAQR3 is a new adiponectin recep tor, another control experiment was performed. The membrane pr ogestin receptors PAQR5, PAQR7, and PAQR8 were expressed in S. cerevisiae and treated with adiponectin and progesterone. PAQR5, PAQR7 and PAQR8 responded to their ligand progesterone but not to adiponectin meaning that these receptors are specific to their ligands. Figure 3-25 shows the assay for PAQR7 and PAQR2 in 2% galactose and Figure 3-26 the assay for PAQR5, PAQR8 and PAQR1 in 0.05% galactose. PAQR3 and PAQR4 were also test ed with progesterone to see if the steroid would induce a response in these receptors, but it did not (Figure 3-27). Even a dose response assay was done to test if higher concentrations of progesterone would activate P AQR4 (Figure 3-28). But again no response was seen, meaning that progesterone is not a ligand for PAQR4. Involvement of PKA and AMPK It was previously m entioned that th e mechanism by which the Izhps repress FET3 is unknown, but it is thought to involve PKA and AMPK (Kupchak et al ., 2007). So in order to test if the human PAQRs also require PKA and AMPK for repression of FET3 assays were done in strains lacking subunits of these enzymes. In Saccharomyces cerevisiae there are three catalytic subunits of PKA (Tpk1p, Tpk2p, Tpk3p), and when the assay was done in a strain lacking Tpk2p, repression of FET3 was no longer seen by overexpre ssion (Figure 3-29) or activation (Figure 3-30) of the human cla ss I PAQRs. Also in strains la cking Snf4p, a stimulatory subunit of AMPK in S. cerevisiae there was no longer repression of FET3 by overexpression (Figure 331) or activation (Figure 3-32) of the human class I PAQRs. Th ese results indicate that the human PAQR might follow the same signaling pathway as the Izhps for repression of FET3

PAGE 63

63 Ceramidase Activity The common m echanism by which the PAQRs repress FET3 could be the hydrolysis of ceramide. This hypothesis came from the distant relationship between the PAQRs and alkaline ceramidases and the sharing of three highly cons erved motifs. So to test this hypothesis, an alkaline ceramidase from S. cerevisiae Ypc1p, was used in this assay. It was observed that overexpression of Ypc1p also represses FET3 and when Ypc1p, PAQR1, and PAQR2 were treated with Derythro -MAPP, a known ceramidase inhibitor, repression of FET3 was relieved (Figure 3-33). Dose response assays were done with Derythro-MAPP to confirm this effect on PAQR1 (Figure 3-34) and PAQR2 (F igure 3-35). From this expe riment it cannot be concluded that the PAQRs are alkaline ceramidases, but it is preliminary evidence. To confirm this hypothesis, the PAQRs would have to be purif ied and the ceramidase activity be tested in vitro The problem with this approach is th at the expression levels obtained in S. cerevisiae are not high enough for the proteins to be purified. And even if they were, because these proteins have never been purified before, it would be very challenging to reconstitute these proteins and determine if they have folded prope rly. So this option was not followed. Fluorescence Studies The localization of the h uman class I PAQRs in Saccharomyces cerevisiae was also studied. First the N-terminal GFP la beled proteins were analyzed and -galactosidase assays were performed to see if these prot eins were functionally expressed. The -galactosidase assay was done in 2% galactose, and overexpression of all four proteins caused repression of FET3 except for PAQR2 that required adiponectin for activation (Figure 3-36). The assay was also performed at 0.05% galactose, and all f our proteins, except for PAQR4, repressed FET3 by activation with adiponectin (Figure 3-37).

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64 Then expression of these proteins was checked by western blot using anti-GFP antibodies (Figure 3-38). We saw expression of all human class I proteins except for PAQR2. As loading control the membrane was stripp ed from the anti-GFP antibodies and was probed against porin. Porin was present in all the lanes, meaning that equal amounts of protein had been loaded in each well (Figure 3-38). In this western blot, the memb rane fractions were analyzed and there were no bands seen for the PAQR2 sample. The soluble frac tions were also analy zed by western blot, and a band at 29 KDa was seen for the PAQR2 sample (Figure 3-39). This band corresponds to the soluble GFP protein. So, a possible explanation for this is that the N-terminal GFP tag from the GFP-PAQR2 construct is cleaved and only a band for GFP is seen on the western blot (Figure 339). The fluorescence of these proteins was then analyzed under a fluorescence microscope and the pictures obtained ar e shown in Figure 3-40. PAQR1 is localized to the plasma membrane, as it had been reported in mammalian cells (Yamauchi et al ., 2003). PAQR3 and PAQR4 were also localized to the plasma membrane. This is the fi rst time the localization of these proteins has been reported. PAQR2 on the other hand, seemed to be localized to the cytosol, but the fluorescence obtained for PAQR2 corresponds to the GFP tag and not PAQR2. The explanation for this is that there was no band seen for PAQR2 on the membrane fraction, and only a band on the soluble fraction corresponding to GFP (Figur e 3-39). Also, the fluorescence obtained for PAQR2 is exactly the same as the one obtaine d for the empty vector pGREG575 (Figure 3-41). The empty vector only expresses the GFP tag, and the fluorescence seen in the cytosol is due to the soluble GFP protein (Figure 3-41). The C-terminal GFP tagged proteins were then analyzed. First -galactosidase assays were performed to see if these proteins were func tionally expressed. In 2% galactose overexpression

PAGE 65

65 of all four proteins caused repression of FET3 except for PAQR2 that required adiponectin for activation (Figure 3-42). And in 0.05% galactose all four protei ns, except for PAQR4, repressed FET3 by activation with adipone ctin (Figure 3-43). Expression of these proteins was then analyzed by western blot and no bands were seen for any of the proteins with anti-GFP antibodies (Figure 3-44). The proteins are still expressed, because they repress FET3 by either overexpression or activation, but we cannot detect them with anti-GFP antibodies because the GFP tag might be degraded. Again as a loading control, the membrane was stripped from the anti-GFP and wa s probed against porin. Po rin was present in all the lanes, meaning that equal amounts of total protein had been loaded in each well (Figure 344). The western blot of the soluble fractions was also run to ch eck if the PAQR-GFP constructs were present in the supernatants after ultracen trifugation. Again, no protein was detected with anti-GFP antibodies (Figure 3-45). If the GFP tag had been expr essed and was later cleaved, a band for GFP would have been seen in the western blot of the soluble fr actions, but it was not, meaning that the GFP tag was probably degraded. The proteins were then observed under a fl uorescence microscope and none of the PAQRGFP constructs fluoresced (Figure 3-46). Again, th is correlates with what was seen in the western blot. If the GFP tag is degraded, the PAQR-GFP constructs would not be expected to fluoresce. It is known that the size of the tag can sometimes affect the stability of the protein, and the bigger the tag, the more unsta ble the construct. Maybe for th ese proteins, a large GFP tag on the C-terminus can affect folding and the tag mi ght be removed to allow proper folding. Another possibility could be that when the GFP tag is extracellular, as it is the case of these constructs, it would not fluoresce. The lack of fluorescence for an extracellular GFP tag has been reported in Escherichia coli (Daley et al ., 2005).

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66 Adiponectin Binding To dem onstrate binding of PA QR3 to adiponectin and further prove that PAQR3 is a new adiponectin receptor, flow cytometry with FAM labeled adiponectin was used. Whole cells were first used in this experiment, but there was no difference in binding between the cells overexpressing these proteins and the empty vector (Figure 3-47). A reason for this might have been that the cell wall prevented the fluorescentl y labeled adiponectin from reaching the plasma membrane and binding to the receptors. So yeast cells lacking their cell wall, also known as spheroplasts, were used instead. But again, no difference in binding was obtained for cells with the empty vector and overexpresso rs. Different concentrations of adiponectin were used, from as low as 10 pM to as high as 20 nM. Binding should have been seen at around 1nM, which is the reported Kd for the human adiponectin receptors (Yamauchi et al ., 2003). A possible problem could have been that long incubations with Zymolyase or Lyticase could have degraded the membrane proteins. To account for this, the amount of enzyme and incubation times was decreased, but still no difference in binding was s een. The binding assay was also performed in a strain lacking the four IZH genes to reduce non-specific binding, although adiponectin has been shown to have no effect on the Izhps (Figure 3-48 ), and again no difference in binding was seen. Because we could not measure binding to Saccharomyces cerevisiae, a different model system was developed for the next chapter.

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67 Table 3-1. Number of rare c odons of human PAQR genes in Saccharomyces cerevisiae These were calculated with Gene Designer with a threshold of 5% (Villalobos et al ., 2006). PAQR gene Rare codons PAQR1 7 PAQR2 6 PAQR3 3 PAQR4 6 PAQR5 2 PAQR6 4 PAQR7 4 PAQR8 5 PAQR9 5 PAQR10 4 PAQR11 4

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68 VectorIzh1pIzh2pIzh3pIzh4p -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 1 M Fe3+ 1mM Fe3+ Figure 3-1. Repression of FET3 by overexpression of Izh1-4p in S. cerevisiae. In low iron, 1 M Fe3+, overexpression of the Izh pr oteins causes repression of FET3 whereas in high iron, 1 mM Fe3+, both the empty vector and the overexpressors repress FET3 This was first shown by Brian Kupchak.

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69 VectorIzh1pIzh2pIzh3pIzh4p -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 700 Figure 3-2. Loss of repression of FET3 by Izhp expression in 0.05% ga lactose. This assay was done in low iron (1 M Fe3+). VectorIzh1pIzh2pIzh3pIzh4p -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 700 Untreated 1 M Thaumatin Figure 3-3. Repression of FET3 by Izh2p when 1 M thaumatin is added.

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70 Vector -Galactosidase Acti vity (Miller Units) 0 100 200 300 400 500 600 700 Untreated 10 M Thaumatin Figure 3-4. Repression of FET3 in cells carrying vector control at 10 M thaumatin. Thaumatin activates Izh2p produced fr om the genomic copy of IZH2 Vector -Galactosidase Activity (Miller Units) 0 200 400 600 800 Untreated 100 M Thaumatin Figure 3-5. Repression of FET3 in cells lacking all four IZH genes ( IZH1-4 ) at 100 M thaumatin.

PAGE 71

71 VectorPAQR1PAQR2 -Galactosidase Activity (Miller Units) 0 100 200 300 400 Figure 3-6. Repression of FET3 by overexpression of PAQR1. PAQR2 does not repress FET3 VectorPAQR1PAQR2 -Galactosidase Activity (Miller Units) 0 100 200 300 400 Untreated 100pM AdipoQ Figure 3-7. Repression of FET3 by PAQR2 upon addition of adiponectin.

PAGE 72

72 [Adiponectin] pM 1 10 100 1000 10000 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 700 PAQR2 Vector Figure 3-8. Dose response assay with adiponectin. As the concentr ation of adiponectin increases in the media the repression of FET3 by PAQR2 increases. -HA -porin Figure 3-9. Expression of PAQR1 and PAQR2 in Saccharomyces cerevisiae. Lane 1 Vector; Lane 2 PAQR1; Lane 3 PAQR2. The exp ected molecular weights for the proteins are 51.5 KDa for PAQR1, 52.8 KDa for PAQR2. In the first western blot, the membrane was probed against the HA tag. The membrane was then stripped and probed against porin. The second western blot shows that porin was present in all three lanes, confirming that equal amounts of total protein had been loaded in each well.

PAGE 73

73 % Galactose 0.01 0.1 1 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 PAQR1 Vector Figure 3-10. Repression of FET3 by PAQR1 dependent on the concentration of galactose. Vector PAQR1 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 700 Untreated 100pM AdipoQ Figure 3-11. Repression of FET3 by PAQR1 upon addition of adiponectin. This assay was done in 0.05% galactose.

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74 [Adiponectin] pM 1 10 100100010000 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 PAQR1 Vector Figure 3-12. Dose response assay of PAQR1 with adiponectin. As the concentration of adiponectin increases in th e media the repression of FET3 by PAQR1 increases. Vector PAQR1 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 Untreated 100 pM AdipoQ 3 nM C1q Figure 3-13. Activation of PAQR1 by C1q. The con centration required for activation with C1q is much higher than the one required with adiponectin.

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75 [C1q] nM 024681 01 2 -Galactosidase Assay (Miller Units) 100 150 200 250 300 350 400 Vector PAQR1 Figure 3-14. Dose response assay of PAQR1 to C1q. Vector PAQR1 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 700 Untreated 500 nM TNF Figure 3-15. Inhibiti on of PAQR1 by TNF. This was first shown by Brian Kupchak.

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76 VectorPAQR3PAQR4 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 Figure 3-16. Repression of FET3 by overexpression of PAQR3 and PAQR4. -HA -porin Figure 3-17. Expression of human class I PAQRs in Saccharomyces cerevisae. Lane 1 Vector; Lane 2 PAQR1; Lane 3 PAQR2; Lane 4 PAQR3; Lane 5 PAQR4. The expected molecular weights are 45.0 KDa for PAQR 3 and 38.0 KDa for PAQR4. In the first western blot, the membrane was probed against the HA tag. The membrane was then stripped and probed against porin. The s econd western blot shows that porin was present all lanes, confirming that equal am ounts of total protein had been loaded in each well.

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77 % Galactose 0.01 0.1 1 -Galactosidase Activity (Miller Units) 0 200 400 600 800 1000 PAQR3 Vector Figure 3-18. Repression of FET3 by PAQR3 dependent on the concentration of galactose. % Galactose 0.01 0.1 1 -Galactosidase Assay (Miller Units) 0 100 200 300 400 500 600 700 800 PAQR4 Vector Figure 3-19. Repression of FET3 by PAQR4 dependent on the concentration of galactose.

PAGE 78

78 VectorPAQR3PAQR4 -Galactosidase Assay (Miller Units) 0 100 200 300 400 500 600 Figure 3-20. Loss of repression of FET3 by PAQR3 and PAQR4 at 0.05% galactose. VectorPAQR3PAQR4 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 Untreated 100pM AdipoQ Figure 3-21. Activation of PAQR3 by adiponect in. PAQR4 did not respond to adiponectin. Assay done at 0.05% galactose.

PAGE 79

79 [Adiponectin] pM 1 10 100 1000 10000 Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 700 PAQR3 Vector Figure 3-22. Dose response assay of PAQR3 with adiponectin. As the concentration of adiponectin increases in th e media the repression of FET3 by PAQR3 increases. [Adiponectin] pM 1 10 100 1000 10000 -Galactosidase Activity (Miller Units) 0 200 400 600 800 1000 PAQR4 Vector Figure 3-23. Dose response assay of PAQR4 with adiponectin. PAQR4 does not repress FET3 even when the concentration of ad iponectin is increased to 10000 pM.

PAGE 80

80 Figure 3-24. Dose response with adi ponectin of human class I PAQRs in S. cerevisae PAQR3 requires higher concentrations of adipon ectin than PAQR1 and PAQR2 to repress FET3 PAQR4 does not repress FET3 with any of the concentrations of adiponectin tested. Table 3-2. EC50 values for PAQR1, PAQR2 and PAQR3 with adiponectin in Saccharomyces cerevisiae. PAQR EC50 (pM) R2 PAQR1 0.686 0.997 PAQR2 2.377 0.997 PAQR3 162 0.995

PAGE 81

81 VectorPAQR7PAQR2 -Galactosidase Assay (Miller Units) 0 200 400 600 800 1000 Untreated 100 pM AdipoQ 100 nM Progesterone Figure 3-25. Repression of FET3 by PAQR7 and PAQR2 by add ition of progesterone and adiponectin respectively. Adiponectin ha s no effect on PAQR7 and progesterone has no effect on PAQR2. Assay done at 2% galactose. VectorPAQR5PAQR8PAQR1 -Galactosidase Assay (Miller Units) 0 200 400 600 800 1000 Untreated 100 pM AdipoQ 100 nM Progesterone Figure 3-26. Repression of FET3 by PAQR5, PAQR8 and PAQR1 by addition of progesterone and adiponectin respectively. Adiponectin has no effect on PAQR5 and PAQR8 and progesterone has no effect on PAQR 1. Assay done at 0.05% galactose.

PAGE 82

82 VectorPAQR3PAQR4 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 700 Untreated 100nM Progesterone Figure 3-27. No repression of FET3 by PAQR3 and PAQR4 by addi tion of progesterone. Assay done at 0.05% galactose. [Progesterone] nM 0.1 1 10 100 100010000 -Galactosidase Activity (Miller Units) 0 200 400 600 Vector PAQR4 Figure 3-28. Dose response of PAQR4 with proge sterone. PAQR4 did not respond to any of the concentrations of progesterone test ed. Assay done at 0.05% galactose.

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83 VectorPAQR1PAQR2PAQR3PAQR4 -Galactosidase Activity (Miller Units) 0 200 400 600 800 1000 1200 WT tpk2 Figure 3-29. Loss of repression of FET3 in tpk2 strain by overexpressi on of human class I PAQRs. Assay done at 2% galactose. VectorPAQR1PAQR2PAQR3PAQR4 -Galactosidase Activity (Miller Units) 0 200 400 600 800 WT + 100pM AdipoQ tpk2 + 100pM AdipoQ Figure 3-30. Loss of repression of FET3 in tpk2 strain by activation with adiponectin of human class I PAQRs. Assay done at 0.05% galactose.

PAGE 84

84 VectorPAQR1PAQR2PAQR3PAQR4 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 700 WT snf4 Figure 3-31. Loss of repression of FET3 in snf4 strain by overexpressi on of human class I PAQRs. Assay done at 2% galactose. VectorPAQR1PAQR2PAQR3PAQR4 -Galactosidase Assay (Miller Units) 0 200 400 600 800 1000 WT + 100pM AdipoQ snf4 + 100pM AdipoQ Figure 3-32. Loss of repression of FET3 in snf4 strain by activation with adiponectin of human class I PAQRs. Assay done at 0.05% galactose.

PAGE 85

85 VectorPAQR1PAQR2Ypc1p -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 700 Untreated 100 pM AdipoQ 200 nM Derythro -MAPP 200 nM Derythro -MAPP + 100 pM AdipoQ Figure 3-33. Loss of repression of FET3 by PAQR1, PAQR2 and Ypc1p by addition of Derythro -MAPP.

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86 [D-erythro -MAPP] nM 0.1 1 10 100 -Galactosidase Assay (Miller Units) 100 200 300 400 500 600 700 800 Vector + 100 pM AdipoQ PAQR1 + 100 pM AdipoQ Figure 3-34. Dose response assay of PAQR1 with Derythro -MAPP. Repression of FET3 by PAQR1 activation by adiponectin is lost as the concentration of D-erythro-MAPP increases. Assay done at 0.05% galactose. [Derythro -MAPP] nM 0.1 1 10 100 -Galactosidase Activity (Miller Units) 50 100 150 200 250 300 350 400 450 Vector + 100pM AdipoQ PAQR2 + 100pM AdipoQ Figure 3-35. Dose response assay of PAQR2 with Derythro -MAPP. Repression of FET3 by PAQR2 activation by adiponectin is lost as the concentration of D-erythro-MAPP increases. Assay done at 0.05% galactose.

PAGE 87

87 VectorPAQR1PAQR2PAQR3PAQR4 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 Untreated 100 pM AdipoQ Figure 3-36. Repression of FET3 by N-terminal GFP tagged huma n class I PAQRs. All four proteins are functionally expressed. Assay performed at 2% galactose. VectorPAQR1PAQR2PAQR3PAQR4 -Galactosidase Activity (Miller Units) 0 200 400 600 800 Untreated 100pM AdipoQ Figure 3-37. Repression of FET3 by activation with adiponectin of the N-terminal GFP tagged human class I PAQRs. All four proteins ar e activated with adiponectin except for PAQR4. Assay done at 0.05% galactose.

PAGE 88

88 -GFP -porin Figure 3-38. Western blot of th e expression of N-terminal GFP tagged human class I PAQRs in S. cerevisiae Lane 1 PAQR4; Lane 2 PAQR 3; Lane 3 PAQR2; Lane 4 PAQR1; Lane 5 Vector. PAQR1, PAQR 3 and PAQR4 are expressed but not PAQR2. The expected molecular weight s are 71.9 KDa for PAQR1, 73.2 KDa for PAQR2, 65.5 KDa for PAQR3 and 58.5 KDa fo r PAQR4. The sizes of the proteins are lower than expected. In the first west ern blot the membrane was probed against GFP. The membrane was then stripped a nd probed against porin. The second western blot shows that porin was present all lane s, confirming that equal amounts of total protein had been loaded in each well. Figure 3-39. Western blot of the soluble fract ion of N-terminal GFP tagged PAQR2 sample probed with anti-GFP antibodies. A band co rresponding to GFP (29 KDa) is present in the soluble fraction of the PAQR2 samp le. The N-terminal GFP tag of PAQR2 is cleaved and that is why no band is presen t in the western blot for the membrane fraction, but there is a band present in the soluble fraction corresponding to the soluble GFP protein.

PAGE 89

89 PAQR1 PAQR2 PAQR3 PAQR4 Figure 3-40. GFP fluorescence of human class I PAQRs in S. cerevisiae. PAQR1, PAQR3, and PAQR4 are localized to the plasma membra ne. The fluorescence seen for PAQR2 is localized to the cytosol. This different lo calization for PAQR2 is due to GFP tag. The GFP tag is cleaved and the fluorescence obs erved is due to the soluble GFP protein, which is localized to the cytosol. These images were obtained in collaboration with Jessica Smith. Figure 3-41. GFP fluorescence of empty vector pGREG575 in S. cerevisiae. In the empty vector only the GFP tag is expressed and there is fluorescence observed in the cytosol. The sample fluorescence was observed for the PAQR2 sample (Figure 3-39).

PAGE 90

90 VectorPAQR1PAQR2PAQR3PAQR4 -Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 Untreated 100 pM AdipoQ Figure 3-42. Repression of FET3 by C-terminal GFP tagged huma n class I PAQRs. All four proteins are functionally expresse d. Assay done at 2% galactose. VectorPAQR1PAQR2PAQR3PAQR4 -Galactosidase Assay (Miller Units) 0 100 200 300 400 500 600 700 Untreated 100 pM AdipoQ Figure 3-43. Repression of FET3 by activation with adiponectin of the C-terminal GFP tagged human class I PAQRs. All four proteins ar e activated with adiponectin except for PAQR4. Assay done at 0.05% galactose.

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91 -GFP -porin Figure 3-44. Western blot for th e expression of C-terminal GFP tagged human class I PAQRs in S. cerevisiae. Lane 1 Vector; Lane 2 PAQR1; Lane 3 PAQR2; Lane 4 PAQR3; Lane 5 PAQR4. None of the C-te rminal GFP constructs were expressed. The expected molecular weights are 71.9 KDa for PAQR1, 73.2 KDa for PAQR2, 65.5 KDa for PAQR3 and 58.5 KDa for PAQR 4. In the first western blot the membrane was probed against GFP. The me mbrane was then stripped and probed against porin. The second western blot s hows that porin was present all lanes, confirming that equal amounts of total protein had been loaded in each well. Figure 3-45. Western blot of the soluble fract ion of C-terminal GFP tagged human class I PAQRs. No band for GFP was seen this tim e, meaning that the GFP tag might be degraded. Lane 1 Vector; Lane 2 PAQR 1; Lane 3 PAQR2; Lane 4 PAQR3; Lane 5 PAQR4.

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92 PAQR1 PAQR2 PAQR3 PAQR4 Figure 3-46. Fluorescence of C-terminal GFP tagged human class I PAQRs in S. cerevisiae. There was no fluorescence observed for any of the proteins, meaning that GFP is not expressed. The cloning for PAQR2 into pGREG600 was performed by Jessica Smith.

PAGE 93

93 VectorPAQR1PAQR2PAQR3PAQR4 % FAM-Adiponectin bound to cells 0 2 4 6 8 10 Figure 3-47. Binding of whole cells to FAM-labe led adiponectin. Assay done with 1 nM FAMAdiponectin. No significant binding was observed for a ny of the PAQR proteins. VectorIzh1pIzh2pIzh3pIzh4p Galactosidase Activity (Miller Units) 0 100 200 300 400 500 600 Untreated 100pM AdipoQ Figure 3-48. Effect of adiponect in on Izhps. Adiponectin does not activate any of the Izh proteins. Assay done at 0.05% galactose.

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94 CHAPTER 4 EXPRESSION AND CHARACTERIZATION OF THE HUMAN CLASS I PAQR PROTEINS IN SF9 INSECT CEL LS Introduction The hum an class I PAQR proteins were heterologously expressed in Sf9 insect cells to demonstrate binding of these recepto rs to adiponectin and validate the finding that PAQR3 is a new adiponectin receptor. In this organism, th ere are no rare codons for the human PAQR genes (Table 4-1). They were used in stead of mammalian cells, because Sf9 cells do not have adiponectin, so it is easier to study bindi ng to adiponectin in this organism. Sf9 cells derive from the parent strain Sf21, officially called IPLB-Sf21AE (Vaughn et al ., 1977). This cell line was developed fr om ovaries of the fall armyworm, Spodoptera frugiperda a moth species that is a pest on co rn and other grass species (Vaughn et al ., 1977). Both Sf9 and Sf21 cells have been extensively used for pr oducing recombinant proteins. These expression systems are better than Saccharomyces cerevisiae for producing human proteins because their posttranslational modifications are more similar to the ones in the mammalian cells. Several human membrane proteins have be en heterologously expressed in Sf9 insect cells: the human histamine H2-receptor (Houston et al ., 2002), the human opioi d receptor (Wei et al ., 2000), the human substance P receptor (Nishimura et al ., 1998), and the human M2 muscarinic receptor (Heitz et al ., 1995), among others. This project started from the collaborati on with Dr. Chasta Parker, from Winthrop University in South Carolina. Dr. Parker had previously expressed the human adiponectin receptors PAQR1 and PAQR2 in Sf9 cells. The proteins had a Cterminal 6x His tagged and expression was verified by western blot. The plasmid used for expressions was pIEX-4 [Novagen]. This plasmid drives gene expressi on through a combination of two transcription elements: the hr5 enhancer and the IE1 immediate early promoter (Rodems and Friesen, 1993;

PAGE 95

95 Jarvis et al ., 1996). This enhancerpromoter combinat ion allows the recruitment of endogenous insect cell transcription machinery, and avoids the use of baculovirus. This is really convenient because the time involved in crea ting, titering and maintaining th e viral recombinants is saved. With the pIEX-4 plasmid, recombinant protein is produced in as little as two days. Materials and Methods Cloning of Human Class I PAQR Proteins The pIEX-4 plasm ids encoding for PAQR1 a nd PAQR2 were gener ously provided by Dr. Parker. These clones contain a C-terminal 6 x His tag that allows for detection and purification. PCR was used to clone out the coding regions of PAQR3 and PAQR4 from their cDNA [Table 2-1]. The primers used are listed in Appendix C. The PCR products and pIEX-4 plasmid were restricted with NcoI and NotI [New England Biolabs] and ligated with T4 DNA ligase. These clones were later veri fied by DNA sequencing. Growth of Sf9 Insect Cells Sf9 insect cells [Novagen #71104-3] were grow n in serum -free BacVector Insect Cell Medium [Novagen #70590-3] in a T75-flask [Fis her] at 28C until the monolayer was 85-90% confluent. Once confluency was reached, cells we re passaged into 2 T75flasks by detaching the cells from the bottom of the flask and transferring half of the cells to a new flask and leaving the other half in the same flask. Cells were grown again until a confluency of 85-90% was reached. The cells were then detached from the two flas ks using 10 mL of fresh medium and transferred to a sterile 125 mL Erlenmeyer flask. Fresh medi um was added to a final volume of 25 mL and the cells were brought into suspension by growi ng the culture at 28C at 150 rpm. They were grown until a density of 2-3 x 106 cells/mL was reached.

PAGE 96

96 Transfection of Sf9 Insect Cells Once the ab ove density was reached, 1 x 107 cells were transferred to another sterile 125 mL Erlenmeyer flask. In a sterile tube, 20 g of plasmid containing th e human PAQR genes was diluted in 1 mL of serum-free me dium. In another sterile tube, 100 L of Insect GeneJuice Transfection Reagent [Novagen # 71259-4] was diluted in 1 mL of serum-free medium. The diluted DNA was then added dropwise to the t ube containing the Insect GeneJuice and it was incubated for 15 minutes at room temperature. The transfection mixture was then added to the Erlenmeyer flask that containe d the cells and they were inc ubated at 28 C for 48 hours with shaking at 150 rpm. Cell Extract Preparation and Western Blot Once transfection ended, 0.5 m L of Insect PopCulture Transfection Reagent [Novagen #71187-3] was added to the culture and incubated at room temperature for 15 min. The protein concentrations of all samples were calculated and equal amounts of total protein loaded on an SDS-PAGE. The proteins were th en transferred onto a nitrocellulose membrane and blocked overnight in 1% BSA in TBST. The membrane wa s washed twice with TBST and incubated at room temperature for 1 hour with HisProbeHRP [Pierce #15165] at a ratio of 1:5000 in TBST. The membrane was washed four times with TBST incubated with chemiluminescent substrate. Finally the x-ray f ilm was exposed and developed. Adiponectin Binding Transfected Sf9 cells were incubated with different concentrations of FAM-adiponectin for 1 hour at 4C. The cells were then run on a Flow Cytom etry Instrume nt (FACS Vantage SE Turbosort) [BD Biosciences].

PAGE 97

97 Results and Discussion Expression of PAQRs in Sf9 cells Out of the four hum an class I PAQR proteins, only the expression for PAQR1 was detected (Figure 4-1).This was a major problem in the quest of identifying PAQR3 as a new adiponectin receptor. Without de tectable expression, binding to adiponectin was not guaranteed to work. Adiponectin binding Binding was perform ed the same way as in Saccharomyces cerevisiae but this time without the need for prepar ing spheroplasts because Sf9 cells do not have cell wall. This is a clear advantage over yeast cells because these steps could damage the membrane proteins. After transfection, the density of the suspension cultures was calcu lated and equal number of cells was incubated with different concentrations of FAM-adiponectin at 4C for 1 hour. Binding was observed for PAQR1 (Figure 42), but not for PAQR2, PAQR3 or PAQR4 (Figure 4-3), which was expected from the undetect able expression levels of these proteins. If no binding had been seen for any of the proteins, a different binding assay could have been used. Kadowaki and colleagues reported binding of PA QR1 and PAQR2 to adiponectin in mammalian cells using 125I labeled adiponectin (Yamauchi et al ., 2003). In this assay, monolayer cultures were incubated with 125I-labeled adiponectin for 1 hour at 4 C and washed twice with cold PBS to remove any unbound adiponectin. The cells were then harvested and the radioactivity was read in a scintillation counter. This method was not followed in the first place because 125I is a gamma emitter and requires great precautions to work with.

PAGE 98

98 Table 4-1. Number of rare codons of the human PAQR genes in Sf9 These were calculated with Gene Designer with a thre shold of 5% (Villalobos et al ., 2006). PAQR gene Rare codons PAQR1 0 PAQR2 0 PAQR3 0 PAQR4 0 PAQR5 0 PAQR6 0 PAQR7 0 PAQR8 0 PAQR9 0 PAQR10 0 PAQR11 0 Figure 4-1. Western blot of expression of Human Class I PAQRs in Sf9 insect cells. Lane 1 PAQR4; Lane 2 PAQR3; Lane 3 PAQR 2; Lane 4 PAQR1; Lane 5 Vector. Only PAQR1 was expressed. The expected molecular weights are 41.4 KDa for PAQR1, 42.8 KDa for PAQR2, 35.0 KDa for PAQR3 and 28.0 KDa for PAQR4. The proteins had C-terminal Hi s tags and were detected with HisProbe-HRP [Pierce #15165].

PAGE 99

99 VectorAdipoR1 % Cells bound to FAM-Adiponectin 0 20 40 60 80 100 Figure 4-2. Binding of PAQR1 to FAM-Adiponectin in Sf9 cells. Assay done with 1nM FAMAdiponectin. Binding was observed for PAQR1. VectorPAQR2PAQR3PAQR4 % Cells bound to FAM-Adiponectin 0.0 0.5 1.0 1.5 2.0 2.5 Figure 4-3. Binding of PAQR2, PAQR3 a nd PAQR4 to FAM-Adiponectin in Sf9 cells. Assay done with 1 nM FAM-Adiponectin. No binding was observed for any of these proteins.

PAGE 100

100 CHAPTER 5 CONCLUSIONS In an effort to prove the ceram idase activ ity of the human PAQRs in vitro the expression of these proteins was first attempted in Escherichia coli, because it is an organism known to provide high enough expression leve ls for later purification. Unfortunately, we could not detect expression of these proteins and their purification was unsuccessful (Figure 2-3). If these proteins had been highly expressed, purified and properly reconstituted, ceramidase assays could have been tested in vitro with pure protein. Instead ceramidase assays were tested with whole cell lysates and no ceramidase activity was obser ved (Figure 2-4). Undetected ceramidase activity could have been due to the use of inappr opriate substrate, assay conditions, or detection systems. Other possible explanations could be that the human PAQRs are not alkaline ceramidases or that the expression levels of these proteins in E. coli are not high enough for the ceramidase activity to be detected. These low expression levels in Escherichia coli prompted us to switch to a different organism, the yeast Saccharomyces cerevisiae In S. cerevisiae the adiponectin receptors were shown to be functional without the presence of other human proteins (Figure 3-7). Expression was detected for PAQR1, but not for PAQR2 (Figur e 3-9). A possible explanation for this would be that the N-terminal HA tag of PAQR2 is cleaved, so PAQR2 cannot be detected with anti-HA antibodies. PAQR2 is clear ly being expressed in S. cerevisiae because it responds to adiponectin and causes repression of FET3 (Figure 3-8). Purification of these proteins was not attempte d because of the low levels of expression obtained, only detectable by we stern blot. Instead the furthe r characterization of human adiponectin receptors was performed in vivo The study in vivo led to the discovery of new possible inhibitors and activator s of the human adiponectin receptors. The complement factor

PAGE 101

101 C1q was found to activate PAQR1 (Fi gure 3-13). On the other hand, TNFwas shown to inactivate the repression of FET3 caused by PAQR1 (Figure 3-15). Th is assay is in the process of being patented because it can be used in the design of antidiabetic a nd anti-atherogenic drugs. Through the study of the other members of the human class I PAQR family in Saccharomyces cerevisiae, it was found that PAQR3 is also activated by adiponectin (Figure 321). To confirm this result a dose response a ssay was done with adiponectin, and as the concentration of adiponectin incr eases, so does the repression of FET3 by PAQR3 (Figure 3-22). This could implicate that PAQR3 is a new adipon ectin receptor. This could also have great implications in the fight against Type 2 diabetes and cardiovascular diseases. PAQR4 on the other hand, showed no response to adiponectin (Figure 3-23). When all four human class I PAQRs are compared in the same graph, we can see that PAQR3 requires higher concentrations of adiponectin than PAQR1 and PAQR2 to repress FET3 (Figure 3-24). The reported EC50 of these proteins in S. cerevisiae is 0.686 pM for PAQR1, 2.377 pM for PAQR2 and 162 pM for PAQR3 (Table 3-2). To further validate that the effect seen with adiponectin is not due to overexpression of any membrane protein, the human membrane progestin receptors were expressed in S. cerevisiae. They were tested with both adiponectin a nd their natural ligand progesterone. They were activated by progesterone but show ed no response to adiponectin (Figures 3-25 and 3-26). This clearly indicates that adiponectin has no effect on any membrane receptor and these receptors only respond to their ligand. The localization of the human class I PAQRs was also studied in Saccharomyces cerevisiae It was found that PAQR1, PAQR3 and PAQR4 are localized to the plasma membrane (Figure 3-40). Unfortunately the localization of PAQR2 could not be determined because the

PAGE 102

102 GFP tag from the GFP-PAQR2 construct was cl eaved. The fluorescence seen for PAQR2, which was localized to the cytosol, corresponded to the soluble GFP protein (Figure 3-41). To further prove that PAQR3 is a new adipon ectin receptor, binding of these proteins to adiponectin was studied in S. cerevisiae Both whole yeast cells and spheroplasts, cells lacking their cell wall, were used in these assays. Different concentrations of adiponectin and assay conditions were tried, but in all cases no diffe rence in binding was detected for the cells expressing the empty vector or the overexpressors (Figure 3-47). In order to show binding of these proteins to adiponectin, the human class I PAQRs were expressed in Sf9 insect cells. Unfortunately binding of PAQR3 to adiponectin could not be detected (Figure 4-3), which could have been due to low expression levels (Figure 4-1). Future directions of this project would be proving bindi ng of PAQR3 to adiponectin, through a different assay or expression system. Als o, an important future experiment would be to prove that the results obtained in Saccharomyces cerevisiae can be reproduced in human cells, further proving that PAQR3 is a new adiponectin receptor. Although not being the original goal of this project, the st udy of the human class I PAQRs led to the discovery of a new possible adiponectin receptor. This can give further insight into the physiology of adiponectin and expand our knowledge in the quest of comba ting type II diabetes and cardiovascular diseases.

PAGE 103

103 APPENDIX A YEAST STRAINS Table 6-1. S trains used in this study. Strain Mutation Source Genotype BY4742 Wild type EUROSCARF MAT ; his3 ; leu2 ; ura3 ; lys2 TLY23 izh1izh2izh3izh4 TLY22 sporulation MAT ; his3 ; leu2; ura3; lys2 ; izh1:: kanMX4; izh2:: hphMX4; izh3:: natMX4; izh4:: ura3MX4 YPL203W tpk2 EUROSCARF MAT ; his3 ; leu2 ; ura3; lys2 ; YPL203w ::kanMX4 YGL115W snf4 EUROSCARF MAT ; his3 ; leu2 ; ura3; lys2 ; YGL115w ::kanMX4

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104 APPENDIX B MEDIA COMPOSITION Yeast Pepto ne (YP) 10.0 g/L yeast extract 20.0 g/L peptone Synthetic Dextrose (SD) 1.7 g/L yeast nitrogen base without am ino acids or ammonium sulfate 5.0 g/L ammonium sulfate 2% w/v glucose Synthetic Galactose (SGal) 1.7 g/L yeast nitrogen base without am ino acids or ammonium sulfate 5.0 g/L ammonium sulfate 2% w/v galactose Low Iron Media (LIM) (1L) 1.7 g yeast nitrogen base without am ino acids or ammonium sulfate 5.0 g ammonium sulfate 2% w/v galactose 20 mL 1M sodium citrate, pH 4.2 0.2 mL 4 g/L zinc sulfate 0.2 mL 100 mM manganese chloride 2 mL 1 mM EDTA, pH 8.0

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105 APPENDIX C PRIMERS USED FOR CLONING pKM263/pKM260-PAQR1-5 5 GGT GGT GGC GAC CAT CAC GAG AAT CTT TAT TTT CAG GGC GAC ATG TCT TCC CAC AAA GGA TC 3 pKM263/pKM260-PAQR1-3 5 ATA TC T GCA GAA TTC CAG CAC ACT GGC GGC CGT TAC TAG TGG ATC CTC AGA GAA GGG TGT CAT CAG TAC 3 pKM263/pKM260-PAQR2-5 5 GGT GGT GGC GAC CAT CAC GAG AAT CTT TA T TTT CAG GGC GCC ATG GGC ATG TCC CCT CTC TT 3 pKM263/pKM260-PAQR2-3 5 ATA TCT GCA GAA TTC CAG CAC ACT GGC GGC CGT TAC TAG TAG ATC TTC ACA GTG CAT CCT CTT CAC TGC 3 pKM263/pKM260-PAQR3-5 5 GGT GGT GGC GAC CAT CAC GAG AAT CTT TA T TTT CAG GGC GCC ATG GAT CAG AAG CTG CTG AA 3 pKM263/pKM260-PAQR3-3 5 ATA TCT GCA GAA TTC CAG CAC ACT GGC GGC CGT TAC TAG TGG ATC CTC ACA AAT GTG AAA CAT AGT CAG 3 pKM263/pKM260-PAQR4-5 5 GGT GGT GGC GAC CAT CAC GAG AAT CTT TA T TTT CAG GGC GCC ATG GCG TTC CTG GCC GGG CC 3 pKM263/pKM260-PAQR4-3 5 ATA TCT GCA GAA TTC CAG CAC ACT GGC GGC CGT TAC TAG TGG ATC CTC AGT CCC GGG GAC AGG CGT GGT 3 pKM263/pKM260-PAQR5-5 5 GGT GGT GGC GAC CAT CAC GAG AAT CTT TA T TTT CAG GGC GCC ATG GTG AGC CTG AAG CTC CC 3 pKM263/pKM260-PAQR5-3 5 ATA TCT GCA GAA TTC CAG CAC ACT GGC GGC CGT TAC TAG TGG ATC CTC ATG TTT CTT TTT TAT GTA ATT 3 pKM263/pKM260-PAQR6-5 5 GGT GGT GGC GAC CAT CAC GAG AAT CTT TA T TTT CAG GGC GAC ATG TTC AGT CTC AAG CTG CC 3

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106 pKM263/pKM260-PAQR6-3 5 ATA TCT GCA GAA TTC CAG CAC ACT GGC GGC CGT TAC TAG TGG ATC CTT ACT GTT GTT TGG CCT GGG TAC 3 pKM263/pKM260-PAQR7-5 5 GGT GGT GGC GAC CAT CAC GAG AAT CTT TA T TTT CAG GGC GAC ATG TCC ATG GCC CAG AAA CT 3 pKM263/pKM260-PAQR7-3 5 ATA TCT GCA GAA TTC CAG CAC ACT GGC GGC CGT TAC TAG TGG ATC CTC ACT TGG TCT TC T GAT CAA GTT 3 pKM263/pKM260-PAQR8-5 5 GGT GGT GGC GAC CAT CAC GAG AAT CTT TA T TTT CAG GGC GAC ATG TCG ACC GCC ATC TTG GA 3 pKM263/pKM260-PAQR8-3 5 ATA TCT GCA GAA TTC CAG CAC ACT GGC GGC CGT TAC TAG TGG ATC CTC AGG AAT CTT TCT TGG TCA GTC 3 pKM263/pKM260-PAQR9-5 5 GGT GGT GGC GAC CAT CAC GAG AAT CTT TA T TTT CAG GGC GCC ATG GCG CGG CGC CTG CAG CC 3 pKM263/pKM260-PAQR9-3 5 ATA TCT GCA GAA TTC CAG CAC ACT GGC GGC CGT TAC TAG TGG ATC CTC ACT TTT TAC TGC AGA ATT CGG 3 pKM263/pKM260-PAQR10-5 5 GGT GGT GGC GAC CAT CAC GAG AAT CTT TA T TTT CAG GGC GCC ATG GTC GCC CCC CGG CTG CT 3 pKM263/pKM260-PAQR10-3 5 ATA TCT GCA GAA TTC CAG CAC ACT GGC GGC CGT TAC TAG TAG ATC TTC ATT TGG ACA CCT TGG TCT GCA 3 pKM263/pKM260-PAQR11-5 5 GGT GGT GGC GAC CAT CAC GAG AAT CTT TA T TTT CAG GGC GTC ATG AAC CAT CGA GCT CCA GC 3 pKM263/pKM260-PAQR11-3 5 ATA TCT GCA GAA TTC CAG CAC ACT GGC GGC CGT TAC TAG TGG ATC CTC ATA AAT GCC GCA TAA AGT CCG 3 pGREG536/pGREG575/pGREG600-PAQR1-5 5 GAA TTC GAT ATC AAG CTT ATC GA T ACC GTC GAC AAT GTC TTC CCA CAA AGG ATC 3

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107 pGREG536/pGREG575-PAQR1-3 5 GCG TGA CAT AAC T AA TTA CAT GAC TCG AG G TCG ACT CAG AGA AGG GTG TCA TCA G 3 pGREG600-PAQR1-3 5 GTT CTT CTC CTT TAC TCA TTC TCG AGG TCG ACG AGA AGG GTG TCA TCA GTA C 3 pGREG536/pGREG575/pGREG600-PAQR2-5 5 GAA TTC GAT ATC AAG CTT ATC GAT ACC GTC GAC AAT GAA CGA GCC AAC AGA AAA C 3 pGREG536/pGREG575-PAQR2-3 5 GCG TGA CAT AAC TAA TTA CAT GAC TCG AGG TCG ACT CAC AGT GCA TCC TCT TCA C 3 pGREG600-PAQR2-3 5 GTT CTT CTC CTT TAC TCA TTC TCG AGG TCG ACC AGT GCA TCC TCT TCA CTG C 3 pGREG536/pGREG575/pGREG600-PAQR3-5 5 GAA TTC GAT ATC AAG CTT ATC GA T ACC GTC GAC AAT GCA TCA GAA GCT GCT GAA 3 pGREG536/pGREG575-PAQR3-3 5 GCG TGA CAT AAC TAA TTA CAT GAC TCG AGG TCG ACT CAC AAA TGT GAA ACA TAG TCA 3 pGREG600-PAQR3-3 5 GTT CTT CTC CTT TAC TCA TTC TCG AGG TCG ACC AAA TGT GAA ACA TAG TCA GGA 3 pGREG536/pGREG575/pGREG600-PAQR4-5 5 GAA TTC GAT ATC AAG CTT ATC GA T ACC GTC GAC AAT GGC GTT CCT GGC CGG G 3 pGREG536/pGREG575-PAQR4-3 5 GCG TGA CAT AAC TAA TTA CAT GAC TCG AGG TCG ACT CAG TCC CGG GGA CAG GCG TG 3 pGREG600-PAQR4-3 5 GTT CTT CTC CTT TAC TCA TTC TCG AGG TCG ACG TCC CGG GGA CAG GCG T 3 pIEX-4-PAQR3-5 5 CCA AGT GAC CAT GCA TCA GAA GCT GCT GAA 3

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108 pIEX-4-PAQR3-3 5 AGA AGA TGC GGC CGC CAA AT G TGA AAC ATA GTC AGG 3 pIEX-4-PAQR4-5 5 CCA AGT GAC CAT GGC GTT CCT GGC CGG G 3 pIEX-4-PAQR4-3 5 AGA AGA TGC GGC CGC GTC CCG GGG ACA GGC GTG 3

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118 BIOGRAPHICAL SKETCH Ibon Garitaonandia was born in Bilbao, Spai n, on February 24, 1979. He grew up in San Sebastian, where he earned a B.S. in chem istry from the University of the Basque Country. In August of 2002, Ibon began pursuing his Ph.D. in chem istry at the University of Florida, where he joined the research group of Dr. Nicole Ho renstein. In May 2004, Ibon joined the group of Dr. Tom Lyons to work on the PAQR receptors. Upon completion of his program, he will continue doing research as a postdoc under Dr. Kathryn Cr ossin, in the Neurobiology Department at The Scripps Research Institute in San Diego.