THE ISOLATION AND CHARACTERIZATION OF HUNTINGTIN INTERACTING PROTEINS By MICHAEL ANDREW KALCHMAN B.Sc. (Honor's Genetics), University of Western Ontario, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE FACULTY OF GRADUATE STUDIES GENETICS GRADUATE PROGRAMME We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JULY, 1998 © Michael Andrew Kalchman 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Huntington Disease (HD) is an autosomal dominant, neurodegenerative disorder with onset normally occurring at around 40 years of age. This devastating disease is the result of the expression of a polyglutamine tract greater than 35 in a protein with unknown function. The underlying mutation in HD places it in a category of neurodegenerative diseases along with seven other diseases, all of which have widespread expression of the protein with an abnormally long polyglutamine tract, but have disease specific neurodegeneration. Yeast two-hybrid screens were used in an attempt to further elucidate the function of the HD gene product, huntingtin. The results help decipher the biochemical signals that may contribute to the neuronal specific cell death seen in HD patients. Three different cDNA fragments spanning greater than 80 % of the HD cDNA were used to screen for Huntingtin Interacting Proteins (HIPs). Only the N-terminal region of huntingtin produced positive interacting proteins. Of the 14 clones isolated 12 were identical and given the name HIP1. HIP2 and H1P3 were isolated as individual positive clones. Assessment of the expression pattern of each of the HIPs reveal them all to be expressed in most tissues, but preferentially expressed in human brain and subcellular regions similar to that of huntingtin. HJP1 is a novel human gene that shares identity with the yeast Sla2p/End4 protein that is involved in the endocytotic pathway and maintenance of the cytoskeleton. The interaction between HIPl and huntingtin appears to be influenced by the size of the polyglutamine tract, in a manner whereby the larger the CAG tract, the lower the affinity the two proteins have for each other. Biochemical and in vitro assessment of huntingtin and HEP1 place the two proteins in the same cell compartments, providing further evidence that these proteins interact in vivo. HIP2 shares complete identity with the previously cloned bovine E2-25K ubiquitin conjugating enzyme. This protein plays an essential role in the ubiquitin proteolytic pathway, suggesting that huntingtin is degraded via this catabolic mechanism. As part of the investigation into this interaction, huntingtin was shown to coimmunoprecipitate with ubiquitin, without preference for the mutant form of huntingtin. This demonstration of the ubiquitination of huntingtin preceded the description of huntingtin-ubiquitin co- immunoreactive intranuclear inclusions. Presently, four of the eight expanded polyglutamine dependent diseases have been shown to have these ubiquitin positive staining intranuclear inclusions. HEP3 is a protein that is highly expressed in the brain, specifically the caudate nucleus and putamen, regions significantly affected in HD patients. The homology of HEP3 with a membrane associated protein in yeast, Akrlp, places it at the membrane with huntingtin. The involvement of Akrlp in receptor mediated endocytosis is consistent with the role of the SLA2/END4 gene in endocytosis. The data presented in this thesis provides clues into the role huntingtin plays within a cell. It supports data that huntingtin is associated with synaptic vesicles and cytoskeletal components of the cellular membrane. The presence of an expanded polyglutamine tract may alter the ability of huntingtin to either bind its normal cellular target. iii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES ix LIST OF TABLES xi ACKNOWLEDGEMENTS xii CHAPTER 1 - INTRODUCTION 1 1.1 HUNTINGTON DISEASE 2 1.2 THE MOLECULAR GENETICS OF HD AND OTHER CAG REPEAT DISORDERS 3 1.3 HUNTINGTIN: THE HD PROTEIN 8 1.4 HUNTINGTIN AND OTHER POLYGLUTAMINE DISEASES: THE FORMATION OF INCLUSIONS AND AGGREGATES 10 1.5 HYPOTHESIS 14 1.6 RATIONALE AND OBJECTIVES 14 1.7 REFERENCE LIST 16 CHAPTER 2 - METHODOLOGY 24 2.1 THE YEAST TWO-HYBRID SYSTEM 25 2.2 METHODOLOGY 31 2.2.1 GAL4 cDNA constructs 31 iv 2.2.2 Yeast strains, transformations and (3-galactosidase assay 33 2.2.3 Analysis of GAL4 DNA binding domain - huntingtin fusion protein expression in yeast 35 2.2.4 Screening for Huntingtin Interacting Proteins (HEPs) 36 2.2.5 DNA sequencing, cDNA isolation and 5' RACE 37 2.2.5.1 Elucidation of the HIP1 full-length cDNA sequence 37 2.2.5.2 Construction of the CMV-HIP1 expression construct 38 2.2.5.3 HJP2 cDNA sequence 39 2.2.6 DNA and amino acid sequence analyses 39 2.2.7 Generation of anti-HEP antibodies 40 2.2.7.1 Anti-HEPl pepl polyclonal antibody 40 2.2.7.2 Anti-HEPl fusion protein polyclonal antibody 40 2.2.7.3 Anti-HE?3 pep3 polyclonal antibody 42 2.2.8 Northern blot analysis and in situ hybridization of HEP1 43 2.2.9 GST-HIP2 fusion protein expression 44 2.2.10 Generation of HD in vitro transcription-translation products 45 2.2.11 Protein preparation and western blotting for expression studies 46 2.2.12 Biochemical assessment of huntingtin - HE? interactions 47 2.2.12.1 Co-immunoprecipitation of HD?1 with huntingtin 47 2.2.12.2 Subcellular fractionation of huntingtin and HEP1 from brain tissue 49 2.2.12.3 Coaffinity purification of huntingtin with GST-HEP2 50 2.2.12.4 Coimmunoprecipitation of huntingtin and ubiquitin 51 2.2.13 In vitro experiments 52 v 2.2.13.1 Transfection of HD and fflPl cDNA constructs into HEK293T cells 52 2.2.13.2 Immunohistochemistry and immunofluorescence 52 2.2.14 Genome mapping of HIPs: FISH detection system and image analysis.. 53 2.3 REFERENCE LIST 55 CHAPTER 3 - HUNTINGTIN INTERACTING PROTEIN 1 57 3.1 INTRODUCTION 58 3.2 RESULTS 59 3.2.1 Isolation of HIPlpGADIO 59 3.2.2 HIP1 cDNA sequence analysis reveals that it is the human homologue of S. cerevisiae Sla2p and C. elegans ZK370.3 gene product 63 3.2.3 The influence of polyglutamine length on the strength of the huntingtin- fflPl interaction 78 3.2.4 Co-immunoprecipitation of huntingtin and HIPl 82 3.2.5 HIPl mRNA is enriched in the brain 82 3.2.6 HIPl protein is predominately found in the central nervous system 88 3.2.7 Subcellular localization of HIPl protein in adult human and mouse brain 94 3.2.8 HIPl maps to human chromosome 7ql 1.23 102 3.3 DISCUSSION 103 3.4 REFERENCE LIST 108 CHAPTER 4 - HUNTINGTIN INTERACTING PROTEIN 2 Ill vi 4.1 HUNTTNGTIN AND UBIQUITIN 112 4.2 RESULTS 113 4.2.1 Isolation of Huntingtin Interacting Protein 2 (HIP) 113 4.2.2 HIP2 is the human E2-25K ubiquitin conjugating enzyme 117 4.2.3 Interaction between GST-HJP2 and the HD protein 122 4.2.4 The hE2-25K ubiquitin conjugating enzyme is highly expressed in brain... 124 4.2.5 The HD gene product is ubiquitinated 129 4.2.6 hE2-25K Maps to Chromosome 4pl4 132 4.3 DISCUSSION 133 4.4 REFERENCE LIST 139 CHAPTER 5 - HUNTINGTIN INTERACTING PROTEIN 3 143 5.1 HUNTINGTIN AND HIP3 144 5.2 RESULTS 145 5.2.1 Isolation and sequencing of HIP3 145 5.2.2 HJP3 shares identity with the yeast Akrlp protein 145 5.2.3 HIP3 protein is highly expressed in the brain 152 5.2.4 HEP3 maps to a single genomic locus in humans 154 5.3 DISCUSSION 155 5.4 REFERENCE LIST 157 CHAPTER 6 - SUMMARY. FUTURE WORK AND CONCLUSIONS 158 6.1 SUMMARY 159 6.2 HUNTLNGTIN INTERACTING PROTEINS 166 6.3 WHAT DOES THE IDENTIFICATION OF INTERACTING PROTEINS TEACH US ABOUT THE PATHOGENESIS OF HUNTINGTON DISEASE? 170 6.4 FUTURE EXPERIMENTS 177 6.5 CONCLUSIONS 180 6.6 REFERENCE LIST 181 viii LIST OF FIGURES Page Figure 2.1 The yeast two-hybrid system 27 Figure 2.2 Screening for Huntingtin interacting proteins. 30 Figure 3.1 P-galactosidase filter assays demonstrating the interaction between huntingtin and HIPl. 61 Figure 3.2 Western blot of the GAL4 DNA binding domain vectors expressing different sized polyglutamine tracts. 62 Figure 3.3 HIPl cDNA contig. 64 Figure 3.4 DNA and amino acid sequence of HIPl. 65 Figure 3.5 Coiled-coil structure of HIPl, Sla2p and ZK370.3. 73 Figure 3.6 Amino acid alignment of HIPlwith ZK370.3 and Sla2p. 76 Figure 3.7 Liquid P-galactosidase assays performed to assess the interaction strength between huntingtin and HIPl. 80 Figure 3.8 Coimmunoprecipitation of HIPl and huntingtin. 83 Figure 3.9 Northern blot of HIPl mRNA. 85 Figure 3.10 HIPl and Hdh mRNA in situ hybridization. 86 Figure 3.11 HIPl protein expression in brain and peripheral tissues. 89 Figure 3.12 Assessment of CMV-HIP1 construct and comparative analysis of the two anti- fflPl antibodies. 91 Figure 3.13 Biochemical fractionation of huntingtin and HIPl from human cortex.