A PROTEIN IN SEARCH OF FUNCTION: NIPSNAP1 IN MITOCHONDRIAL BRANCHED-CHAIN AMINO ACID METABOLON, BRAIN AND APOPTOSIS BY MANISHA NAUTIYAL A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY in the Molecular Medicine and Translational Science Program December 2008 Winston-Salem, North Carolina Approved by: Susan M. Hutson, Ph.D., Advisor ___________________________________ Examining Committee: Leslie B. Poole, Ph.D., Chair ___________________________________ R. Mark Payne, M.D. ___________________________________ Raymond Penn, Ph.D. ___________________________________ Reidar Wallin, Ph.D. ___________________________________ ACKNOWLEDGEMENTS I would like to acknowledge, my advisor, Dr. Susan Hutson, and my committee members, Dr. Leslie Poole, Dr. Mark Payne, Dr. Raymond Penn and Dr. Reidar Wallin, for their support and guidance during this research. I deeply appreciate the efforts of my committee on improving the quality of this work. I would like to thank all the members of the Hutson lab, past and present, for their assistance and friendship in the laboratory. In particular, I would like to thank Dr. Andrew Sweatt for his guidance and assistance with immunological studies of brain, and Dr. Mohammad Islam for assistance with the enzyme assays. I would also like to thank Dr. Nadeem Wajih for training me in the molecular biology skills that helped start the project. I would also like to thank the faculty and staff in the Departments of Biochemistry and Internal Medicine for providing the resources and supportive environment that has made my graduate career here so enjoyable. I am thankful to the Graduate School and Molecular Medicine program for giving me the opportunity to learn at Wake Forest University and I greatly appreciate the support network provided by my fellow graduate students, friends and colleagues. My deepest gratitude goes to my family: my parents, my brother and my in-laws for their constant love, support and encouragement, and to my husband, Sanjeev, for motivating me to fulfill my dream and for always being there for me. Thank you so much. ii TABLE OF CONTENTS LIST OF ILLUSTRATIONS ….………………………………………….………..…...v ABBREVIATIONS …………..……………………………………………..……...........ix ABSTRACT ….……………………………………………………………..…..………xii Chapter I. INTRODUCTION ….…………….…………………………………………. 1 II. METHODS ………………………………………………………………… 15 A. CLONING IN BACTERIAL AND MAMMALIAN EXPRESSION SYSTEMS …………………………………………………………..15 B. OVEREXPRESSION AND PURIFICATION ……………………...21 C. ANTI-NIPSNAP1 ANTIBODY PRODUCTION AND AFFINITY PURIFICATION ………………………………...…...……………...23 D. ANALYTICAL ULTRACENTRIFUGATION, CIRCULAR DICHROISM SPECTROSCOPY AND SULFHYDRYL GROUP TITRATION ………………...………………………………...…….25 E. ISOLATION OF MITOCHONDRIA, PREPARATION OF EXTRACTS, IMPORT ASSAYS AND MITOCHONDRIAL FRACTIONATION ...……………………………………………….27 F. AFFINITY CHROMATOGRAPHY ………………………………..32 G. IN VITRO ASSAYS FOR BCAA ENZYMES .……………………..34 H. BCAA OXIDATION ASSAY IN HEK 293 CELLS ………...……..37 I. IMMUNOHISTOCHEMISTRY .…......…………………………….38 J. CELL VIABILITY AND CASPASE-3 ACTIVITY ASSAYS ...……………………………………………………….….41 K. DT-DIAPHORASE ACTIVITY ASSAY …..…………………...…..42 iii III. RESULTS …………………...………………………………………………43 A. CHARACTERIZATION OF PURIFIED RECOMBINANT NIPSNAP1 AND ANTI-NIPSNAP1 ANTIBODIES………………..43 B. MITOCHONDRIAL IMPORT AND INTRAMITOCHONDRIAL LOCALIZATION OF NIPSNAP1 ………………………………….48 C. CHARACTERIZATION OF PROTEIN-PROTEIN INTERACTIONS OF NIPSNAP1 IN THE BCAA METABOLON .……………….….57 D. IMMUNOLOCALIZATION OF NIPSNAP1 IN BRAIN ……...…..61 E. ANALYSIS OF NIPSNAP1 LEVELS IN PHENYLKETONURIA MICE ........................................................................................…….70 F. NIPSNAP1 AND APOPTOSIS …………………………………….70 IV. DISCUSSION ……………………………………………………………....76 REFERENCES ……………………………………………......................................89 APPENDIX- STUDIES IN BCATm -/- KNOCKOUT (KO) MOUSE ……..…….100 SCHOLASTIC VITA ………………………………………………………….......110 iv LIST OF ILLUSTRATIONS CHAPTER I: INTRODUCTION Figure 1: Diagram of the first two steps in branched-chain amino acid (BCAA) metabolism ……………………………………………………….................2 Figure 2: The proposed glutamate-glutamine and BCAA shuttle …………………….5 Figure 3: NIPSNAP1 was found in a complex of BCAA metabolizing enzymes …….7 Figure 4: Tissue expression patterns of NIPSNAPs (from GeneCards) ………………9 Figure 5: Primary sequence alignment shows that NIPSNAP proteins are highly conserved through evolution ……………………………………………….10 CHAPTER II: METHODS Figure 6: cDNA sequence for rat NIPSNAP1 ……………………………………….16 Figure 7: The NIPSNAP1 fusion protein constructs for bacterial (A) and mammalian (B) expression systems …………………………………………………….17 Figure 8: cDNA sequence for mouse NIPSNAP2 …………………………………...18 Figure 9: cDNA sequence for mature NIPSNAP1 …………………………………..20 Figure 10: Western blot of nickel affinity chromatography purified recombinant NIPSNAP1 using anti-NIPSNAP1 polyclonal antibodies ………………..25 Figure 11: Measurement of respiratory control ratio (RCR) in isolated rat liver mitochondria ………………………………………………………………28 CHAPTER III: RESULTS Figure 12: PCR product of full length rat NIPSNAP1 ……………………………….43 Figure 13: Overexpression of NH -terminal histidine tagged recombinant rat 2 NIPSNAP1 (~30 kDa) using the dual media system from Zymo Research …………………………………………………………...44 v Figure 14: Amino acid sequence for mature rat NIPSNAP1 ……………….………...45 Figure 15: NIPSNAP1 is expressed in tissues throughout the body …………………45 Figure 16: NIPSNAP1 is a homotetramer with significant helical structure containing two free surface accessible sulfhydryl groups and shows normal sensitivity to urea denaturation ………………………………………………………..48 Figure 17: The mitochondrial targeting sequence of NIPSNAP1 lacks the traditional amphiphilic helical structure ………………………………………………50 Figure 18: The predicted cleavage sites for human NIPSNAP proteins ……………...51 Figure 19: Comparision of the first 60 amino acids between the NIPSNAP homologs (A) and orthologs (B) does not reveal significant conservation of amino acid residues in the import sequence …………………………………………..51 Figure 20: NIPSNAP1 is present in mitochondria ………………………………………52 Figure 21: NIPSNAP1 is imported into the mitochondria using the mitochondrial electropotential gradient ……………………………………………………53 Figure 22: NIPSNAP2, 3, and 4 are also mitochondrial proteins and their import is also dependent on the electropotential gradient ………………………………..54 Figure 23: Scheme showing sub-fractionation of rat liver mitochondria …………….55 Figure 24: Western blotting shows that a fraction of the NIPSNAP1 associates with the inner mitochondrial membrane …………………………………………..56 Figure 25: BCKDC subunits were retained by NIPSNAP1-specific antibodies coupled to Profound Immunoprecipitation coupling beads ………………………..57 Figure 26: NIPSNAP1 binds to the E2 subunit of the BCKDC and Pyruvate Dehydrogenase Complexes ……………………………………………….59 Table I: NIPSNAP1 did not affect the k values of overall BCKDC and individual cat component enzyme activities ……………………………………………...59 Figure 27: NIPSNAP1 message and protein expression in HEK 293 cells …………..60 Figure 28: NIPSNAP1 overexpression resulted in decrease in BCAA oxidation by 25% which increases to 50% upon a 15% increase in transfection efficiency ...61 Figure 29: NIPSNAP1 shows widespread neuronal expression in rat brain and spinal cord ………………………………………………………………………..63 vi Figure 30: NIPSNAP1 is found in mitochondria of neurons ………………….……..65 Figure 31: NIPSNAP1 is not found in astrocytes ……………………………………67 Figure 32: In the hippocampus, NIPSNAP1 primarily stains glutamatergic neurons with some staining of GABAergic neurons of the dentate gyrus ……..…..68 Figure 33: NIPSNAP1 is found in catecholaminergic neurons …………………..…..69 Figure 34: NIPSNAP1 protein levels are reduced in PKU ………………………..….71 Figure 35: Activation of apoptosis through mitochondrial pathway …………..……..72 Figure 36: NIPSNAP1, cytosolic mature lacking the mitochondrial import sequence (CM) and fifty amino acid deletions of the carboxyl-terminal amino acids of NIPSNAP1 (ΔC ) promote Bax-mediated cell death .…………………73 50 Figure 37: NIPSNAP1, CM and ΔC antagonize XIAP function independent of 50 caspase-3 activation ………………………………………………………75 CHAPTER IV: DISCUSSION Figure 38: Secondary structural analysis of rat NIPSNAP1 …………………………78 Table II: NIPSNAP1 does not affect liver DT-diaphorase (quinone reductase) activity …………………………………….………………………………79 Figure 39: Protein structure homology-modeling (SWISS-MODEL) identifies two separate domains in NIPSNAP1 ………….……………………………….80 Figure 40: PCR products of Fragments 1 & 2 of rat NIPSNAP1 …………………….81 Figure 41: Western blotting using anti-HIS antibodies show the expression (A) and purification (B) of Fragments 1 (~19 kDa) and 2 (~15 kDa) ……………..82 Figure 42: PCR product of full length rat NIPSNAP2 …………………………….....82 Figure 43: NIPSNAP1 and BCKDC are found in similar neuronal populations ….….83 APPENDIX Table I: State 3 and State 4 respiration rates in isolated kidney mitochondria from vii wild type (WT) and BCATm -/- (KO) mouse fed on choice diets ………101 Table II: State 3 and State 4 respiration rates in isolated liver mitochondria from wild type (WT) and BCATm -/- (KO) mouse fed on choice diets .……..101 Table III: State 3 and State 4 respiration rates in isolated liver mitochondria from wild type (WT) and BCATm -/- (KO) mouse fed on choice and high fat diets ……….……………………………………………………….…….102 Figure 1: Quantification of Western in tissue mitochondria of male mice on choice diet …………………………………………………………………..……103 Figure 2: Quantification of Western in tissue mitochondria of female mice on choice diet ………………………………………………………………….….…104 Figure 3: Quantification of Western in liver mitochondria of male mice on high fat diet …………………………………………………………………….….105 Figure 4: Fatty acid oxidation in tissue homogenates from wild type (WT) and BCATm-/- (KO) mice fed on choice diets …………………………….….108 Figure 5: Western blot showing the expression of NIPSNAP1 in livers of wild type (WT) vs. BCATm-/- knockout (KO) mouse ………………………….….109 viii ABBREVIATIONS APP Amyloid precursor protein BCAA Branched-chain amino acid BCKA Branched-chain α-keto acid BCATc Cytosolic branched-chain aminotransferase BCATm Mitochondrial branched-chain aminotransferase BCKDC Branched-chain α-keto-acid dehydrogenase complex Bax B-cell lymphoma-2 family protein CCCP Carbonyl cyanide m-chlorophenylhydrazone CD Circular dichroism spectroscopy CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate CNS Central nervous system CyOx-1 Cytochrome oxidase subunit I DCPIP 2, 6-Dichlorophenolindophenol DTNB 5,5’-Dithiobis-(2-nitrobenzoate) DTT Dithiothreitol E1 Branched-chain α-keto acid decarboxylase/dehydrogenase E2 Branched-chain α-keto acid dihydrolipoyl transacylase E3 Dihydrolipoyl dehydrogenase EB Expansion broth GABA -Aminobutyric acid GAD Glutamic acid decarboxylase ix GCL Granule cell layer GDH Glutamate dehydrogenase GFP Green fluorescent protein GFAP Glial fibrillary acidic protein HEK Human embryonic kidney IAP Inhibitor of apoptosis protein IPTG Isopropyl β-D-1-thiogalactopyranoside KIC α-Ketoisocaproate KIV α-Ketoisovalerate MDH Malate dehydrogenase MSE Mannitol sucrose EDTA buffer MSUD Maple syrup urine disease NIPSNAP1 4-Nitrophenylphosphatase domain and non-neuronal SNAP25- like protein homolog 1 OB Overexpression broth PAH Phenylalanine hydroxylase PBS Phosphate buffered saline PCR Polymerase chain reaction PDC Pyruvate dehydrogenase complex pI Isoelectric point PKU Phenylketonuria PL Polymorphic layer PSD Post synaptic density PVDF Polyvinylidene fluoride x