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.
95
Figure 3.14 Immunolocalization of HIPl and huntingtin in mouse brain. 98
Figure 3.15 Transfection of HD and HIPl cDNA constructs into HEK293T cells. 100 Figure 3.16 Genomic mapping of HIPl locus. 102
IX Figure 4.1 Specific interaction of HJP2 with the 5' region of the HD gene. 114
Figure 4.2 Liquid P-galactosidase assays showing the interaction between huntingtin and HJP2. 116
Figure 4.3 DNA and amino acid sequences of HJP2 (hE2-25K). 119
Figure 4.4 GST-H1P2 fusion protein is detected with the anti-bE2-25K antibody.
121
Figure 4.5 Interaction of HEP2 with the HD protein (western blots). 123
Figure 4.6 Tissue and regional specificity of E2-25K expression. 126
Figure 4.7 The HD protein co-immunoprecipitates with ubiquitin. 130 Figure 4.8 Fluorescent in-situ hybridization localized the hE2-25K protein to cytogenetic band4pl4. 132
Figure 5.1 The interaction of huntingtin with HIP3. 147
Figure 5.2 cDNA sequence of HD?3. 148
Figure 5.3 Alignment of HBP3 with Akrlp. 150
Figure 5.4 HIP3 and other ankyrin repeats. 151
Figure 5.5 Western blot showing expression of HIP3 protein. 153
Figure 5.6 Fluorescence In Situ Hybridization of HJP3 shows a single genomic locus for the HJP3 gene at 12ql2-14. 154
Figure 6.1 Model of potential pathway leading to increased susceptibility to cell death. 170
x LIST OF TABLES
Table 1.1 Diseases caused by the presence of an expanded polyglutamine tract. page 6
Table 6.1 Interacting proteins of huntingtin and other diseases with a polyglutamine tract. page 161
xi ACKNOWLEDGEMENTS
I would like to take this opportunity to thank all those who made my graduate experience extraordinary. Firstly, without my parents' support I could never have stretched my wings and ventured out to Vancouver to pursue my PhD. They have always been there for me and I can't thank them enough. Secondly, I would like to thank Michael Hayden for his continual support both intellectually and spiritually, without it this thesis would not have materialized. Furthermore, I would like to thank the members of my thesis committee, Drs.
Humphries, Jirik and Mager for their input throughout my degree.
Also important to me over the years were some very special friendships I established.
Eric Gagne, thank you for putting up with my craziness and idiosynchrocies. Graeme
Hodgson (aka sizzle chest), Keith Fichter, Susan Andrew, Kerrie Nichol, Dave Spear and
Maria Hubinette. To Paul Goldberg, I never could have made it through the first few years without your encouragement and support, emotionally and professionally. Dr. Keppie
Pimstone, I am incredibly indebted to you for your compassion and friendship. To Dr.
Elisabeth Almqvist, I will miss the massages.
Throughout my PhD I had tremendous friendly, technical and intellectual support from Rona Graham, Krista McCutcheon, Brook Koide and many summer students especially
Francis Lynn. Furthermore, I also want to thank Dr. Cecile Pickart for her tremendous work on the ubiquitin assays, and Dr. Dan Geitz for letting me come to his lab in Winnipeg to perform the yeast two-hybrid screen. Chapter 1: Introduction
CHAPTER 1 - INTRODUCTION
Huntington Disease and other disorders associated with polyglutamine expansion
1 Chapter 1: Introduction
1.1 HUNTINGTON DISEASE
Huntington Disease (HD) is a fatal disease with a frequency of approximately lxlO"5.
Although variable in its expression, normally onset is in adulthood (average 35-40 years) as noted by the development and progression of uncontrolled movements, altered behaviour and cognitive decline (Hayden, 1981). It was the first inherited disease to be mapped to an autosomal chromosome by linkage using restriction fragment length polymorphism analysis (4p) (Gusella et al., 1983). More refined mapping placed it within the cytogenetic band 4pl6.3. However, it took ten years before the actual underlying CAG expansion in a gene (IT 15 - interesting transcript 15), with still unresolved biological function (Huntington's Disease Collaborative Research Group,
1993), was discovered as the mutation associated with the disease.
HD manifests in individuals who express an expanded CAG (>35) tract near the 5' end of the gene, in exon 1. The age of onset in HD patients, as well as with other diseases with a CAG expansion, is inversely correlated to the length of the polyglutamine tract i.e. the longer the polyglutamine tract the younger the age of onset (Matsuyama et al., 1997; David et al., 1997; Maciel et al., 1995; Maruyama et al., 1995; Takiyama et al.,
1995; Jodice et al., 1994; Koide et al., 1994; Nagafuchi et al., 1994; Ranum et al., 1994;
La Spada et al., 1992; Andrew et al., 1993).
Two mRNAs of 10.3 kb and 13.7 kb for the HD gene are known to be expressed and differ only in the size of the 3' untranslated region. Both messages are found in all tissues with the highest amount seen in brain and testes (Lin, et al., 1993a). The HD mRNA or regions of it have been cloned from different model organisms, including nonhuman primates, mouse, rat and pufferfish (Djian, et al., 1996; Lin, et al., 1994b;
2 Chapter 1: Introduction
Schmitt, et al, 1995; Baxendale, et al., 1995). Interestingly, the HD gene and protein are highly conserved through evolution. In the human, mouse and pufferfish the HD gene is encoded by 67 exons (Ambrose, et al., 1994; Lin, et al, 1994b; Baxendale, et al., 1995).
Over the entire length of the HD protein there is a high degree of conservation with cloned HD genes from other organisms. The human and mouse share 91 % amino acid identity (Lin, et al., 1994b), the rat has 90 % identity with human huntingtin (Schmitt, et al., 1995) and the pufferfish is 67 % (Baxendale, et al., 1995) conserved with the human protein.
Although the huntingtin protein is conserved through evolution over the entire length of the protein, the polyglutamine tract itself appears to be less conserved. In humans, the normal size of the polyglutamine ranges between 11 - 35, whereas nonhuman primates, such as chimpanzees, gorillas and the gibbon have between seven and nine CAG codons at a similar place within its transcript (Djian, et al, 1996). The mouse and rat HD genes have seven and eight CAG repeats, respectively (Lin, et al., 1994b; Schmitt, et al., 1995), whereas the pufferfish has four (Baxandale, et al., 1996).
1.2 THE MOLECULAR GENETICS OF HD AND OTHER CAG REPEAT
DISORDERS
The field of neurodegenerative diseases has been highlighted recently as eight such diseases (Table 1.1) have the same underlying mutation. These diseases have an expanded CAG trinucleotide repeat resulting in the mutant protein expressing a pathologically significant polyglutamine tract. Each of the disorders, with the exception of SBMA (X-linked recessive), is inherited in an autosomal dominant fashion. Similar to Chapter 1: Introduction the other diseases caused by the presence of an expanded polyglutamine tract, HD results in a very particular pattern of neuronal loss. The mRNA and protein (huntingtin) is widely expressed with no differential expression noted between affected and unaffected individuals (De Rooij et al., 1996; Trottier et al., 1995; Aronin et al., 1995; DiFiglia et al.,
1995).
Studies have shown that the HD mRNA expression is high in the dentate gyrus and pyamidal neuron subfields of the hippocampal formation, within the cerebellar granule cell layer, periaqueductal gray, medial habenula, entorhinal cortex and pontine nuclei (Strong et al., 1993). High levels of the transcript are seen in neuronal tissue and lower levels seen in the glia (Landwehrmeyer et al., 1995; Strong et al., 1993).
Patients with HD have specific neuronal loss, with significant atrophy specifically seen in the striatum. The medium spiny neurons of the caudate nucleus and putamen are affected, along with astrocytes (Ferrante et al., 1997; Vonsattel et al., 1985). The specific neuronal loss is seen in these tissues where the HD gene product (huntingtin) is highly expressed. HD patients do not show a cellular phenotype in the large and medium sized aspiny neurons, where analysis has demonstrated low levels of huntingtin expression
(Ferrante et al., 1997).
Anticipation is observed in HD, and a preferential expansion through the paternal germ line is known (Telenius et al., 1994). The phenomenon of genetic anticipation now has a molecular explanation. As the length of the polyglutamine tract increases, a decrease in the mean age of onset is observed in HD families (Telenius et al., 1994).
Although both expansions and contractions can occur on trinucleotide repeats, expansions appear to be more frequent (La Spada, 1997). Parental effects of the
4 Chapter 1: Introduction expansion of the CAG repeat are also noted in some of the other CAG repeat disorders including SBMA, DRPLA, SCA1 and SCA3 (La Spada, 1997).
There are other factors yet undetermined, independent from CAG size that influence the age of onset for HD patients. The CAG tract itself only contributes ultimately 70 % of the variation seen in the age of onset (^=0.73) (Brinkman, et al.,
1997). Statistical analysis of a large cohort comprised of both HD affected and asymptomatic (at risk) individuals predicted the probability of a particular person manifesting with a HD phenotype with a particular CAG size at a particular age
(Brinkman et al., 1997). For example, the probability of developing HD by age 45 is 13
% with 40 CAG repeats, 32 % with 42 CAG repeats, and 100 % for an individual with 46
CAG repeats (Brinkman et al., 1997).
5 1) C co co co .2 CM E NO • CM CM s CM CM OH co § CM a, a, NO ON OH o 3 co
a CM g 2 g « c 00 2 '53 o a, o •4-» is S g o G ^ o OH C
•a r- o r-i NOi oo CM CO ON NO oo CO CO NO CM oo co CO CO o T3
O O CO ON NO CO CO CO i O I I I i •»-» ON ON NO II a <0 _o 9 a. '•4-1 >>» OH o ca. 4) o c o 0) cd on -o 'C 43 u a- OH "5b > p O .2-a 71 •c 60 O c o i -s s " ca H 1£ ca o a a i .2 S ° o ° X on y o ca C ea on 3 on £ £ -9 X c 43 Q a. D 2 < w Q < .5 < co a < >-) u l l l l ca c3 i i c S 00 T3 _o a 75 2 T! S ^ 3 fill o .2 3 O (D The properties of CAG repeat instability are similar to those seen with other trinucleotide repeats, such as the CGG repeat in the FRAXA gene. In this case AGG is interspersed within the FRAXA CGG repeat, and as a result the most unstable allele is that which contains the longest pure CGG repeat (Oberle et al., 1991; Verkerk et al., 1991; Fu et al., 1991). Understanding the molecular genetics of the CAG repeat in HD and other CAG repeat disease genes has been advanced by the utilizing different model systems. Lower organisms such as bacteria (Eschericia coli) and yeast (Saccharomyces cerevisiae) have been used to demonstrate that tracts of CAG trinucleotide repeats are prone to changes in size, either expansions or contractions (Freudenreich et al., 1997; Maurer et al., 1996; Jaworski et al., 1995). In the bacterial strain DH5-0C, a CTG (CAG) tract is more prone to deletions (contractions) when the CTG is on the lagging strand, whereas expansions are more frequent when the CTG is on the leading strand (Freudenreich et al., 1997; Maurer et al., 1996). Further investigation into this model revealed the mismatch repair system is responsible for moderating this property. This is in contrast with FRAXA where the mismatch repair system is not responsible for the integrity of the CGG repeat. The budding yeast S. cerevisiae has also been used to assess trinucleotide repeat stability (Freudenreich et al., 1997; Maurer et al., 1996). As found in E. coli, and with the same consequences, orientation of the CAG/CTG tract was the major determinant of the type of stability observed (expansions vs. contractions) (Freudenreich et al., 1997; Maurer et al., 1996) . Interestingly, as in the human diseases (Andrew et al., 1993), in yeast, the larger the size of the trinucleotide repeat the more unstable the trinucleotide repeat (Freudenreich et al., 1997) . 7 Chapter 1: Introduction The study of CAG trinucleotide repeat instability in model organisms has not been limited to prokaryotes or single cell eukaryotes. The HD exon 1 transgenic mouse model (Mangiarini et al., 1996) was generated with CAG repeats between 116 to 156 as a result of instability during propagation of the genomic clone used for injection. Subsequently, when these mice were bred and offspring assessed for trinucleotide stability, trinucleotide repeat instability through the male germline was seen. This is in contrast with a tendency to contract when passed through the mouse female germline (Mangiarini et al., 1996). 1.3 HUNTINGTIN: THE HD PROTEIN The understanding of the molecular genetics of HD is a critical keystone in determining the etiology of the disease. However, without knowledge of the function of the HD gene product significant progress toward treating the disease is improbable. Sequence analysis of the HD cDNA predicts a protein 3144 amino acids with an approximate molecular weight of 348 000 (348 kDa). Outside the polyglutamine tract, at the amino terminus of the protein, primary sequence analysis of huntingtin reveals that it does not have significant similarities to other known proteins. The huntingtin protein has been found to be essential for normal mouse embryonic development (White et al., 1997; Nasir et al., 1995; Duyao et al., 1995; Zeitlin et al., 1995). Embryos homozygous for a targeted disruption of the murine homologue of the HD gene, Hdh, are unable to develop in utero past day 8.5. These embryos demonstrated increased apoptosis (Zeitlin et al., 1995) initiated gastrulation but did not continue to form somites or proceed to organogenesis (Duyao et al., 1995; Nasir et al., 1995). 8 Chapter 1: Introduction Huntingtin has been localized to neurons throughout the brain, including all cortical regions. However, lower levels are seen in the cerebellum, a region almost exclusively spared in HD (Ross et al., 1997). Furthermore, at the substructural level, Purkinje and granule cells of the cerebellum and caudate nucleus are immunoreactively positive for huntingtin (Schilling et al., 1995). Axons, cell bodies and axon termini demonstrate immunoreactive positive signals for huntingtin (DiFiglia et al., 1995). Further biochemical analyses using sucrose density gradients and immunoprecipitation studies have demonstrated that huntingtin is preferentially distributed to the somatodendritic cytoplasm and is associated with vesicle membranes, microtubules (DiFiglia et al., 1995) and cytoskeletal components (see Chapter 3) (Kalchman et al., 1997; Wood et al., 1996). Huntingtin is found in soluble and membranous fractions after biochemical fractionation of neurons. It has been found in the PI (cell debris and nuclei), P2 (mitochondria and synaptosomes) and P3 (microsomes and plasma membranes) fractions (see Chapter 3) (Kalchman et al., 1997; Wanker et al., 1997; Wood et al., 1996). These findings suggest that huntingtin may be involved in protein or vesicle trafficking along microtubule tracks, membrane cycling or maintaining structural integrity of cells in which it is expressed. The generation of various antibodies to different epitopes throughout huntingtin has allowed further investigation into the possible function of huntingtin through electron and confocal microscopy. The use of these various antibodies resulted in the recent description of a possible pathogenic outcome via the formation of aggregates (inclusions) that are made up of at the very least, huntingtin and ubiquitin (Martindale et al., 1998; DiFiglia et al., 1997; Davies et al., 1997; Mangiarini et al., 1996). 9 Chapter 1: Introduction 1.4 HUNTINGTIN AND OTHER POLYGLUTAMINE DISEASES: THE FORMATION OF INCLUSIONS AND AGGREGATES The question arises, how does a polyglutamine tract, in the context of a particular protein, cause cell death? It has been suggested that long stretches of polyglutamine tracts can form anti-parallel "polar zippers" (Perutz, 1996; Stott et al., 1995). These polar zippers may then act to recruit other molecules to form a stable P-pleated sheet. As a result by its hydrophobic nature, the P-pleated sheet may precipitate in the form of para or intranuclear aggregates. Interestingly, in vitro experiments using only exon 1 of the HD gene (Scherzinger et al., 1997) to assess the ability of huntingtin to form P-pleated sheets, produced results consistent with the polar zipper hypothesis (Perutz et al., 1994). These p- sheets, when examined by electron microscopy, resembled the P-amyloid fibrils seen in Alzheimer's disease (AD) and the scrapie prions (Scherzinger et al., 1997). It has been shown that mice transgenic for exon 1 of the HD gene (expressing (CAG) 115 to (CAG) 156) do develop neuronal intranuclear inclusions (Davies et al., 1997). These inclusions may contribute to the progressive clinical phenotype apparent in these mice. The HD exon 1 transgenic mice have a distinct neurological phenotype characterized by weight loss, involuntary movements, seizures, and premature death (Davies et al., 1997). Further in vivo data demonstrated intranuclear inclusions form in neurons of HD patients (Becher et al., 1998; DiFiglia et al., 1997). Intranuclear and perinuclear huntingtin aggregation are seen in the neurons of both juvenile and adult HD patients but are absent in control tissue (DiFiglia et al., 1997). Intracellular inclusions are more prominent in the tissue 10 Chapter 1: Introduction of juvenile HD patients whereas perinuclear aggregation is seen more frequently in adult patients (DiFiglia et al., 1997; Sapp et al., 1997). The intranuclear inclusions seen in HD transgenic mice and patients also stain for ubiquitin immunoreactivity, suggesting that huntingtin is coupled to ubiquitin and targeted for degradation through that proteolytic pathway (see Chapter 4) (Kalchman et al., 1996). It is known when a substrate becomes ubiquitinated the substrate-linked multi-ubiquitinated chain facilitates the 26S proteasome degradative pathway (Varshavsky, 1997). The coupling of huntingtin to ubiquitin may be the signal required for the 26S proteasome to degrade huntingtin and trigger downstream events necessary for the selective cell death seen in HD brains. Transfection experiments using various length HD cDNA constructs demonstrate that the localization of huntingtin in HEK293T cells is dependent upon the length of the cDNA construct. Smaller gene products can enter the nucleus and form the intranuclear inclusion. As the length of the HD protein expressed increases above approximately 47 kDa, the pattern of aggregation appears to be predominately perinuclear (Martindale et al., 1998; A. Hackam, submitted). Huntingtin, atrophinl, ataxin3 and the androgen receptor (Wellington et al., 1998) are substrates for proteolytic cleavage by caspases (Goldberg et al., 1996; Miyashita et al., 1997). Therefore, the questions of what are the underlying mechanism, outcome and significance of the caspase specific cleavage of these proteins? The diseases associated with polyglutamine expansion possess intranuclear aggregates in the disease state. It is possible that cleavage of the disease proteins produces a toxic fragment that can enter the nucleus and form the intranuclear inclusions. 11 Chapter 1: Introduction The possibility of a toxic fragment of huntingtin was first presented in the description of a homozygous targeted disruption of the murine homologue of the HD gene (Nasir et al., 1995). The phenotype of aggregates seen in the nuclei of HD transgenic mice (Davies et al., 1997) and human neurons (DiFiglia et al., 1997; Sapp et al., 1997) is also seen in other diseases caused by the presence of an expanded polyglutamine tract. Ataxinl, 3, atrophin and the androgen receptor have been shown to be associated with perinuclear aggregate or intranuclear inclusion formation (Becher et al., 1998; Butler, 1998; Paulson et al., 1997; Skinner et al., 1997). Could the intranuclear inclusions be a result of the polyglutamine tract alone, or is the surrounding sequence important in the development of aggregates? In order to address this issue, the mouse hypoxanthine phosphoribosyltransferase gene (Hpri) gene was manipulated in such a way as to introduce a 146 CAG trinucleotide repeat (polyglutamine) in-frame with the entire Hprt gene (Ordway et al., 1997). There was a progressive neurological phenotype present in the aged mice expressing this chimeric protein. The mice had many abnormal characteristics including, but not limited to, seizures, ataxia, resting tremor and premature death (Ordway et al., 1998). These mice also have a cellular phenotype of intranuclear aggregates that stain positive for ubiquitin. Therefore, ubiquitinated intranuclear inclusions may be fundamental in the development of a cellular phenotype in diseases associated with a polyglutamine expansion. The presence of the expanded polyglutamine tract may be a signal for targeted degradation through the 26S proteasome ubiquitin catabolic pathway. The presence of a polyglutamine expansion is detrimental even when expressed in an in vitro model. Constructs that fused the coding region of Glutathione-S-transferase with a piece of DNA encoding long polyglutamine tracts were generated. Expanded polyglutamine 12 Chapter 1: Introduction tracts (59-81), when expressed in E. coli had a significantly slower growth curve when compared to that of a smaller (10-35) polyglutamine tract, or even an expanded polyalanine tract (61) (Onodera et al., 1996). Therefore, an expanded polyglutamine tract is deleterious when expressed independent of other sequences, suggesting that the contribution to the human diseases may be similar in each of the diseases. The specificity of each of the diseases may arise from specific protein-protein interactions that occur as a result of unique sequences that are present within each of the disease proteins. At the time of commencing the work presented in this thesis two different proteins had been shown to interact with huntingtin; Huntingtin Associated Protein 1 (HAP1) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Li et al., 1995; Burke et al., 1996). HAP1 was identified through the yeast two-hybrid system early in the search for huntingtin protein partners (Li et al., 1995). HAP1 shows a positive correlation in the strength of the interaction between huntingtin and the length of the polyglutamine tract. Various studies have demonstrated that HAP1 is associated with membranes and may be involved with movement of signals down neuronal axons (Colomer et al., 1997; Li et al., 1995; Li etal., 1996). Biochemical purification experiments demonstrated GAPDH interacts with huntingtin and atrophin (Burke et al., 1996). The region of GAPDH shown to interact is that of amino acids 1-149. This region contains the nicotinamide adenine dinucleotide (NAD) binding domain (and the first 21 amino acids of the catalytic domain), suggesting that it is the NAD binding domain primarily involved in the interaction. In addition GAPDH has been shown to interact with both ataxinl and the androgen receptor (Koshy et al, 1996). This suggests that GAPDH may be an important protein involved in all the diseases associated with a 13 Chapter 1: Introduction polyglutamine expansion. Koshy et al. (1996) suggest that a slow decline in energy metabolism of a neuronal cell may trigger the degenerative process. The fact that GAPDH is so widely and highly expressed, extrapolating the results must be taken cautiously. If the glycolytic role of GAPDH can influence neuronal survival, which are known to be highly responsive to metabolic changes, especially reductions, in ATP levels, it is feasible to envision GAPDH as a protein intricately involved in the pathogenesis of these diseases. Although HAP1 and GAPDH were shown to associate with huntingtin a strong understanding of the function the HD gene product performs in a cell remained unclear. Further studies into identifying huntingtin protein partners were essential when I began investigating Huntingtin Interacting Proteins. 1.5 HYPOTHESIS Huntingtin interacts with novel proteins that play critical roles in the development of Huntington disease. The identification of such proteins will help elucidate the role huntingtin plays in cells expressing both the normal and mutant form of huntingtin and give insight into HD pathogenesis. 1.6 RATIONALE AND OBJECTIVES The cloning of the HD gene was a major step forward in the understanding of this tragic disease. However, the lack of similarity the HD gene has with known genees could not allow a specific biological roll to be assigned to huntingtin. 14 Chapter 1: Introduction The distinct HD neuropathology may be explained if proteins with an expression pattern similar to huntingtin confer a specific susceptibility to the effects of mutant huntingtin. Proteins that exclusively and/or differentially interact with mutant huntingtin could provide insight into function of huntingtin and provide valuable information toward the understanding of the premature cell death seen in HD patients. The following chapters will describe the cloning and characterization of huntingtin interacting proteins (HIPs). The specific objectives for this thesis were to: 1. Identify Huntingtin Interacting Proteins (HIPs) using the yeast two-hybrid system from a human brain GAL4 activating domain cDNA library. By cloning different regions of the HD cDNA into the GAL4 DNA binding domain vector a survey of interacting proteins over the entire length of huntingtin was possible. 2. Sequence and assess the similarities or identities of the isolated HIPs with known clones or genes. 3. Determine the distribution of the HIP mRNA and protein. 4. Isolate and analyze full-length cDNAs of the partial HIP cDNAs isolated from the two-hybrid screen. 5. Confirm the interaction observed in the yeast two-hybrid system using biochemical techniques, such as coimmunoprecipitation or coaffinity purification. 6. Assess the influence the size of the polyglutamine tract has on the interaction between huntingtin and the identified HIP. 7. Identify the chromosomal location of each of the HIP genes to assess if any of the HIPs can be responsible for the observation of HD families without CAG expansion. 15 Chapter 1: Introduction 1.7 REFERENCE LIST Andrew, S.E., Goldberg, Y.P., Kremer, B., Telenius, H., Theilmann, J., Adam, S., Starr, E., Squitieri, F., Lin, B., and Kalchman, M.A., Goldberg, Y.P., and Hayden, M.R. (1993). The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington's disease. Nat.Genet. 4, 398-403. Aronin, N., Chase, K., Young, C, Sapp, E., Schwarz, C, Matta, N., Kornreich, R., Landwehrmeyer, B., Bird, E., and Beal, M.F. (1995). CAG expansion affects the expression of mutant Huntingtin in the Huntington's disease brain. Neuron 15, 1193-1201. Baxendale,S., Abdulla,S., Elgar,G., Buck,D., Berks,M., Micklem,G., Durbin, R, Bates,G., Brenner,S., Beck,S., Lehrach,H. (1995). Comparative sequence analysis of the human and pufferfish Huntington's disease genes. Nat. Genet. 10, 67-76. Becher, M., Kotzuk, J.A., Sharp, A.H., Davies, S.W., Bates, G.P., Price, D.L., and Ross, CA. (1998). Intranuclear neuronal inclusions in Huntington's disease and Dentatorubral Pallidoluyssian Atrophy: correlation between the density of inclusions and IT15 triplet repeat length. Neurobio. Dis. in press, Brinkman, R. R., Mezei, M. M., Thielmann, J., Almqvist, E., and Hayden, M. R. The liklihood of being affected with Huntington disease by a particular age for a specific CAG size. Amer. J. Hum. Genet. 60, 1202-1210. 1997. Burke, J.R., Enghild, J.J., Martin, M.E., Jou, Y.S., Myers, R.M., Roses, A.D., Vance, JM, and Strittmatter, W.J. (1996). Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH. Nat. Med. 2, 347-350. Butler, R Leigh PN. Mcphaul MJ. Gallo JM. (1998). Truncated forms of the androgen receptor are associated with polyglutamine expansion in X-linked spinal and bulbar muscular atrophy. Hum. Mol. Genet. 7, 121-127. Chung, M. Y., Ranum, L. P. W., Duvick, L. A., Servadio, A., Zoghbi, H. Y., and Orr, H. T. Evidence for a mechanism predisposition to intergenerational CAG repeat instability in spinocerebellar ataxia type I. Nat. Genet. 5, 254-258. 1993. Colomer, V., Engelender, S., Sharp, A.H., Duan, K., Cooper, J.K., Lanahan, A., Lyford, G., Worley, P., and Ross, CA. (1997). Huntingtin-associated protein 1 (Hapl) Binds to a trio• like polypeptide, with a rad guanine nucleotide exchange factor domain. Hum. Mol. Genet. 6, 1519-1525. 16 Chapter 1: Introduction David, G., Abbas, N., Stevanin, G., Durr, A., Yvert, G., Cancel, G., Weber, C, Imbert, G., Saudou, F., Antoniou, E., Drabkin, H., Gemmill, R., Giunti, P., Benomar, A., Wood, N., Ruberg, M., Agid, Y., Mandel, J.L., and Brice, A. (1997). Cloning of the sca7 gene reveals a highly unstable cag repeat expansion. Nat. Genet. 17, 65-70. Davies, S.W., Turmaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L., and Bates, G.P. (1997). Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90, 537-548. De Rooij, K.E., Dorsman, J.C., Smoor, M.A., Den Dunnen, J.T., and van Ommen, G.J. (1996). Subcellular localization of the Huntington's disease gene product in cell lines by immunofluorescence and biochemical subcellular fractionation. Hum. Mol. Genet. 5, 1093- 1099. DiFiglia, M., Sapp, E., Chase, K., Schwarz, C, Meloni, A., Young, C, Martin, E., Vonsattel, J.P., Carraway, R., and Reeves, S.A. (1995). Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron 14, 1075-1081. DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Vonsattel, J.P., and Aronin, N. (1997). Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277, 1990-1993. Djian,P., HancockJ.M., Chana,H.S. (1996) Codon repeats in genes associated with human diseases: fewer repeats in the genes of nonhuman primates and nucleotide substitutions concentrated at the sites of reiteration.. Proc. Natl. Acad. Sci. USA, 93, 417-421. Duyao, M.P., Auerbach, A.B., Ryan, A., Persichetti, F., Barnes, G.T., McNeil, S.M., Ge, P., Vonsattel, J.P., Gusella, J.F., and Joyner, A.L. (1995). Inactivation of the mouse Huntington's disease gene homologue Hdh. Science 269, 407-410. Ferrante, R.J., Gutekunst, C.A., Persichetti, F., McNeil, S.M., Kowall, N.W., Gusella, J.F., MacDonald, M.E., Beal, M.F., and Hersch, S.M. (1997). Heterogeneous topographic and cellular distribution of huntingtin expression in the normal human neostriatum. J. Neuro. 17, 3052-3063. Freudenreich, C.H., Stavenhagen, J.B., and Zakian, V.A. (1997). Stability of a CTG/CAG trinucleotide repeat in yeast is dependent on its orientation in the genome. Mol. and Cell. Biol. 77,2090-2098. Fu, Y.H., Kuhl, D.P., Pizzuti, A., Pieretti, M., Sutcliffe, J.S., Richards, S., Verkerk, A.J., Holden, J.J., Fenwick, R.G.J., and Warren, S.T. (1991). Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 67, 1047- 1058. 17 Chapter 1: Introduction Goldberg, Y.P., Andrew, S.E., Clarke, L.A., and Hayden, M.R. (1993). A PCR method for accurate assessment of trinucleotide repeat expansion in Huntington disease. Hum. Mol. Genet. 2(6), 635-636. Goldberg, Y.P., McMurray, C.T., Zeisler, J., Almqvist, E., Sillence, D., Richards, F., Gacy, A.M., Buchanan, J., Telenius, H., and Hayden, M.R. (1995). Increased instability of intermediate alleles in families with sporadic Huntington disease compared to similar sized intermediate alleles in the general population. Hum. Mol.Genet. 4(10), 1911-1918. Goldberg, Y.P., Nicholson, D.W., Rasper, D.M., Kalchman, M.A., Koide, H.B., Graham, RK, Bromm, M., Kazemi-Esfarjani, P., Thornberry, N.A., Vaillancourt, J.P., and Hayden, M.R. (1996). Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract. Nat.Genet. 13, 442-449. Gusella, J.F., Wexler, N.S., Conneally, P.M., Naylor, S.L., Anderson, M.A., Tanzi, R.E., Watkins, P.C., Ottina, K., Wallace, M.R., and Sakaguchi, A.Y. (1983). A polymorphic DNA marker genetically linked to Huntington's disease. Nature 306, 234-238. Hayden, M.R. (1981). Huntington's chorea (London, Berlin, Heidelberg: Springer-Verlag). Hoogeveen, A.T., Willemsen, R., Meyer, N., De Rooij, K.E., Roos, R.A., van, O., GJ, and Galjaard, H. (1993). Characterization and localization of the Huntington disease gene product. Hum. Mol. Genet. 2, 2069-2073. Huntington's Disease Collaborative Research Group (1993). A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell 72, 971-983. Imbert, G., Saudou, F., Yvert, G., Devys, D., Trottier, Y., Gamier, J.M., Weber, Mandel, J.L., Cancel, G., Abbas, N., Durr, A., Didierjean, O., Stevanin, G., Agid, Y., and Brice, A. (1996). Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats. Nat. Genet. 14, 285-291. Jaworski, A., Rosche, W.A., Gellibolian, R., Kang, S., Shimizu, M., Bowater, R.P., Sinden, R.R., and Wells, R.D. (1995). Mismatch repair in Escherichia coli enhances instability of (CTG)n triplet repeats from human hereditary diseases. Proc. Natl. Acad. Sci. USA. 92, 11019-11023. Jodice, C, Malaspina, P., Persichetti, F., Novelletto, A., Spadaro, M., Giunti, P., Morocutti, C, Terrenato, L., Harding, A.E., and Frontali, M. (1994). Effect of trinucleotide repeat length and parental sex on phenotypic variation in spinocerebellar ataxia 1. Am. J. Hum. Genet. 54, 959-965. Kalchman, M.A., Graham, R.K., Xia, G., Koide, H.B., Hodgson, J.G., Graham, K.C., Goldberg, Y.P., Gietz, R.D., Pickart, CM., and Hayden, M.R. (1996). Huntingtin is 18 Chapter 1: Introduction ubiquitinated and interacts with a specific ubiquitin-conjugating enzyme. J. Biol. Chem. 271, 19385-19394. Kalchman, M.A., Koide, H.B., McCutcheon, K., Graham, R.K., Nichol, K., Nishiyama, Kazemi-Esfarjani, P., Lynn, F.C., Wellington, C, Metzler, M., Goldberg, Y.P., Kanazawa, I., Gietz, R.D., and Hayden, M.R. (1997). HIPl, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nat. Genet. 16, 44-53. Koide, R., JJceuchi, T., Onodera, O., Tanaka, H., Igarashi, S., Endo, K., Takahashi, H., Kondo, R., Ishikawa, A., Hayashi, T., Saito, M., Tomoda, A., Miike, T., Naito, H., Dcuta, F., and Tsuji, S. (1994). Unstable expansion of CAG repeat in hereditary dentatorubral- pallidoluysian atrophy (DRPLA). Nat. Genet. 6, 9-13. La Spada, A.R. (1997). Trinucleotide repeat instability: genetic features and molecular mechanisms. Brain Path. 7, 943-963. La Spada, A.R., Roling, D.B., Harding, A.E., Warner, C.L., Spiegel, R., Hausmanowa- Petrusewicz, I., Yee, W.-C, and Fischbeck, K.H. (1992). Meiotic stability and genotype- phenotype correlation of the trinucleotide repeat in X-linked spinal and bulbar muscular atrophy. Nat. Genet. 2, 301-304. Lin,B., RommensJ.M., Graham,R.K., Kalchman,M., MacDonald,H., Nasir,J., Delaney,A., Goldberg,Y.P., (1993a) Hayden,M.R. Differential 3' polyadenylation of the Huntington disease gene results in two mRNA species with variable tissue expression. Hum. Mol. Gen. 2, 1541-1545 Landwehrmeyer, G.B., McNeil, S.M., Dure, L.S.4., Ge, P., Aizawa, H., Huang, Q., Ambrose, CM., Duyao, M.P., Bird, E.D., Bonilla, E., and et al. (1995). Huntington's disease gene: regional and cellular expression in brain of normal and affected individuals. Ann. Neurol. 37, 218-230.. Li, X.J., Li, S.H., Sharp, A.H., Nucifora, F.C.J., Schilling, G., Lanahan, A., Worley, P., Snyder, S.H., and Ross, CA. (1995). A huntingtin-associated protein enriched in brain with implications for pathology. Nature 378, 398-402. Li, X.J., Sharp, A.H., Li, S.H., Dawson, T.M., Snyder, S.H., and Ross, CA. (1996). Huntingtin-associated protein (HAP1): discrete neuronal localizations in the brain resemble those of neuronal nitric oxide synthase. Proc. Natl. Acad. Sci. USA 93, 4839-4844. Lin,B., Nasir,J., MacDonald,H. Hutchinson,G.B., Graham,R.K., RommensJ.M., and Hayden,M.R. (1994b) Sequence of the murine Huntington disease gene: evidence for conservation, alternate splicing and polymorphism in a triplet (CCG) repeat. Hum. Mol. Genet., 3, 85-92. 19 Chapter 1: Introduction Maciel, P., Gaspar, C., DeStefano, A.L., Silveira, I, Coutinho, P., Radvany, J., Dawson, D.M., Sudarsky, L., Guimaraes, J., Loureiro, J.E.L., Nezarati, M.M., Corwin, L.I., Lopes- Cendes, I., Rooke, K., Rosenberg, R., MacLeod, P., Fairer, L.A., Sequeiros, J., and Rouleau, G.A. (1995). Correlation between CAG repeat length and clinical features in Machado- Joseph disease. Am. J. Hum. Genet. 57, 54-61. Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., hetherington, C, Lawton, M., Trottier, Y., Lehrach, H., Davies, S.W., and Bates, G.P. (1996). Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87, 493-506. Martindale, D., Hackam, A.S., Wieczorek, A., Ellerby, L., Wellington, C.L., McCutcheon, K., Singaraja, R., Kazemi-Esfarjani, P., Devon, R., Bredesen, D.E., Tufaro, F., and Hayden, M.R. (1998). Length of the protein and polyglutamine tract influence localization and frequency of intracellular aggregates of huntingtin. Nat.Genet. 18 (2), 150-154. Maruyama, H., Nakamura, S., Matsuyama, Z., Sakai, T., Doyu, M., Sobue, G., Seto, M., Tsujihata, M., Oh-i, T., Nishio, T., Sunohara, N., Takahaski, R., Hayashi, M., Nishino, I., Ohtake, T., Oda, T., Nishimura, M., Saida, T., Matsumoto, H., Baba, M., Kawaguchi, Y., Kakizuka, A., and Kawakami, H. (1995). Molecular features of the CAG repeats and clinical manifestation of Machado-Joseph disease. Hum. Mol. Genet. 4(5), 807-812. Matsuyama, Z., Kawakami, H., Maruyama, H., Izumi, Y., Komure, O., Udaka, F., Kameyama, M., Nishio, T., Kuroda, Y., Nishimura, M., and Nakamura, S. Molecular features of the CAG repeats of spinocerebellar ataxia 6 (SCA6). Hum. Mol. Genet. 6(8), 1283-1287. 1997. Maurer, D.J., O'Callaghan, B.L., and Livingston, D.M. (1996). Orientation dependence of trinucleotide CAG repeat instability in Saccharomyces cerevisiae. Mol. and Cell. Biol. 16, 6617-6622. Myers, R.H., Leavitt, J., Karner, L.A., Farrer, L., Jagedeesh, J., McFarlane, H., Mastromouro, C.A., Mark, R.J., and Gusella, J.F. (1989). Homozygote for Huntington disease. Am. J. Hum. Genet. 45, 615-618. Nagafuchi, S., Yanagisawa, H., Ohsaki, E., Shirayama, T., Tadokoro, K., Inoue, and Yamada, M. (1994). Structure and expression of the gene responsible for the triplet repeat disorder, dentatorubral and pallidoluysian atrophy (DRPLA). Nat. Genet. 8, 177-182. Nasir, J., Floresco, S.B., O'Kusky, J.R., Diewert, V.M., Richman, J.M., Zeisler, J., Borowski, A., Marth, J.D., Phillips, A.G., and Hayden, M.R. (1995). Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell 81, 811-823. 20 Chapter 1: Introduction Oberle, I., Rousseau, F., Heitz, D., Kretz, C, Devys, D., Hanauer, A., Boue, J., Bertheas, M.F., and Mandel, J.L. (1991). Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 252, 1097-1102. Onodera, O., Roses, A.D., Tsuji, S., Vance, J.M., Strittmatter, W.J., and Burke, J.R. (1996). Toxicity of expanded polyglutamine-domain proteins in Escherichia coli. FEBS Letters 399, 135-139. Ordway, J.M., Tallaksen-Greene, S., Gutekunst, C.A., Bernstein, E.M., Cearley, J.A., Wiener, H.W., Dure TV, L.S., Lindsey, R., Hersch, S.M., Jope, R.S., Albin, R.L., and Detloff, P.J. (1997). Ectopically expressed CAG repeats cause intranuclear inclusions and a progressive late onset neurological phenotype in the mouse. Cell 91, 753-763. Paulson, H. L., Perez, M. K., Trottier, Y., Trojanowski, J. Q., Subramony, S. H., Das, S. S., Vig, P., Mandel, J.-L., Fischbeck, K. H., and Pittman, R. N. Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3. Neuron 19, 333-334. 1997. Perutz, M.F. (1996). Glutamine repeats and inherited neurodegenerative diseases: molecular aspects. Curr. Opin. Struct. Bio. 6, 848-858. Perutz, M.F., Johnson, T., Suzuki, M., and Finch, J.T. (1994). Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases. Proc. Natl. Acad. Sci. USA 97,5355-5358. Pulst, S.M., Nechiporuk, A., Nechiporuk, T., Gispert, S., Chen, X.-N., Lopes-Cendes, I., Pearlman, S., Starkman, S., Orozco-Diaz, G., Lunkes, A., Dejong, P., Rouleau, G.A., Auburger, G., Korenberg, J.R., Figueroa, C, and Sahba, S. (1996). Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2. Nat. Genet. 14(3), 269-276. Ranum, L.P., Chung, M.Y., Banfi, S., Bryer, A., Schut, L.J., Ramesar, R., Duvick, LA, McCall, A., Subramony, S.H., and Goldfarb, L. (1994). Molecular and clinical correlations in spinocerebellar ataxia type I: evidence for familial effects on the age at onset. Am. J. Hum. Genet. 55, 244-252. Ross,C.A., Becher,M.W., Colomer.V., Engelender,S., WoodJ.D., and Sharp,A.H. (1997). Huntington's disease and dentatorubral-pallidoluysian atrophy: proteins, pathogenesis and pathology. Brain Pathology. 7(3), 1003-1016. Sanpei, K., Takano, H., Igarashi, S., Sato, T., Oyake, M., Sasaki, H., Wakisaka, Tashiro, K., Ishida, Y., Dceuchi, T., Koide, R., Saito, M., Sato, A., Tanaka, Hanyu, S., Takiyama, Y., Nishizawa, M., Shimizu, N., Nomura, Y., Segawa, M., Iwabuchi, K., Eguchi, I., Tanaka, H., Takahashi, H., and Tsuji, S. (1996). Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique, DIRECT. Nat. Genet. 14, 277-284. 21 Chapter 1: Introduction Sapp, E., Schwarz, C, Chase, K., Bhide, P.G., Young, A.B., Penney, J., Vonsattel, J.P., Aronin, N., and DiFiglia, M. (1997). Huntingtin localization in brains of normal and huntingtons-disease patients. Ann. Neurol. 42, 604-612. Scherzinger, E., Lurz, R., Turmaine, M., Mangiarini, L., Hollenbach, B., Hasenbank, R., Bates, G.P., Davies, S.W., Lehrach, H., and Wanker, E.E. (1997). Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell 90, 549-558. Schilling,G., Sharp,A.H., Loev,S.J., Wagster,M.V., Li,S.H., Stine,O.C, and Ross; CA (1995). Expression of the Huntington's disease (IT 15) protein product in HD patients. Hum. Mol. Genet. 4(8), 1365-1371. SchmittJ., Bachner,D., Megow,D., Henklein,P., Hameister,P., EpplenJ., Reiss,0. (1995). Expression of the Huntington disease gene in rodents: cloning the rat homologue and evidence for downregulation in non-neuronal tissues during development. Hum. Mol. Genet. 4, 1173-1182. Skinner, P.J., Koshy, B.T., Cummings, C.J., Klement, I.A., Helin, K., Servadio, A., Zoghbi, H.Y., and Orr, H.T. (1997). Ataxin-1 with an expanded glutamine tract alters nuclear matrix- associated structures. Nature 389, 971-97'4. Stott, K., Blackburn, J.M., Butler, P.J., and Perutz, M. (1995). Incorporation of glutamine repeats makes protein oligomerize: implications for neurodegenerative diseases. Proc. Natl. Acad. Sci. USA 92, 6509-6513. Strong, T.V., Tagle, D.A., Valdes, J.M., Elmer, L.W., Boehm, K., Swaroop, M., Kaatz, K.W., Collins, F.S., and Albin, R.L. (1993). Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues. Nat. Genet. 5(3), 259-265. Takiyama, Y., Igarashi, S., Rogaeva, E.A., Endo, K., Rogaev, E.I., Tanaka, H., Sherrington, R., Sanpei, K., Liang, Y., Saito, M., Tsuda, T., Takano, H., Ikeda, M., Lin, C, Chi, H., Kennedy, J.L., Lang, A.E., Wherrett, J.R., Segawa, M., Nomura, Y., Yuasa, T., Weissenbach, J., Yoshida, M., Nishizawa, M., Kidd, K.K., Tsuji, S., and St. George-Hyslop, P.H. (1995). Evidence for inter-generational instability in the CAG repeat in the MJD1 gene and for conserved haplotypes at flanking markers amongst Japanese and Caucasian subjects with Machado-Joseph disease. Hum.Mol.Genet. 4(7), 1137-1146. Telenius, H., Kremer, H.P.H., Goldberg, Y.P., Theilmann, J., Andrew, S.E., Zeisler, J., Adam, S., Greenberg, C, Ives, E.J., Clarke, L.A., and Hayden, M.R. (1994). Somatic and gonadal mosaicism in the Huntington disease gene CAG repeat in brain and sperm. Nat. Genet. 6, 409-414. Trottier, Y., Devys, D., Imberg, G., Saudou, F., An, I., Lutz, Y., Weber, C, Agid, Y., Hirsch, E.C., and Mandel, J.-L. (1995). Cellular localization of the Huntington's disease protein and discrimination of the normal and mutated form. Nat. Genet. 10, 104-110. 22 Chapter 1: Introduction Varshavsky, A. (1997). The ubiquitin system. T.I.B.S. 6, 362-387. Verkerk, A.J., Pieretti, M., Sutcliffe, J.S., Fu, Y.H., Kuhl, D.P., Pizzuti, A., Reiner, O., Richards, S., Victoria, M.F., and Zhang, F.P. (1991). Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65, 905-914. Vonsattel, J.P., Myers, R.H., Stevens, T.J., Ferrante, R.J., Bird, E.D., Richardson, and Jr, E.P. (1985). Neuropathological classification of Huntington's disease. Journal of Neuropathology & Experimental Neurology 44, 559-577. Wanker, E.E., Rovira, C, Scherzinger, E., Hasenbank, R., Walter, S., Tait, D., Colicelli, J., and Lehrach, H. (1997). HIP-I: a huntingtin interacting protein isolated by the yeast two- hybrid system. Hum. Mol. Genet. 6, 487-495. White, J.K., Augood, S.J., Duyao, M.P., Vonsattel, J.-P., Gusella, J.F., Joyner, A.L., and MacDonad, M.E. (1997). Huntingtin is required for neurogenesis and is not impaired by the Huntington's disease CAG expansion. Nat. Genet. 17, 404-410. Wood, J.D., MacMillan, J.C., Harper, P.S., Lowenstein, P.R., and Jones, A.L. (1996). Partial characterisation of murine huntingtin and apparent variations in the subcellular localisation of huntingtin in human, mouse and rat brain. Hum. Mol. Genet. 5, 481-487. Zeitlin, S., Liu, J.P., Chapman, D.L., Papaioannou, V.E., and Efstratiadis, A. (1995). Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue. Nat. Genet. 11, 155-163. 23 Chapter 2: Methodology CHAPTER 2 - METHODOLOGY 24 Chapter 2: Methodology 2.1 THE YEAST TWO-HYBRID SYSTEM The protocol used to isolate the proteins described in this thesis was the yeast two- hybrid system (Fields and Song, 1989). The yeast two-hybrid system is based upon the function of the yeast GAL4 protein, which has distinct and separable domains that serve as DNA binding and transcriptional activation functions. The yeast two-hybrid system was the chosen mode to isolate huntingtin interacting proteins for three principal reasons. First, nothing was known regarding proteins that interact with huntingtin at the time this project was started. Secondly, at the time of embarking on this project HD cDNA constructs were being generated in Dr. Hayden's laboratory that would be amenable to using the with yeast two-hybrid system. Thirdly, as part of the Canadian Genetics Diseases Network a collaborative effort with yeast geneticist Dr. Dan Geitz (University of Manitoba) was established to expedite the teaching of the yeast two- hybrid. This design of the yeast two-hybrid system is outlined in Fig 2.1. The function of the GAL4 protein can be reconstituted to bind to its normal sequence, called the upstream activator sequence (UAS) driving a specific reporter gene in a particular yeast host. The system has been constructed so that multiple UAS fragments drive genes to allow for auxotrophic selection (Histidine) as well as chromogenic selection (LacT), The yeast two-hybrid system has both advantages and disadvantages. It is a system that should be considered as a method to identify proteins that interact with a protein of interest (the bait). Subsequent biochemical and functional analyses must be used to confirm the interaction between the potential interactors. A limitation is that in order to identify the 25 Chapter 2: Methodology interacting proteins, they must be found within the yeast nucleus in order to drive transcription of the reporter genes. Unfortunately, some proteins that are not normally found within the nucleus may contain sequences that can trans-activate the reporter constructs independent of interacting with any other protein. Some bait constructs appear to be able to activate transcription in the presence of the GAL4 activating domain alone, with no insert fused in-frame (Fritz and Green, 1992). The false-positive dilemma may be circumvented by fusing only portions of the bait in-frame with the GAL4 DNA binding domain, eliminating the region that causes the spurious result. The yeast two-hybrid system vectors use the 2 micron origin of replication on the vectors in order for appropriate in vivo replication. An inconsistent number of plasmids are maintained with each division of the yeast with the 2 micron origin of replication. This caveat results in a tremendous amount of variability in using quantitative p-galactosidase assays as a measure for interaction strength. The yeast two-hybrid approach provides an in vivo system to assess the interaction between two proteins. Biochemical techniques such as affinity purification, immunoprecipitation and far-western blots have the limitation of being in vitro. By using the yeast host translational machinery the yeast two-hybrid system ensures that the proteins causing the expression of the reporter genes play a role in such. However, it should be noted that even though the yeast system is in vivo, when assaying for an interaction between two human genes, some post-translational modifications that take place in the human cell might not be mimicked in yeast. 26 Chapter 2: Methodology Huntingtin (1-540; 44Q) GAL4 (1-147 1) (UAS)n LacZ/His HIPs GAL4 (768-881) 2) (UAS)n LacZ/His 3) (UAS)n LacZ/His 27 Chapter 2: Methodology Figure 2.1 The yeast two-hybrid system. The search for HIP cDNAs using the yeast two- hybrid system produced interacting proteins from a screen with only the first 540 amino acids of huntingtin. Panel 1 shows that the first 540 amino acids of huntingtin (with 44 glutamines) were fused in-frame with the DNA binding domain of the yeast GAL4 protein (residues 1-147). Alone the DNA BD cannot activate transcription of the reporter gene from the GAL upstream activator sequence (UAS). A human cDNA library constructed in a GAL4 activation domain vector (panel 2) allows for many times coverage of cDNAs to be screened for potential interacting proteins. The function of GAL4 can be reconstituted by the fusion of two proteins that bring the GAL4 BD, and the GAL4 AD in close proximity to each other (panel 3). A yeast host that has UAS upstream of a reporter gene can be used to test for this reconstitution of function. In the yeast two-hybrid system the auxotrophic histidine marker is used and an additional marker, LacZ, is used to chromogenically select for interacting proteins. 28 Chapter 2: Methodology The popularity of the two-hybrid system lies in its ease of use. Molecular biology techniques such as cloning, generation of libraries and protein assays, coupled with the power of yeast genetics provide a highly sensitive assay to screen an entire library for proteins that interact with the bait protein. Biochemical fractionation methods are arduous and not as sensitive as the yeast two-hybrid system (Guarente, 1993). In order to perform the yeast two-hybrid screens I cloned three different regions of the HD cDNA into the GAL4 DNA binding (BD) yeast two-hybrid vector, pGBT9. Greater than 80 % of the coding region was cloned into yeast two-hybrid DNA binding domain vectors in order to screen for HIPs (Fig 2.2). The only bait that isolated positive clones was the clone expressing the amino terminal region of huntingtin, where 14 positive clones were isolated, and 12 of such were identical (HIPl). The different pGBT9 bait constructs were chosen as a result of the availability of a full-length cDNA construct generated in Michael Hayden's lab by Dr. Paul Goldberg. Nco I restriction sites throughout the HD cDNA facilitated the cloning of a 5' fragment containing the CAG repeat, as well as other portions of the HD cDNA into either the Nco I or Sma I site of the commercially available pGBT9 or pAS2-l vectors (Clontech). 29 Chapter 2: Methodology 44pGBT9 70-2pAS2-l 5.3.10pAS2-l 44Q 540 729 1244 1244 3023 3144 I 1 1 4xl07 lxlO6 4xl06 clones clones clones 1 1 1 14 positives: 0 positives 0 positives 12 HIP1 1HIP2 1HIP3 Figure 2.2 Screening for Huntingtin interacting proteins. The screening of the yeast two- hybrid library resulted in only the amino terminal bait (44pGBT9) generating positive interactors. The clones 70-2pAS2-l and 5.3.10pAS2-l did not generate any positive HIPs. Only two small regions of huntingtin, 540 - 729 and 3023 - 3144, were not assessed in a two-hybrid screen. The number of transformants screened is listed as the number of Tryptophan/Leucine positive clones. The numbers listed on the top of the hatched boxes represent amino acid residue position within huntingtin. 30 Chapter 2: Methodology 2.2 METHODOLOGY 2.2.1 GAL4 cDNA constructs A huntingtin cDNA construct, with 44 CAG repeats, was generated encompassing amino acids 1 - 540 of the published huntingtin sequence (nucleotide 314-1955) (Goldberg et al., 1996). This cDNA fragment was fused in-frame into the Sma I site of the GAL4 DNA- binding domain (BD) of the yeast two-hybrid shuttle vector pGBT9 (Clontech) generating 44pGBT9. The clone 16pGBT9 was constructed by blunt-ending an Nco I fragment from the full length HD cDNA construct (Goldberg et al., 1996) into the Sma I site of pGBT9. 80pGBT9 and 128pGBT9 were constructed by blunt end cloning an Nco l-Not I fragment (HD amino acids 1-540) into the Sma I site of pGBT9 (Fig 2.2). The clones 70-2pAS2-l and 5.3.10pAS2-l (Fig 2.2) were generated by digesting a full-length HD cDNA construct with Nco I and gel purifying the regions encoding residues 729 - 1244 and 1244 - 3023, respectively. These DNA fragments were ligated into the Nco I site of the pAS2-1 vector (Clontech). The HJPlpGBT9 clone was produced by releasing the HIPl cDNA from the pGADIO vector with Not I and subsequently blunt ending that insert with the large fragment of Klenow. This blunt ended fragment was then ligated into a Sma I digested pGBT9 vector backbone. The A Pst clones were generated by digesting the parent 16, 44 and 128 clones with Pst I and ligated back onto itself. Deleted constructs were isolated by restriction digest analysis. This restriction digest released nucleotide 1007 through to 1955, subsequently allowing assessment of the minimal region of interaction between amino acids 1 - 240 of 31 Chapter 2: Methodology huntingtin. The clone HXpGBT9 spans from nucleotide 788 through 1955 and was generated by digesting the respective 1955 plasmid with Hind Eland Xho I. This fragment was then blunt ended using the large Klenow fragment of DNA polymerase, according to the manufacturers recommendations and ligated into a Sma I digested pGBT9 vector. The 16,44 and 128pGAD constructs were generated by digesting the HD-pGBT9 constructs with Eco RI and Bam HI and ligating into the same sites of pGADlO. All pGBT9 clones were proven to be in-frame with the GAL4 binding domain by DNA sequencing across the cloning fusion junction with the primer GAL4p ( 5' TCA TCG GAA GAG AGT AG 3'). As a test of evolutionary conservation between Sla2p and HIP1, the first 575 amino acids of Sla2p were tested in the two-hybrid system for its ability to interact with the first 540 amino acids of huntingtin. The Sla2pAS2-l GAL4 BD construct was a generous gift from D. Drubin. The parental 16/44 pGBT9 and 80/128 clones differed in their open reading frame 3' to the 1955 nucleotide. Oligonucleotides 15/44 (5' GAC CCT GCC ATG TGA GAT CCT CTA GAG 3') and 80/128 (5' GAC CCT GCC ATG TGA GGT ACC GAG CTC 3') were used to generate a stop codon (underlined) at the first codon 3' of the GAL4-BD-HD open reading frame. The site-directed mutagenesis was performed using the Transformer Site- Directed Mutagenesis kit (Clontech) as recommended by the manufacturer. Positive clones were verified by sequencing, restriction analysis, and PCR analysis of CAG repeat length. Clones containing 16,44 or 128 CAG repeats were generated using the site-directed mutagenesis approach. 32 j Chapter 2: Methodology Another clone (DMKA5amHIpGBT9) containing the first 544 amino acids of the DM kinase gene (a gift from R. Korneluk) was fused in-frame with the GAL4-BD of pGBT9 and was used as a negative control. The clones JTT5-23Q and IT15-44Q and HAP1 (Li et al., 1995), which represent the known huntingtin HAP1 interaction, were generous gifts from C. Ross (Johns Hopkins) and were used as a positive control for 8-galactosidase activity. 2.2.2 Yeast strains, transformations and p-galactosidase assay The yeast strain Y190 (MA 7a leu2-3,\\2, wrai-52, trpl-901, his3-D200, ade2-\Q\, gal4Agal80A, URA3::GAL-/acZ, LYS2::GAL-///S3,cvcr) (Durfee et al., 1993) was used for all transformations and assays. Yeast transformations were performed using a modified lithium acetate transformation protocol (Gietz et al., 1996) and grown at 30 °C using appropriate synthetic complete (SC) dropout media. The p-galactosidase chromogenic filter assays were performed by transferring the yeast colonies onto Whatman #3 filters. The filters were submerged in liquid nitrogen for 15-20 seconds to lyse the cells. The filters were allowed to dry at room temperature for at least five minutes and placed onto filter paper presoaked in Z-buffer (100 mM sodium phosphate (pH 7.0), 10 mM KC1, 1 mM MgS04) supplemented with 50 mM 2- mercaptoethanol and 0.07 mg/ml 5-bromo-4-chloro-3-indolyl P-D-galactoside. Filters were placed at 37 °C for up to 8 hours. Liquid P-galactosidase assays were performed by inoculating a single yeast colony into appropriate SC dropout media and grown to OD600 0.6-1.0. Five millilitres of overnight culture was pelleted and washed once with 1 ml of Z-Buffer, then resuspended in 100 (xl Z- 33 Chapter 2: Methodology Buffer supplemented with 38 mM 2-mercaptoethanol, and 0.05 % SDS. Acid washed glass beads (-100 (il) were added to each sample and vortexed for four minutes, by repeatedly alternating periods of vortexing (30 seconds) and incubation on ice (30 seconds). Each sample was pelleted and 10 |il of lysate was added to 500 \i\ of Z- buffer. The samples were incubated in a 30 °C water bath for 30 seconds and then 100 (il of a 4 mg/ml o-nitrophenyl P- D galactopyranoside solution was added to each tube. The reaction was allowed to continue for 20 minutes at 30 °C and stopped by the addition of 500 \i\ of 1 M Na2CC>3 and placing the samples on ice. Subsequently, OD420 was measured in order to calculate the p- galactosidase activity with the equation 1 000 x OD420 / (t x V x OD595) where t is the elapsed time (minutes) and V is the amount of lysate used (|il) (Paetkau et al., 1994). Each huntingtin-HIP assay was performed with at least 9 replicates. 34 Chapter 2: Methodology 2.2.3 Analysis of GAL4 DNA binding domain - huntingtin fusion protein expression in yeast Yeast colonies transformed with 16, 44 or 128 CAG repeats and HIP1 were picked and grown separately in 10 ml selective (-Trp/-Leu) liquid media overnight, shaking (300rpm) at 30 °C. The 10 ml cultures were each used to inoculate two 25 ml YPAD cultures and grown with shaking at 30 °C until OD600 of approximately 1.0. Non- transformed yeast colonies were grown in parallel as negative controls. Cells were centrifuged for 5 minutes at 1000 x g and 4 °C. Cell pellets were washed once with 25 ml of ice-cold PBS then transferred to eppendorf tubes and a final wash using 1ml PBS was performed. Pellets were frozen in liquid nitrogen and stored at -70 °C. Pellets were thawed and suspended in 5 volumes (1 ml) of 50 mM Tris-HCl, pH 8, 0.1 % Triton X-100, 0.5 % SDS, 1 mM PMSF, 1 mM benzamidine, 5 ug/ml leupeptin and 10 ug/ml soybean trypsin inhibitor. The cells were mechanically lysed by a brief sonication followed by five periods of 20 seconds vortexing with glass beads filling the suspension to the meniscus. The suspension was cooled on ice between each vortexing cycle. The cell extract was recovered by a 5 minutes 2 000 x g centrifugation of the suspension through a 1 ml pipette tip secured by an adapter (the top half of an eppendorf tube) to a 6 ml falcon collection tube nested in a 14 ml falcon tube. The collected extract was briefly sonicated and then centrifuged at 12 000 x g for 5 minutes at 4 °C. The cleared supernatant was concentrated lOx using Centricon-30 units. 7.5 % SDS-PAGE was performed using 375 (j,g of protein transferred to PVDF membranes at 30 V overnight. The GAL4-BD-huntingtin 35 Chapter 2: Methodology fusion protein was detected with ECL (Amersham) using a GAL4 DNA binding domain monoclonal primary antibody (Clontech) or an anti-huntingtin polyclonal antibody (not shown), BKP1 (Kalchman et al., 1996) with a horseradish-peroxidase labeled IgG mouse specific secondary antibody. 2.2.4 Screening for Huntingtin Interacting Proteins (HIPs) A human adult brain Matchmaker cDNA library (Clontech) was transformed into the yeast strain Y190 already harboring the 44pGBT9 construct. The transformants were plated onto one hundred 150 mm x 15 mm circular culture dishes containing SC media deficient in Trp, Leu and His. The herbicide 3-amino-triazole (3-AT) (25mM) was used to limit the number of false His+ positives (Durfee et al., 1993). The yeast transformants were placed at 30 °C for 5 days and (3-galactosidase filter assays were performed on all colonies found after this time, as described above, to identify p-galactosidase"1" clones. Primary His+/(3- galactosidase+ clones were then orderly patched onto a grid on SC -Trp/-Leu/-His (25 mM 3AT) plates and assayed again for His+ growth and the ability to turn blue with a filter assay. Secondary positives were identified for further analysis. Proteins encoded by positive cDNAs were designated as HIPs. The HIP cDNA plasmids were isolated by growing the His+/P-galactosidase+ colony in SC -Leu media overnight, lysing the cells with acid-washed glass beads and electroporating the bacterial strain, KC8 (leuB auxotrophic) with the yeast lysate. The KC8 ampicillin resistant colonies were replica plated onto M9 (-Leu) plates. The plasmid DNA from M9+ colonies was transformed into DH5-a for further manipulation. 36 Chapter 2: Methodology 2.2.5 DNA sequencing, cDNA isolation and 5' RACE 2.2.5.1 Elucidation of the HIP1 full-length cDNA sequence Oligonucleotide primers were synthesized on an ABIPCR-MATE oligo-synthesizer. DNA sequencing was performed using an ABI 373A fluorescent automated DNA sequencer. The HIP cDNAs were confirmed to be in-frame with the GAL4-AD by sequencing across the AD-HIP cloning junction using an AD oligonucleotide (G4AD2) (5' GAA GAT ACC CCA CCA AAC 3'). Subsequently, primer walking was used to determine the remaining sequences. A human frontal cortex > 4.0 kb cDNA library (a gift from S. Montal) was screened to isolate longer HIP1 cDNAs. Fifty nanograms of a 558 bp Eco RI fragment from the original HIP1 cDNA was radioactively labeled with [a 32P] - dCTP using random- priming and the probe allowed to hybridize to filters containing > 105 pfu/ml of the cDNA library overnight at 65 °C in Church buffer (see Northern blot protocol). The filters were washed at 65 °C for 10 minutes with 1 X SSPE, 15 minutes at 65 °C with 1 X SSPE, 0.1 % SDS, then for 30 minutes and 15 minutes with 1 X SSPE, 0.1 % SDS. The filters were exposed to X-ray film (Kodak, XAR5) overnight at -70 °C. Primary positives were isolated, replated and subsequent secondary positives were hybridized and washed as for the primary screen. The resulting positive phage were converted into plasmid DNA by conventional methods (Stratagene) and the cDNA was isolated and sequenced. In order to obtain the most 5' sequence of the HIP1 gene, Rapid Amplification of cDNA Ends (RACE) was performed according to the manufacturers recommendations 37 Chapter 2: Methodology (Gibco-BRL). First strand cDNA was synthesized using the oligo HIP1-242R (5' GCT TGA CAG TGT AGT CAT AAA GGT GGC TGC AGT CC 3'). After dCTP tailing the cDNA with terminal deoxynucleotidyl transferase, two rounds of 35 cycles (94 °C for 1 minute; 53 °C for 1 minute; 72 °C for 2 minutes) of PCR using HJP1-R2 (5' GGA CAT GTC CAG GGA GTT GAA TAC 3') and an anchor primer (5' (CUA)4 GGC CAC GCG TCG ACT AGT ACG GGI IGG Gil GGG IIG3') (Gibco-BRL) were performed. The subsequent 650 bp PCR product was cloned using the TA cloning system (Invitrogen) and sequenced using T3 and T7 primers. 2.2.5.2 Construction of the CMV-HIP1 expression construct The CMV-H1P1 DNA was prepared on a Qiagen miniprep column after overnight growth at 37 °C in LB broth (100 u,g/ml ampicillin). The HIPl protein was synthesized in the TnT-Rabbit Reticulocyte Lysate (RRL) system (Promega) as recommended by the manufacturer. Aliquots of the in vitro translation were separated on a 10 % SDS-PAGE system (Bio-Rad), and western blotted onto PVDF membrane. The PVDF membrane was subsequently exposed to X-ray film for 4 hours and an autoradiogram produced. After exposure to the film, the same membrane was immunoreacted with either anti-HIPl-pepl or anti-HIPl-FP. Detection of translated HIPl proteins was then detected after 1 second exposure using ECL via a goat-anti-rabbit HRP conjugated secondary antibody. In order to perform in vitro experiments a full length HIPl construct was generated. A Kpn I - Hpa I fragment was released from the HIPl 5' RACE product in the TA cloning vector. This fragment was subsequently ligated into a Kpn I - Hpa I digested cHIP3 clone in 38 Chapter 2: Methodology pBluescript (Stratagene). Finally, the full length H1P1 cDNA was digested from pBluescript with Kpn I and Sma I and ligated into same sites of the mammalian expression vector pCI (Promega). 2.2.5.3 HIP2 cDNA sequence In order to obtain the most 5' sequence of the hE2-25K gene, direct sequencing of a gel purified RT-PCR product was performed. First strand cDNA was generated using Superscript II reverse transcriptase, according to the manufacturers recommendations (BRL) following annealing of the anti-sense oligonucleotide 5' CCG TGC GGA GAG TCA TTG CAG CTG3 ' to total RNA. Subsequent PCR was performed using the same reverse primer used for the RT reaction and a forward primer (5' GAC ATG GCC AAC ATC GCG GTG CAG 3') derived from the bE2-25K nucleotide sequence. 2.2.6 DNA and amino acid sequence analyses Overlapping DNA sequence was assembled using the program Mac Vector or GeneRunner and sent via e-mail or Netscape to the BLAST server at NIH (http://www.ncbi.nlm.nih.gov) to search for sequence similarities with known DNA (blastra) or protein (blast/?) sequences. Amino acid alignments were performed with the program ClustalW. The alignment in GCG format was transferred to the program GeneDoc to produce the resulting figure. 39 Chapter 2: Methodology 2.2.7 Generation of anti-HIP antibodies 2.2.7.1 Anti-HIPl pepl polyclonal antibody The HJP1 peptide (VLEKDDLMDMDASQQN, amino acids 379-394) was synthesized with Cys on the N-terminus for the coupling, and coupled to Keyhole limpet hemocyanin (KLH) (Pierce) with succinimidyl 4-(N-maleimidomethyl) cyclohexame-1- carboxylate (Pierce). Female New Zealand White rabbits were injected with HIP1 peptide- KLH and Freund's adjuvant. Antibodies against the HIP1 peptide were purified from rabbit sera using affinity column with low pH elution. The affinity column was made by incubation of HIP1 peptide with activated Thiol-Sepharose (Pharmacia). 2.2.7.2 Anti-HIPl fusion protein polyclonal antibody The anti-HJPl fusion protein antibody was generated by releasing the 1.2 kb HIP1 cDNA isolated from pGADIO with Not I and ligating it into the Not I site of the GST expression vector, pGEX4T2 (Promega) and transformed into the host BL21 or UTC5600. In order to prepare purified HJP1 protein for injection, 5 ml of LB culture was inoculated with GST-HIP 1 and grown overnight at 37 °C. This 5 ml culture was subcultured into 1 litre of LB with 200 (Ig/L of ampicillin and grown to an OD60o of 0.8 - 1.2 at 37 °C. To induce expression of the GST-HIP 1 fusion protein, IPTG was added to a final concentration of 0.1 mM and allowed to shake for 3 hours to overnight at 30 °C. After induction, the cells were spun down at 5000 rpm for 10 minutes. The pellet was resuspended in 50 ml of 1 X PBS (pH 7.2), 1 mM PMSF and spun down at 5000 rpm for 10 minutes. 40 Chapter 2: Methodology The fusion protein was extracted from the host bacteria by resuspending the culture in 50 ml of extraction buffer (1 % Triton X-100, 1 mM EDTA, 0.2 mM EGTA, 1 mM PMSF/PBS buffer). Two hundred milligram of lysozyme was added to the 50 ml of extraction buffer and placed on ice for 20 minutes, with occasional vortexing. The cell suspension was sonicated in 15 ml Falcon tubes, centrifuged at 11 000 rpm for 10 minutes and the supernant saved. Glutathione-Sepharose 4B beads were prepared as per the manufacturers recommendations (Sigma). The bacterial lysate was added to 750 |ll of beads and placed rotating at 4 °C for 30 minutes in a 50 ml conical tube. After the adsorption was complete the beads were washed twice with 2 x excess amounts of PBS (pH 7.2), once with 1 x volume of 1 % Triton X-100 / PBS, and again washed with twice 2 x excess amounts of PBS. The 750 |il of beads was then transferred to an eppendorf tube and again washed with 2 x excess of PBS. The HIPl portion of the GST-HIP 1 fusion protein was cleaved from the Glutathione- GST complex by adding 50 units of thrombin (Promega) to the mix and placed at room temperature overnight. The cleaved HIPl protein was collected by centrifuging the beads and collecting the supernant. The purified HIPl protein was denatured by adding 1:1 4 M guanidine-HCl/PBS and heating for 4 hours at 60 °C. Aliquots were placed at -70 °C. The protein (-20 |ig) was coupled to keyhole limpet hemocyanin using succinimidyl 4-(Af-maleimidomethyl) cyclohexane-l-carboxylate. Two female New Zealand White rabbits 41 Chapter 2: Methodology were immunized with Freund's adjuvant. Antibodies were purified on an affinity column with the purified HIP1 protein coupled to activated CH Sepharose 4B beads. The H1P1 protein was dialyzed in coupling buffer (0.1 M NaHCC>3, pH 8, 0.5 M NaCl). The protein and Sepharose beads were mixed together in an affinity column, rotating at 4 °C for 4 hours. Excess active groups were blocked for one hour with 0.1 M Tris buffer (pH 8). Excess protein was washed through the column with coupling buffer followed by salt buffers of high pH (0.1 M Tris buffer, pH 8, 0.5 M NaCl) and low pH (acetic acid added drop-wise to 0.5 M NaCl in water until pH4). The beads were then equilibrated in PBS (pH 12)10.1 % sodium azide and stored at 4 °C. Diluted serum (1:1) in PBS was first passed through a 0.22 |im siring filter and then allowed to flow through the Sepharose-HIPl coupled column. After flow-through, the beads were washed with 5 x with excess PBS. The antibody was eluted using 3 column volumes of 0.1 M glycine (pH 2.5) and then neutralized by adding concentrated Tris base until the pH of the fraction was approximately 7. ELISA was performed to assess the titre of the antibody. 2.2.7.3 Anti-HIP3 pep3 polyclonal antibody The HIP3 peptide (RKTHIDDYSTWD, amino acids 31-42) was synthesized with Cys on the N-terminus for the coupling, and coupled to Keyhole limpet hemocyanin (KLH) (Pierce) with succinimidyl 4-(N-maleimidomethyl) cyclohexame-l-carboxylate (Pierce). Female New Zealand White rabbits were injected with H1P1 peptide-KLH and Freund's adjuvant. Antibodies against the HJP1 peptide were purified from rabbit sera using affinity column with low pH elution. The affinity column was made by incubation of HJP1 peptide with activated Thiol-Sepharose (Pharmacia). 42 Chapter 2: Methodology 2.2.8 Northern blot analysis and in situ hybridization of HIPl RNA was isolated using the single step method of homogenization in guanidinium isothiocyanate and fractionated on a 1.0 % agarose gel containing 0.6 M formaldehyde. The RNA was transferred to a Hybond-N membrane (Amersham) and cross-linked with ultraviolet radiation. Hybridization of the Northern blot with (3-actin as an internal control probe provided confirmation that the RNA was intact and had transferred. The 1.2 kb HIPl cDNA was labeled using random-priming and incorporation of [a32 P] -dCTP. Hybridization of the original 1.2 kb HIPl cDNA was carried out in Church buffer (0.5 M sodium phosphate buffer (pH 7.2), 2.7 % SDS, 1 mM EDTA) at 60 °C overnight. Following hybridization, Northern blots were washed once for 10 minutes in 0.2 X SSPE, 0.1 % SDS at room temperature and twice for 10 minutes in 0.15 X SSPE, 0.1 % SDS. Autoradiography was carried out from one to three days using Hyperfilm (Amersham) at -70 °C. For in situ hybridization the RNA probes were prepared using the plasmid gtl49 for HD (Lin et al., 1993) or a 558 bp subclone of HIPl. The anti-sense and sense single-stranded RNA probes were synthesized using T3 and T7 RNA polymerases and the In Vitro Transcription Kit (Clontech) with the addition of [a35 SJ-CTP (Amersham) to the reaction mixture. Sense RNA probes were used as negative controls. For HIPl studies normal C57BL/6 mice were used. Huntingtin probes were tested on two different transgenic mouse strains expressing full-length huntingtin, cDNA HD 10366 (44CAG) C57BL/6 mice and YAC HD10366 (18CAG) FVB/N mice. Frozen brain sections (10 um thickness) were placed onto silane-coated slides under RNase-free conditions. The hybridization solution 43 Chapter 2: Methodology contained 40 % formamide, 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.3 M NaCl, 10 mM sodium phosphate (pH 7.0), 1 x Denhardt's solution, 10 % dextran sulfate (pH 7.0), 0.2 % w/v sarcosyl, yeast tRNA (500 ug/ml) and salmon sperm DNA (200 ug/ml). The radiolabelled RNA probe was added to the hybridization solution to give 1 x 106 cpm/200 ul/section. Sections were covered with hybridization solution and incubated on formamide paper at 65 °C for 18 hours. After hybridization, the slides were washed for 30 minutes sequentially with 2 X SSC, 1 X SSC and high stringency wash solution (50 % formamide, 2 X SSC and 0.1 M dithiothreitol) at 65 °C, followed by treatment with RNase A (1 ug/ml) at 37 °C for 30 minutes, then washed again and air-dried. The slides were first exposed on autoradiographic film (p-max, Amersham) for 48 hours and developed for 4 minutes in Kodak D-19 followed by a 5 minute fixation in Fuji-fix. For longer exposures, the slides were dipped in autoradiographic emulsion (50 % in distilled water, NR-2, Konica, Japan), air-dried and exposed for 20 days at 4 °C then developed as described. Sections were counter-stained with methyl-green or Giemsa solutions. 2.2.9 GST-HJP2 fusion protein expression The HJP2 cDNA was released from the GAL4-AD library plasmid, pGADIO, by digestion with Not I, ligated into the Not I site of pGEX4T-2 (Pharmacia) and electroporated into DH5-a. A clone in the correct orientation was electroporated into the E. coli host BL21 (Pharmacia) for expression of the GST protein. A single colony of both GST-FflP2 and GST alone were inoculated into 5 ml of LB liquid media supplemented with 100 ug/ml of ampicillin and grown overnight at 37 °C with good aeration. The 5 ml culture was 44 Chapter 2: Methodology subcultured into a 30 ml LB culture supplemented with 100 |ig/ml of ampicillin and grown shaking overnight at 37 °C. The 30 ml culture was poured into 500 ml of 2 X YT media supplemented with 0.1 mM IPTG and 100 |lg/ml of ampicillin and grown shaking overnight at 26 °C. Two hundred and fifty millilitres aliquots of culture were pelleted and resuspended in 12.5 ml of ice cold 1 X PBS. The bacterial suspension was sonicated with 30 second intervals for 10 minutes. The supernatant was passed through a Glutathione-Sepharose (Pharmacia) column (500 The column was washed 3 times with 10 ml of ice cold 1 X PBS. One millilitre of 10 mM Glutathione in 50 mM Tris-HCl (pH 8.0) was used to elute the GST protein from the Glutathione beads. The eluted protein was subsequently diluted to a concentration of 1 mg/ml and dialyzed overnight against 1 X PBS to remove the Glutathione. To assess that the HIP2 protein was the human E2-25k homologue, an anti bovine E2-25k (bE2-25K) antibody was immunoreacted against purified GST-HIP2 protein on a western blot (1:5000), and detected using ECL of an HRP-conjugated goat-anti-rabbit secondary antibody, as suggested by the manufacturer (Amersham). 2.2.10 Generation of HD in vitro transcription-translation products Clones containing either 44 or 16 glutamine repeats, from amino acid 1 through 540 were cloned (Goldberg et al., 1996) into the RcCMV vector (Invitrogen). The in vitro HD products were synthesized according to the manufacturer's directions for the TnT-Rabbit Reticulocyte (Invitrogen). 45 Chapter 2: Methodology 2.2.11 Protein preparation and western blotting for expression studies Frozen human tissues were homogenized using a Polytron homogenizer in a buffer containing 0.25 M sucrose, 20 mM Tris-HCl (pH 7.5), 10 mM EGTA, 2 mM EDTA supplemented with 10 ug/ml of leupeptin, soybean trypsin inhibitor and 1 mM PMSF, then centrifuged at 4 000 rpm for 10 minutes at 4 °C to remove cellular debris. One hundred to 150 (ig/lane of protein was separated on SDS - PAGE (8 % acrylamide) mini-gels and then transferred to Immobilon-P membranes (Millipore) overnight in transfer buffer (25 mM Tris, 0.192 M glycine (pH 8.3), 10 % methanol) at 30 V as described (Towbin et al., 1979). Membranes were blocked for 1 hour at room temperature in 5 % skim milk/TBS (10 mM Tris-HCl (pH 7.5), 0.15 M NaCl). Antibodies against huntingtin (BKP1 - 1:500; GHM1 - 1:500), actin (1:500) or HJP1 (1:200) were added to blocking solution and incubated for 1 hour at room temperature. After 3 x 10 minutes washes in TBS-T (0.05 % Tween-20 in TBS), secondary antibody (1:10 000) (horseradish peroxidase conjugated IgG, Bio-Rad) was applied in blocking solution for 1 hour at room temperature. Membranes were washed and then incubated in chemiluminescent ECL solution and visualized using Hyperfilm-ECL (Amersham). In order to confirm that hE2-25K in fact encoded the hE2-25K protein, an affinity- purified polyclonal anti-bE2-25K antibody (Haldeman et al., 1995) was immunoreacted against the H1P2 fusion protein after transfer onto a PVDF membrane from a 10 % SDS- PAGE gel. Although this antibody is highly specific for detection of the E2-25K protein, it has been shown not to be useful as an immunoprecipitating antibody. The membranes were blocked in 5% skim milk powder (Carnation) and immunoreacted in blocking buffer with the anti-bE2-25K polyclonal antibody (1:5000) for one hour. After washing the membrane three 46 Chapter 2: Methodology times in TBS-T (Tris-Buffered Saline (pH 7.4); 0.05 % Tween-20), an HRP-conjugated secondary antibody (1:10000) (Bio-Rad) was immunoreacted against the blots for one hour followed by washing as described above. The blot was subsequently incubated with ECL solution (Amersham) and exposed to ECL-Hyperfilm (Amersham). An aliquot of purified bE2-25K was used as a positive control. The human embryonic kidney cell line HEK293 was grown in DMEM-F12 media. Cultured cells, human, mouse and rat tissues were sonicated in a lysis buffer containing protease inhibitors (0.25 mM sucrose, 20 mM Tris-HCl (pH 7.5), 10 mM EGTA, 2 mM EDTA, 1 mM Na3"V04, 20 mM p-glycerophosphate; with 10 ng/ml each of leupeptin, aprotinin, antipain, soybean trypsin inhibitor, pepstatin and 100 mM PMSF). Protein extracts (as specified in figure legends) were separated on 10 % SDS PAGE mini-gels and transferred to PVDF membrane (Millipore). Filters were then probed with an affinity-purified anti-bE2- 25K polyclonal antibody (Haldeman et al., 1995), with detection by enhanced chemiluminescence. 2.2.12 Biochemical assessment of huntingtin - HIP interactions 2.2.12.1 Co-immunoprecipitation of HIPl with huntingtin Control human brain (frontal cortex) lysate was prepared in the same manner as for the subcellular localization study (see below). Prior to immunoprecipitation, tissue lysate was centrifuged at 5 000 rpm for 2 minutes at 4 °C, then the supernatant was pre-cleared by incubating with excess amount of Protein A-Sepharose (Pharmacia) for 30 minutes at 4 °C, 47 Chapter 2: Methodology and centrifuged at the same condition. Fifty microlitres of supernatant (500 [ig protein) was incubated with or without antibodies (10 pig of GHM1) in the total 500 |il of incubation buffer (20 mM Tris-HCl (pH 7.5), 40 mM NaCl, 1 mM MgCl2) for 1 hour at 4 °C. Twenty microlitres of Protein A-Sepharose (1:1 suspension) was added and incubated for 1 hour at 4 °C. The beads were washed with washing buffer (incubation buffer containing 0.5 % Triton X-100) three times. The samples on the beads were separated using SDS-PAGE (7.5 % acrylamide) and transferred to Immobilon-P membrane. The membrane was cut at about 150 kDa after transfer for western blotting. The upper piece was probed with anti-huntingtin BKP1 (1:1 000) and lower piece with anti-HIPl antibody (1:300). 48 Chapter 2: Methodology 2.2.12.2 Subcellular fractionation of huntingtin and HIPl from brain tissue Cortical tissue (20-100 mg/ml) was homogenized on ice in a 2 ml Pyrex-teflon (IKA- RW15, Tekmar Company) homogenizer in a buffer containing 0.303 M sucrose, 20 mM Tris-HCl (pH 6.9), 1 mM MgCl2,0.5 mM EDTA, 1 mM PMSF, leupeptin, soybean trypsin inhibitor and benzamidine (Wood et al., 1996). Crude membrane vesicles were isolated by two cycles of a three-step differential centrifugation protocol in a Beckman TLA 120.2 rotor at 4 °C as described (Wood et al., 1996). The first step precipitated cellular debris and nuclei from tissue homogenates for 5 minutes at 1300 x g (PI). The 1300 x g supernatant was subsequently centrifuged for 20 minutes at 14 000 x g to isolate synaptosomes and mitochondria (P2). Finally, microsomal and plasma membrane vesicles were collected by centrifugation for 35 minutes at 142 000 x g (P3). The remaining supernatant was defined as the cytosolic fraction. Aliquots of P3 membranes were twice suspended at 2 mg/ml in 0.5 M NaCl, 10 mM Tris- Cl (pH 7.2), 2 mM MgCl2, containing protease inhibitors (see above). The same buffer without NaCl was used as a control. The membrane suspensions were incubated on ice for 30 minutes and then centrifuged at 142 000 x g for 30 minutes. To extract cytoskeletal proteins, crude membrane vesicles from the P3 fraction membrane were suspended in a volume of Triton X-100 extraction buffer to give a protein:detergent ratio of 5:1. Triton X-100 extraction buffer contained 2 % Triton X-100,10 mM Tris-HCl (pH 7.2), 2 mM MgCl2,1 mM leupeptin, soybean trypsin inhibitor, PMSF and benzamidine (Arai and Cohen, 1994). Membrane pellets were suspended by hand with a round-bottom teflon pestle, and placed on ice for 40 minutes. Insoluble cytoskeletal matrices were precipitated for 35 minutes at 142 000 x g. The supernatant was defined as non-cytoskeletal associated membrane or membrane- associated protein and was removed. The pellet was extracted with Triton X-100 a second time 49 Chapter 2: Methodology using the same conditions. The final pellet was defined as cytoskeletal and cytoskeletal-associated protein. Membrane and cytoskeletal protein was solubilized in a minimum volume of 1 % SDS, 3 M urea, 0.1 mM dithiothreitol in TBS and sonicated. Protein concentration was determined using the Bio-Rad DC Protein assay and samples were diluted at least 1 X with 5 X sample buffer (250 mM Tris-HCl (pH 6.8), 10 % SDS, 25 % glycerol, 0.02 % bromophenol blue and 7 % 2-mercaptoethanol) and were loaded on SDS-PAGE gels (7.5 % acrylamide) without boiling. Western blotting was performed as described above. 2.2.12.3 Coaffinity purification of huntingtin with GST-HJP2 Five microlitres of in vitro translated HD proteins (amino acids 1-540 with either 44 or 16 glutamine repeats) were incubated with GST-HIP2 and GST (10 ug each) in 500 ul of reaction buffer (20 mM Tris-HCl (pH 7.5) 120 mM NaCl) for 2 hours at 4 °C. Glutathione- Sepharose beads (10 ul) were then added and incubated for an additional 2 hrs. The beads were pelleted for 5 minutes, and washed 3 times with reaction buffer containing 3% NP-40. Samples were mixed with Laemmlis sample buffer, applied to 7.5% SDS-PAGE gel and transferred to PVDF membrane. Immunodetection was performed using one of two HD N- terminal polyclonal antibodies (AP78 or BKP1). For the experiments with 293 cell lysates, GST-HJP2 and GST were incubated with 300 ul of cell lysate (-500 ug of total protein) and 200 ul of reaction buffer. 50 Chapter 2: Methodology 2.2.12.4 Coimmunoprecipitation of huntingtin and ubiquitin An Epstein-Barr virus transformed cell line was used to determine if the HD protein is a substrate for ubiquitin conjugation. Cells from lymphoblasts of a heterozygote for HD were lysed in buffer containing NP-40, and supplemented with N-ethylmaleimide to inactivate endogenous de-ubiquitinating enzymes (Haas and Bright, 1985). Fifty micrograms of cell lysate was mixed with dilution buffer (50 mM Tris-HCl, pH 7.6, 1 mM EDTA, 1 % Triton X-100) to give a final volume of 50 |il. Five microlitres of affinity-purified rabbit polyclonal antibodies against ubiquitin were added and the mixture rotated at 4 °C for 3 hours. Protein-A-Sepharose (Sigma), 50 JLLI of a 1:1 slurry in dilution buffer, was then added, and the suspension was rotated at room temperature for 30 minutes. The resin was pelleted, then washed four times with 500 |xl of dilution buffer. The beads were suspended in 40 \i\ of 2 X SDS-PAGE sample buffer and boiled for one minute. The resin was pelleted and 20 |il aliquots were electrophoresed on 5.5 % and 10 % mini-gels. Proteins in each gel were transferred to PVDF membrane in a buffer containing 10 mM CAPS (pH 10) and 10 % methanol. The blot derived from the 10 % gel was probed with anti-ubiquitin antibodies. The blot derived from the 5.5 % gel was probed with the anti-HD monoclonal antibody GHM1. In both cases, detection was by ECL using a commercial secondary antibody. Samples with either no cell lysate or no anti-ubiquitin antibody were used as negative controls. A non-immunoprecipitated aliquot was used as a positive control for the detection of the HD protein. 51 Chapter 2: Methodology 2.2.13 In vitro experiments 2.2.13.1 Transfection of HD and HIP1 cDNA constructs into HEK293T cells Plasmid DNA for LacZ, pCI, HIP1 and/or HD were grown in DH5-a cells overnight in LB media (with ampicillin - 100 ug/ml) and purified over gravity columns as recommended by the manufacturer (Qiagen). HEK293T human embryonic kidney cells were seeded at approximately 4 X 105 cells per well in a 6 well 30 mm plate. The purified DNA was transiently transfected either alone, or co-transfected, into the HEK293-T cells by lipofection using Lipofectamine, as recommended by the manufacturer (Life Technologies). 2.2.13.2 Immunohistochemistry and immunofluorescence Twenty four to forty eight hours after transfection, HEK293T cells were washed 3 x in IX PBS, then fixed in 4 % paraformaldehyde for 15 minutes. The paraformaldehyde was removed by 3 washes in 1 X PBS (pH 7.2). The cells were permeabalized by exposing the cells to IX PBS (pH 7.2)/l % BSA / 0.25 % Saponin. A commercially available monoclonal antibody MAb2166 (1:2000) (Chemicon) for the N-terminal portion of huntingtin produced from a fusion protein and the polyclonal anti-HIPl fusion protein antibody (1:50) were used as primary antibodies for immunofluorescence. The secondary antibodies were goat anti- mouse IgG fraction coupled to Texas-red (1:100), or goat anti-rabbit IgG fraction coupled to FITC (1:100). Immunofluorescence was captured digitally using a CCD camera at either 60 or 100 times magnification and overlaid electronically. Brain tissue obtained from a normal C57BL/6 adult (6 months old) male mouse sacrificed with chloroform were perfusion-fixed with 4 % v/v paraformaldehyde 52 Chapter 2: Methodology (PFA)/10 mM phosphate buffer (4 % PFA). The brain tissues were removed, immersion fixed in 4 % paraformaldehyde for 1 day, washed in 10 mM phosphate buffered saline (pH 7.2) (PBS) for 2 days, and then equilibrated in 25 % sucrose PBS for a few days. The samples were then snap-frozen in Tissue Tek molds by isopentane cooled in liquid nitrogen. After warming to -20 °C, frozen blocks derived from frontal cortex, caudate/putamen, cerebellum and brainstem were cut into 14 Jim sections. Following washing in PBS, the tissue sections were processed for immunofluorescence. First, sections were blocked using 1 % BSA for 2 hours at room temperature then primary antibodies diluted with PBS (0.1 % BSA) were applied to sections overnight at 4 °C. Optimal dilutions for the polyclonal antibodies BKP1 and HIPl were 1:50. Using washes of 3 x 5 minutes in PBS (0.1 % BSA) at room temperature, sections were probed with biotinylated secondary antibody and then either streptavidin-fluorescein (HIPl) or Texas Red (BKP1) conjugates for 60 minutes each at room temperature. Sections developed using 3'-3'-diamino-benzidine-tetrahyrochloride and ammonium nickel sulfate were processed using the Vecta Stain Elite ABC kit (Vector). For controls, sections were treated as described above except that HIPl antibody aliquots were pre-absorbed with an excess of HIPl peptide as well as a peptide unrelated to HIPl prior to incubation with the tissue sections. 2.2.14 Genome mapping of HIPs: FISH detection system and image analysis The HIPl, HJP2 and HJP3 cDNAs isolated from the two-hybrid screen were mapped by fluorescent in situ hybridization (FISH) to normal human lymphocyte chromosomes counter-stained with propidium iodide and DAPI. Biotinylated probe was detected with avidin-fluorescein isothiocyanate (FITC). Images of metaphase preparations were captured 53 Chapter 2: Methodology by a thermoelectrically cooled charge coupled camera (Photometries). Separate images of DAPI banded chromosomes and FITC targeted chromosomes were obtained. Hybridization signals were acquired and merged using image analysis software and pseudo coloured blue (DAPI) and yellow (FITC) as described and overlaid electronically. 54 Chapter 2: Methodology 2.3 REFERENCE LIST Arai, M. and Cohen, J.A. (1994). Subcellular localization of the F5 protein to the neuronal membrane-associated cytoskeleton. J. Neurosc. Res. 38, 348-357. Durfee, T., Becherer, K., Chen, P.-L., Yeh, S.-H., Yang, Y., Kilburn, A.E., Lee, W.-H., and Elledge, S.J. (1993). The retinoblastoma protein associates with the protein phosphatase type 1 catalystic subunit. Genes and Develop. 7, 555-569. Fields, S. and Song, O. (1989). A novel genetic system to detect protein-protein interacts. Nature 340, 245-246. Fritz, C.C. and Green, M.R. (1992). Fishing for partners: A method for studying protein- protein interactions in vivo is beginning to bear fruit. Curr. Biol. 2, 403-405. Gietz, R.D., Woods, R.A., Manivasakam, P., and Schiestl, R.H. (1996). Yeast growth and yeast transformation. In Cell Biology: A Laboratory Manual. D. Spector, R. Goldman, and L. Leinwand, eds. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press),) Goldberg, Y.P., Kalchman, M.A., Metzler, M., Nasir, J., Zeisler, J., Graham, R., Koide, H.B., O'Kusky, J., Sharp, A.H., Ross, C.A., Jirik, F., and Hayden, M.R. (1996). Absence of disease phenotype and intergenerational stability of the CAG repeat in transgenic mice expressing the human Huntington disease transcript. Hum. Mol. Genet. 5, 177-185. Guarente, L. (1993). Strategies for the identification of interacting proteins. Proc. Natl. Acad. Sci. USA 90, 1639-1641. Haas, A.L. and Bright, P.M. (1985). The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. J. Biol. Chem. 260, 12464-12473. Haldeman, M.T., Finley, D., and Pickart, CM. (1995). Dynamics of ubiquitin conjugation during erythroid differentiation in vitro. J. Biol. Chem. 270, 9507-9516. Kalchman, M.A., Graham, R.K., Xia, G., Koide, H.B., Hodgson, J.G., Graham, K.C, Goldberg, Y.P., Gietz, R.D., Pickart, CM., and Hayden, M.R. (1996). Huntingtin is ubiquitinated and interacts with a specific ubiquitin-conjugating enzyme. J. Biol. Chem. 271, 19385-19394. Li, X.J., Li, S.H., Sharp, A.H., Nucifora, F.C.J., Schilling, G., Lanahan, A., Worley, P., Snyder, S.H., and Ross, CA. (1995). A huntingtin-associated protein enriched in brain with implications for pathology. Nature 378, 398-402. Lin, B., Rommens, J.M., Graham, R.K., Kalchman, M., MacDonald, H., Nasir, J., Delaney, A., Goldberg, Y.P., and Hayden, M.R. (1993). Differential 3' polyadenylation of the 55 Chapter 2: Methodology Huntington disease gene results in two mRNA species with variable tissue expression. Hum. Mol. Genet. 2, 1541-1545. Paetkau, D.W., Riese, J.A., MacMorran, W.S., Woods, R.A., and Gietz, R.D. (1994). Interaction of the yeast RAD7 and SIR3 proteins: implications for DNA repair and chromatin structure. Genes and Develop. 8, 2035-2045. Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from poly aery 1 amide gels to nitrocellulose sheets: procedure and some applications Proc. Natl. Acad. Sci. USA 76, 4350-4354. Wood, J.D., MacMillan, J.C., Harper, P.S., Lowenstein, P.R., and Jones, A.L. (1996). Partial characterisation of murine huntingtin and apparent variations in the subcellular localisation of huntingtin in human, mouse and rat brain. Hum. Mol. Genet. 5, 481-487. 56 Chapter 3: Huntingtin-HIPl and the cytoskeleton CHAPTER 3 - HUNTINGTIN INTERACTING PROTEIN 1 A majority of the data presented in this chapter contributed to the manuscript: Kalchman MA, Koide HB, McCutcheon K, Graham RK, Nichol K, Nishiyama K, Kazemi- Esfarjani P, Lynn FC, Wellington CL, Metzler M, Goldberg YP, Kanazawa, Gietz RD, Hayden MR HJP1, a Human Homolog of S. cerevisiae Sla2p, Interacts with Membrane- Associated Huntingtin in the Brain, Nature Genetics (1997). 16,1: 44-53. 57 Chapter 3: Huntingtin-HIPl and the cytoskeleton 3.1 INTRODUCTION HIPl was isolated 12 times from the yeast two-hybrid screen for huntingtin interacting proteins. The original cDNA sequence of HIPl revealed that it shares identity with a protein from S. cerevisiae, Sla2p/End4. Sla2p/End4 is a membrane associated protein involved in the maintenance of the yeast cytoskeleton and also plays a significant role in vesicle formation and/or receptor mediated endocytosis. HIPl is highly expressed in neuronal tissue and shares similar biochemical properties as huntingtin. Both HIPl and huntingtin associate with the cytoskeleton and are co-purified with cellular vesicles. The data presented in this chapter provides the first molecular link between huntingtin and the neuronal cytoskeleton and suggests that, in HD, loss of normal huntingtin-HIPl interaction may contribute to a defect in membrane-cytoskeletal integrity in the brain. 58 Chapter 3: Huntingtin-HIPl and the cytoskeleton 3.2 RESULTS 3.2.1 Isolation of HIPlpGADIO Of the approximately 4.0 x 107 Trp/Leu auxotrophic transformants screened, 12 identical HJP1 clones were isolated as Trp/Leu/His prototrophs and LacZ positive. The 12 HIP1 cDNA clones were deduced by restriction analysis and DNA sequencing to be identical. The HIPl-GAL4-Activating Domain (AD) cDNA activated both the LacZ and HIS3 reporter genes in the yeast strain Y190 when co-transformed with the GAL4-Binding Domain (BD)-HD construct, but not with the negative controls (Fig 3.1). Constructs that contained the additional 40 amino acid tail (with 80 or 128 repeats) turned blue more slowly than both the 16 or 44 repeats with the tail or even when compared to the 128 construct with no tail. HAP1 (AD), a protein previously shown to interact with huntingtin (Li et al., 1995) was co-transformed with HD yeast two-hybrid constructs IT15-23Q (BD) and 1T15-44Q (BD) (a generious gift from Dr. C. Ross, Johns Hopkins University) were used as positive controls. The BD vector alone (pGBT9) or a non-related fusion protein (DMKAfiam HIpGBT9) were used as negative controls. Neither the APst IpGBT9 clones nor the HXpGBT9 clones showed a positive interaction. Previous findings that huntingtin interacts with GAPDH (Burke et al., 1996) was attempted using the yeast two-hybrid system but no interaction was within the first 540 amino acids of huntingtin. Furthermore, although huntingtin aggregate formation is observed in cultured cells and neurons (Martindale et al., 1998; DiFiglia et al., 1997; Sapp et al., 1997), again the yeast two-hybrid approach failed to detect homodimerization of the region of huntingtin containing the polyglutamine tract (Fig 3.1). 59 Chapter 3: Huntingtin-HIPl and the cytoskeleton The first 575 amino acids of the S. cerevisiae homologue of HIPl, Sla2p, was cloned into the GAL4BD vector pAS2-l (a generous gift from Dr. Dave Drubin, Stanford University) (Clontech) and assessed for its ability to interact with huntingtin. In order to assess if the interaction between huntingtin and HIPl is a conserved interaction, a [3- galactosidase filter assay was performed on yeast harbouring both the amino terminus of huntingtin (in the pGAD vector) and the first 575 amino acids of Sla2p. No interaction was observed between huntingtin and Sla2p. 60 Chapter 3: Huntingtin-HIPl and the cytoskeleton 16pGBT9 + HIPl USAPst IpGBT9 + HIPl 44pGBT9 + HIPl SLA2pAS2-l + 128pGAD424 128pGBT9 + HIPl jj HIPlpGBT9 + 128pGAD424 pGBT9 + HIPl 16pGBT9 + 16pGAD424 DMKpGBT9 + HIPl | 16pGBT9 + 128pGAD424 g IT15-23Q + HAP1 128pGBT9 + GAPDH , ~] IT15-44Q + HAP1 | ] SCAlpAS2-l + GAPDH Figure 3.1 P-galactosidase filter assays demonstrating the interaction between huntingtin and HIPl. The interaction occurs when huntingtin is expressed in either the GAL4BD or the GAL4AD. Huntingtin failed to interact with the yeast homolog of HIPl, Sla2p. HIPl did not interact with any of the negative controls. Although huntingtin aggregates have been shown to be produced in vitro, the two-hybrid system failed to detect an interaction when BD and AD constructs with 16 and 16 or 16 and 128 polyglutamines were co-transformed into the yeast reporter strain. Furthermore, no interaction with GAPDH could be detected with a huntingtin construct containing 128 polyglutamines, eventhough the SCA1-GAPDH interaction was detected. Failure to detect the huntingtin-GAPDH interaction may be a result of the low levels of expression of the two constructs in the yeast host or due to the nature of the conformation of huntingtin within the yeast host. 61 Chapter 3: Huntingtin-HIPl and the cytoskeleton To ensure that equal levels of expression of each of the constructs were not influenced by the size of the polyglutamine tract, a western blot was performed on yeast extracts using an anti- GAL4 monoclonal antibody (Clontech) from hosts carrying the 16,44 or 128pGBT9 constructs (Fig 3.2). No immunoreaction was observed in the yeast control. Figure 3.2 Western blot of the GAL4 DNA binding domain vectors expressing different sized polyglutamine tracts. The western blot of yeast extracts from cells expressing indicated GAL4BD-huntingtin fusion proteins (250 p:g) using an anti-GAL4BD monoclonal antibody (Clontech). The results show no differences in CAG dependent expression in the yeast host. 62 Chapter 3: Huntingtin-HIPl and the cytoskeleton 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 Subsequent screening of a human adult frontal cortex cDNA library using the HJP1 cDNA resulted in the isolation of a 4392 bp cDNA (cHIP3). Open reading frame analysis revealed a long open reading frame. 5' RACE and other 5' genomic sequence was added and a putative full-length cDNA sequence was assembled. The H1P1 clone isolated from the yeast two-hybrid screen corresponds to nucleotides 1414-2573 of the full-length HJP1 cDNA (Fig 3.3). The cHJP3 cDNA isolated corresponds to nucleotides 1161-5553 of the full length HJP1 cDNA. Rapid Amplification of cDNA Ends (RACE) was used to extend available cDNA sequence as far 5' as possible. However, the most 5' in-frame (ATG) that was identified within the RACE product generated a truncated HIP1 cDNA as later determined by in vitro translation assessment. The sequence surrounding this putative ATG initiation codon (at nucleotide 1031) is GAGTGACatgA. The Kozak consensus sequence stipulates that a purine at -3 is essential and a G at +4 is preferred. The minimum Kozak consensus sequence is RNNatgG (where R is a purine and N is any of the four nucleotides) and the most preferential context for an ATG start codon is CCAGGatgG (Kozak, 1996; Kozak, 1995). Just recently, genomic sequence made available from Dr. Stephen Schearer contains a 5' ATG start codon (nucleotide 503) in the context AATTGCCatgT that was not seen in the RACE product nor in any cDNA cloned. Although this is not the best Kozak consensus sequence, it is suitable (Kozak, 1995). Thus, the predicted HJP1 coding region spans 3149 nucleotides, encoding 1049 amino acids with a predicted molecular weight of 114 K (Fig. 63 Chapter 3: Huntingtin-HIPl and the cytoskeleton 3.4). The HJP1 nucleotide and amino acid sequences have been submitted to Genbank (accession # U79734). ATG (503) Stop (3652) 1 7289 I* I 1414 2573 J! HIPlpGADIO 1161 5553 cHIP3 787 |^io3l 1244 HIP1 RACE 1 787 5553 7289 Genomic sequence Cosmid 181G10 Figure 3.3 HJP1 cDNA contig. The HIPlpGADIO cDNA was the cDNA isolated from the yeast two-hybrid screen. Subsequent screening of a cDNA library, 5' RACE and genomic sequence has generated the contig seen above. The putative ATG is at position 503 (black arrow) and the stop codon at 3652, resulting in a 114 kDa protein that is encoded by 3149 nucleotides. The gray arrow represent the CMV-HIP1 ATG start codon that was originally thought to be the most 5' start site, however, genomic sequence has identified a stretch of DNA that has coding potential in-frame with the HJP1 cDNA isolated and used for expression studies. 64 Chapter 3: Huntingtin-HJPl and the cytoskeleton A) 1 TGTGGGAAAACAAAGACTTAGTGACCACCGCCGGTGCTGGCCAGCCGGAGAAGCTCTGTGGA AGGTTTGGAGGGGAGAGAGGGGCAGCTGGATGCTCTTGGGCCACGGTCGCCCCTGATCTCTG CGCCTCTTCCTCCTGCTCCGGGAGAAATAATGTTTCCCTGGGGGATGAAAGCATCTCTTTGT GCGGGCTTTAATTGCCATGTTGTTGTGCCAAGGGAGTGAGTGGCGGCGGGACCAGCAGCTG GGCACAGCCAATGCCAGGCAGTGGTGCCCACTCCCTCAGGACGCCCAGCCAGCTGGCTCCTG GGAGCGCTGCCCACCTCTGCCCCCAGCTGGGCGCCTGCAAGGAACCGACCACCCGTGGGGCT GGGGGAGGTTGGCTGGAGGAGGAGAAAGGGGCGGGCTCTGGGAGGGTCTCAGCCACTCTCAG AGGCTTATTCATCTCATCCTCCTTTCCCTCCCCCTTCTTGTTTTTCAGACTGTCAGCATCAA TAAGGCCATTAATACGCAGGAAGTGGCTGTAAAGGAAAAACACGCCAGAACGTGCATACTGG GCACCCACCATGAGAAAGGGGCACAGACCTTCTGGTCTGTTGTCAACCGCCTGCCTCTGTCT AGCAACGCAGTGCTCTGCTGGAAGTTCTGCCATGTGTTCCACAAACTCCTCCGAGATGGACA CCCGAACGTCCTGAAGGACTCTCTGAGATACAGAAATGAATTGAGTGACATGAGTGACCGCC AGCTGGACGAGGCTGGAGAAAGTGACGTGAACAACTTTTTCCAGTTAACAGTGGAGATGTTT GACTACCTGGAGTGTGAACTCAACCTCTTCCAAACAGTATTCAACTCCCTGGACATGTCCCG CTCTGTGTCCGTGACGGCAGCAGGGCAGTGCCGCCTCGCCCCGCTGATCCAGGTCATCTTGG ACTGCAGCCACCTTTATGACTACACTGTCAAGCTTCTCTTCAAACTCCACTCCTGCCTCCCA GCTGACACCCTGCAAGGCCACCGGGACCGCTTCATGGAGCAGTTTACAAAGTTGAAAGATCT GTTCTACCGCTCCAGCAACCTGCAGTACTTCAAGCGGCTCATTCAGATCCCCCAGCTGCCTG AGAACCCACCCAACTTCCTGCGAGCCTCAGCCCTGTCAGAACATATCAGCCCTGTGGTGGTG ATCCCTGCAGAGGCCTCATCCCCCGACAGCGAGCCAGTCCTAGAGAAGGATGACCTCATGGA CATGGATGCCTCTCAGCAGAATTTATTTGACAACAAGTTTGATGACATCTTTGGCAGTTCAT 65 Chapter 3: Huntingtin-HIPl and the cytoskeleton TC AGC AGTGATC C C TTC AATTTC AAC AGTC AAAATGGTGTG7AAC AAGGATGAG7AAGGAC C AC TTAATTGAGCGACTATACAGAGAGATCAGTGGATTGAAGGCACAGCTAGAAAACATGAAGAC TGAGAGCCAGCGGGTTGTGCTGCAGCTGAAGGGCCACGTCAGCGAGCTGGAAGCAGATCTGG CCGAGCAGCAGCACCTGCGGCAGCAGGCGGCCGACGACTGTGAATTCCTGCGGGCAGAACTG GACGAGCTCAGGAGGCAGCGGGAGGACACCGAGAAGGCTCAGCGGAGCCTGTCTGAGATAGA AAGGAAAGCTCAAGCCAATGAACAGCGATATAGCAAGCTAAAGGAGAAGTACAGCGAGCTGG TTCAGAACCACGCTGACCTGCTGCGGAAGAATGCAGAGGTGACCAAACAGGTGTCCATGGCC AGACAAGCCCAGGTAGATTTGGAACGAGAGAAAAAAGAGCTGGAGGATTCGTTGGAGCGCAT CAGTGACCAGGGCCAGCGGAAGACTCAAGAACAGCTGGAAGTTCTAGAGAGCTTGAAGCAGG AACTTGCCACAAGCCAACGGGAGCTTCAGGTTCTGCAAGGCAGCCTGGAAACTTCTGCCCAG TCAGAAGCAAACTGGGCAGCCGAGTTCGCCGAGCTAGAGAAGGAGCGGGACAGCCTGGTGAG TGGCGCAGCTCATAGGGAGGAGGAATTATCTGCTCTTCGGAAAGAACTGCAGGACACTCAGC TCAAACTGGCCAGCACAGAGGAATCTATGTGCCAGCTTGCCAAAGACCAACGAAAAATGCTT CTGGTGGGGTCCAGGAAGGCTGCGGAGCAGGTGATACAAGACGCCCTGAACCAGCTTGAAGA ACCTCCTCTCATCAGCTGCGCTGGGTCTGCAGATCACCTCCTCTCCACGGTCACATCCATTT CCAGCTGCATCGAGCAACTGGAGAAAAGCTGGAGCCAGTATCTGGCCTGCCCAGAAGACATC AGTGGACTTCTCCATTCCATAACCCTGCTGGCCCACTTGACCAGCGACGCCATTGCTCATGG TGCCACCACCTGCCTCAGAGCCCCACCTGAGCCTGCCGACTCACTGACCGAGGCCTGTAAGC AGTATGGCAGGGAAACCCTCGCCTACCTGGCCTCCCTGGAGGAAGAGGGAAGCCTTGAGAAT GCCGACAGCACAGCCATGAGGAACTGCCTGAGCAAGATCAAGGCCATCGGCGAGGAGCTCCT GCCCAGGGGACTGGACATCAAGCAGGAGGAGCTGGGGGACCTGGTGGACAAGGAGATGGCGG CCACTTCAGCTGCTATTGAAACTGCCACGGCCAGAATAGAGGAGATGCTCAGCAAATCCCGA GCAGGAGACACAGGAGTCAAATTGGAGGTGAATGAAAGGATC C TTGGTTGC TGTAC CAGCCT 66 Chapter 3: Huntingtin-HJPl and the cytoskeleton CATGCAAGCTATTCAGGTGCTCATCGTGGCCTCTAAGGACCTCCAGAGAGAGATTGTGGAGA GCGGCAGGGGTACAGCATCCCCTAAAGAGTTTTATGCCAAGAACTCTCGATGGACAGAAGGA C TTATC TCAGC C TC CAAGGCTGTGGGC TGGGGAGCCAC TGTCATGGTGGATGCAGC TGATC T GGTGGTACAAGGCAGAGGGAAATTTGAGGAGCTAATGGTGTGTTCTCATGAAATTGCTGCTA GCACAGCCCAGCTTGTGGCTGCATCCAAGGTGAAAGCTGATAAGGACAGCCCCAACCTAGCC CAGCTGCAGCAGGCCTCTCGGGGAGTGAACCAGGCCACTGCCGGCGTTGTGGCCTCAACCAT TTCCGGCAAATCACAGATCGAAGAGACAGACAACATGGACTTCTCAAGCATGACGCTGACAC AGATCAAACGCCAAGAGATGGATTCTCAGGTTAGGGTGCTAGAGCTAGAAAATGAATTGCAG AAGGAGCGTCAAAAACTGGGAGAGCTTCGGAAAAAGCACTACGAGCTTGCTGGTGTTGCTGA GGGCTGGGAAGAAGGAACAGAGGCATCTCCACCTACACTGCAAGAAGTGGTAACCGAAAAAG AATAGAGCCAAACCAACACCCCATATGTCAGTGTAAATCCTTGTTACCTATCTCGTGTGTG TTATTTCCCCAGCCACAGGCCAAATCCTTGGAGTCCCAGGGGCAGCCACACCACTGCCATTA CCCAGTGCCGAGGACATGCATGACACTTCCCAAAGACTCCCTCCATAGCGACACCCTTTCTG TTTGGACCCATGGTCATCTCTGTTCTTTTCCCGCCTCCCTAGTTAGCATCCAGGCTGGCCAG TGCTGCCCATGAGCAAGCCTAGGTACGAAGAGGGGTGGTGGGGGGCAGGGCCACTCAACAGA GAGGACCAACATCCAGTCCTGCTGACTATTTGACCCCCACAACAATGGGTATCCTTAATAGA GGAGCTGCTTGTTGTTTGTTGACAGCTTGGAAAGGGAAGATCTTATGCCTTTTCTTTTCTGT TTTCTTCTCAGTCTTTTCAGTTTCATCATTTGCACAAACTTGTGAGCATCAGAGGGCTGATG GATTCCAAACCAGGACACTACCCTGAGATCTGCACAGTCAGAAGGACGGCAGGAGTGTCCTG GCTGTGAATGCCAAAGCCATTCTCCCCCTCTTTGGGCAGTGCCATGGATTTCCACTGCTTCT TATGGTGGTTGGTTGGGTTTTTTGGTTTTGTTTTTTTTTTTTAAGTTTCACTCACATAGCCA ACTCTCCCAAAGGGCACACCCCTGGGGCTGAGTCTCCAGGGCCCCCCAACTGTGGTAGCTCC AGCGATGGTGCTGCCCAGGCCTCTCGGTGCTCCATCTCCGCCTCCACACTGACCAAGTGCTG 67 Chapter 3: Huntingtin-HIPl and the cytoskeleton GCCCACCCAGTCCATGCTCCAGGGTCAGGCGGAGCTGCTGAGTGACAGCTTTCCTCAAAAAG CAGAAGGAGAGTGAGTGCCTTTCCCTCCTAAAGCTGAATCCCGGCGGAAAGCCTCTGTCCGC CTTTACAAGGGAGAAGACAACAGAAAGAGGGACAAGAGGGTTCACACAGCCCAGTTCCCGTG ACGAGGCTCAAAAACTTGATCACATGCTTGAATGGAGCTGGTGAGATCAACAACACTACTTC CCTGCCGGAATGAACTGTCCGTGAATGGTCTCTGTCAAGCGGGCCGTCTCCCTTGGCCCAGA GACGGAGTGTGGGAGTGATTC CCAAC TC CTTTC TGCAGACGTC TGC C TTGGCATCC TC TTGA ATAGGAAGATCGTTCCACTTTCTACGCAATTGACAAACCCGGAAGATCAGATGCAATTGCTC CCATCAGGGAAGAACCCTATACTTGGTTTGCTACCCTTAGTATTTATTACTAACCTCCCTTA AGCAGCAACAGCCTACAAAGAGATGCTTGGAGCAATCAGAACTTCAGGTGTGACTCTAGCAA AGCTCATCTTTCTGCCCGGCTACATCAGCCTTCAAGAATCAGAAGAAAGCCAAGGTGCTGGA CTGTTACTGACTTGGATCCCAAAGCAAGGAGATCATTTGGAGCTCTTGGGTCAGAGAAAATG AGAAAGGACAGAGCCAGCGGCTCCAACTCCTTTCAGCCACATGCCCCAGGCTCTCGCTGCCC TGTGGACAGGATGAGGACAGAGGGCACATGAACAGCTTGCCAGGGATGGGCAGCCCAACAGC ACTTTTCCTCTTCTAGATGGACCCCAGCATTTAAGTGACCTTCTGATCTTGGGAAAACAGCG TCTTCCTTCTTTATCTATAGC7AACTCATTGGTGGTAGCCATC7AAGCACTTCCCAGGATCTGC TC C AAC AGAATATTGC T AGGTTTTGC TAC ATGACGGGTTGTGAGAC TTC TGTTTGATC AC TG TGAACCAACCCCCATCTCCCTAGCCCACCCCCCTCCCCAACTCCCTCTCTGTGCATTTTCTA AGTGGGACATTCAAAAAACTCTCTCCCAGGACCTCGGATGACCATACTCAGACGTGTGACCT CCATACTGGGTTAAGGAAGTATCAGCACTAGAAATTGGGCAGTCTTAATGTTGAATGCTGCT TTCTGCTTAGTATTTTTTTGATTCAAGGCTCAGAAGGAATGGTGCGTGGCTTCCCTGTCCCA GTTGTGGCAACTAAACCAATCGGTGTGTTCTTGATGCGGGTCAACATTTCCAAAAGTGGCTA GTCCTCACTTCTAGATCTCAGCCATTCTAACTCATATGTTCCCAATTACCAAGGGGTGGCCG GGCACAGTGGCTCACGCCTGTAATCCCAGCACTTTGAGAGGCTGAGGTGGTAGGATCACCTG 68 Chapter 3: Huntingtin-HJPl and the cytoskeleton AGGTCAGGAGTTCAAGACCAGCCTGTCCAACATGGTGAAACCCCCATCTCTACTAAAAATAC CAAAAATTAGCCGAGCGTAGTGACGGGTGCCCGTAATCCCAGCTACTCAGGAGGCTGAGACA GGAGAATCACCTGAACCCCAGAGGCAGAGGTTGCAGTGAGCTGAGATCACGCCATTGTACTC CAGCCTGGGCAACAAGAGCAAAACTCCGTCTCAAAAAAAAAAAAAAATTACAAATGGGGCAA ACAGTCTAGTGTAATGGATCAAATTAAGATTCTCTGCCCAGCCGGGCACAGTGGCGCATGCC TGTAATCCCAGAACTTTGGGAGGCCAAGACGGGATGATTGCTTGAGCTCAGGAGTTTGAGAC CAGGCTGGGCATCATAGCAAGACCTCATCTCTACTAAAATTCAAAAACAAAATTAGCCGGGC ATGATGGTGCATGC C TGTAGTCTCAGC TAGTTGGGGAGC TAAGGTGGGAGAATTGC TTGAGC TTGGGAAGTCGAGGCTGCAGTCAGCCCTGATTGTGCCAGTGCACTCCGGCCTGGGTGACAGA GTGAGACCCGTGCTCAAAAAAAAAAAGATTCTGTGTCAGAGCCCAGCCCAGGAGTTTGAGGC TGCAATGAGCCATGATTTCCCACTGCACTCCAGCCTGAGTGACAGAGCGAGACTCCATCTCT TTAAAAACAAACAAAAAATTATC TGAATGATCCTGTC TC TAAAAAGAAGC CACAGAAATGTT TAAAAACTTCATCGACTTAGCCTGAGTCATAACGGTTAAGAAAGCACTTAAACAGAAGCAGA GGCTAATTCAGTGTCACATGAGGAAGTAGCTGTCAGATGTCACATAATTACTTTCGTAATAG CTCAGATTAGAATGGCTACCCCATTCTCTAGACAAAATCAAATTGTCCTATTGTGACTCTTC TAAAAATGAAGATGAAGAGCTATTTAATGACACACCTTGGATTAAAACGGGAATCACATCTT AAAGCTAAAAATGAACCTGCAAGCCTTCTAAATGAGTCACTGAGCATCACTAGTGACAAGTC TCGGGTGAGCGTAAATGGGTCATGACAAGATGGGACAGCAACAAAATCATGGCTTAGGATCG ACAAGAAGTTAAAAAACAGCTGCATCTGTTACTTAAGTTTGTAAGACAGTGCCCTGAGACCT CTAGAGAAAAGATGTTTGTTTACATAAGAGAAAGAAGGCCAGACATGGTGTCTCACACGTTT AATCCCAGCACTTTGGGAGGCAGGGGCGGGTGGATCACCTGAGGTCAGGAGTTCAAGACTAG CCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCATGGTGG CAGGCGCCTATAATCCCAGCTACTGGGGAGGCTGAGGCAGGAGAATC 72 89 69 Chapter 3: Huntingtin-HJPl and the cytoskeleton B) MLLCQGSEWRRDQQLGTANARQWCPLPQDAQPAGSWERCPPLPPAGRLQG 50 TDHPWGWGRLAGGGERGGLWEGLSHSQRLIHLILLSLPLLVFQTVSINKA 10 0 INTQEVAVKEKHARTCILGTHHEKGAQTFWSWNRLPLSSNAVLCWKFCH 15 0 VFHKLLRDGHPNVLKDSLRYRNELSDMSDRQLDEAGESDVNNFFQLTVEM 200 FDYLECELNLFQTVFNSLDMSRSVSVTAAGQCRLAPLIQVILDCSHLYDY 250 TVKLLFKLHSCLPADTLOGHRDRFMEQFTKLKDLFYRSSNLQYFKRL101 300 PQLPENPPNFLRASALSEHISPVWIPAEASSPDSEPVLEKDDLMDMDAS 350 OONLFDNKFDDIFGSSFSSDPFNFNSONGVNKDEKDHLIERLYREISGLK 400 AOLENMKTESORWLOLKGHVSELEADLAEQOHLROQAADDCEFLRAELD 450 ELRROREDTEKAORSLSEIERKAOANEORYSKLKEKYSELVQNHADLLRK 500 NAEVTKOVSMAROAOVDLEREKKELEDSLERISDOGORKTOEQLEVLESL 550 KQELATSQRELQVLQGSLETSAQSEANWAAEFAELEKERDSLVSGAAHRE 600 EELSALRKELODTOLKLASTEESMCOLAKDQRKMLLVGSRKAAEOVIODA 650 LNQLEEPPLISCAGSADHLLSTVTSISSCIEQLEKSWSQYLACPEDISGL 7 00 LHSITLLAHLTSDAIAHGATTCLRAPPEPADSLTEACKQYGRETLAYLAS 750 LEEEGSLENADSTAMRNCLSKIKAIGEELLPRGLDIKQEELGDLVDKEMA 800 ATSAAIETATARIEEMLSKSRAGDTGVKLEVNERILGCCTSLMQAIQVLI 850 VASKDLQREIVESGRGTASPKEFYAKNSRWTEGLISASKAVGWGATVMVD 900 AADLWQGRGKFEELMVCSHEIAASTAQLVAASKVKADKDSPNLAQLQQA 950 SRGVNQATAGWASTISGKSQIEETDNMDFSSMTLTQIKRQEMDSQVRVL 10 00 ELENELQKERQKLGELRKKHYELAGVAEGWEEGTEASPPTLQEWTEKE 1049 Chapter 3: Huntingtin-HIPl and the cytoskeleton Figure 3.4 DNA and amino acid sequence of HIPl. A) DNA sequence of the HIPl message isolated to date. No 3' polyadenylation signal or poly-A tract was noted. B) Amino acid sequence of HIPl. The HIPl amino acid sequence is 1049 amino acids and approximately 114 kDa. The portion of the amino acid sequence that was part of the original GAL4AD- HIP 1 fusion protein isolated from the two-hybrid screen is double underlined. The leucine zipper (coiled-coil domain) is bold print and underlined with a wavy line. The amino acid sequence is part of Genbank accession # U79734. 71 Chapter 3: Huntingtin-HIPl and the cytoskeleton Analysis of the HIPl primary sequence revealed a low pi (5.2), and a highly conserved motif consistent with a leucine zipper encompassing amino acid residues 588 - 609 (LESLKQELGTSQRELQVLQGSL). In Sla2p a leucine zipper is found at approximately the same region, from amino acid 481 - 502 (LAKLYSQLRQEHLNLLPRFKKL). Leucine zippers are known to mediate protein-protein interactions occurring in the cytoskeleton (Pearlman et al., 1994) or to act as transcriptional activators by allowing the formation of homo or hetero dimers (John et al., 1994). Insight into one of the functional domains may be derived from the primary amino acid sequence. When the amino acid sequence of huntingtin, HIPl, ZK370.3 and Sla2p were assessed for the presence of coiled-coiled domains using the algorithim found at http://ulrec3.unil.ch/software (Lupas et al., 1991) each of the proteins have a high probability of containing a coiled-coil domain (Fig 3.5). Northern blot data revealed that the HIPl encodes an approximately 8 kb mRNA. 3 636 bp of 3' UTR has been sequenced and no poly-A signal or poly-A tract has been identified, indicating the remaining portion of the HIPl message to be cloned will represent 3" UTR (~5 kb). 72 a o r- 13 M O -4—* O o c 5 r7" I— c/5 Chapter 3: Huntingtin-HIPl and the cytoskeleton Figure 3.5 Coiled-coil structure of HIP1, Sla2p and ZK370.3. The coiled-coil profile of each of these proteins is quite similar, suggesting a conserved function through evolution. 74 Chapter 3: Huntingtin-HIPl and the cytoskeleton SLA2 was also identified as Mop2p and was described to be required for the accumulation and maintenance of plasma membrane H+-ATPase on the cell surface (Na et al., 1995). SLA2 was previously identified in yeast as END4 where it appears to be crucial for endocytosis (Raths et al., 1993). Furthermore, HIPl also shares significant homology with the C. elegans ZK370.3 gene product which has no known function (Fig 3.6). Pairwise amino acid sequences comparison performed between HIPl and Sla2p (EMBL accession number Z22811) revealed a 20 % identity and 40 % similarity between these two proteins. An amino acid alignment between HIPl and ZK370.3 (Genpept accession number celzk370.3) showed them to have 26 % identity and a 46 % level of conservation (Fig 3.6). These homologies suggest that HIPl is the human homologue of the S. cerevisiae SLA2 gene product and the C. elegans ZK370.3 encoded protein. 75 Chapter 3: Huntingtin-HIPl and the cytoskeleton Sla2p -HSR 5 HIPl HLLCQGSELIRRDQQLGTMARQHCPLPQDAQPAGSLFFIRCPPLPPAGRLQGTDHPHGWGRLAGGGERGGLT^ 9 J zk370.3 |D|RAQAREVF 12 Sla2p 95 HIPl 178 zk370.3 102 51a2p HS—GGgsSSKLSogVJjYSvij GFTOlfTFEYEEYVS LVSV^P] 179 HIPl 228 zk370.3 UKHLHT|G|GPCiES|clLJ •WP&J^HDSQLKn; 191 Sla2p AEGDA 268 HIPl 314 zk370.3 —P3 277 Sla2p 340 Jpg ETPARTPARTPTPTP PWSAI 3 - PESv^-TTSTBTCYfflQTf PM HIPl 404 zk370.3 ' AgAgS PDSEPVLEKDD LMD|1A|QQ|L FSNKFIISIFGS 3 FS SDPFNFMppG'-Piij8SEE0— l 346 E -HGUSLSGHSGELLBIAEIIIIQQ—AS|SJ Sla2p : TGAR^IFP^TApQPJF¥AHQI^.^^^M^--gRV|[Q| POBCTOELFCfflLi 428 iiaLRRo! 489 Zk370.3 : R1]^---RSB1!JYE^L^^^ACTI^HRL. ILRDASSTHDD! 429 |s LAK|YS Q^P3HLHL1P1FKKQBLKB?HAO5 514 576 :QLE*LEsBmATSQREL|v{tegSA zk370.3 : ---FP^^^A^rlLG^^^^^5sKr^2ji •598 Sla2p : iKEQLEH^QKDlOilAlLVKSDRARLE LSU GPLTP 604 HIPl : HTOAAgFA^E^EI^SHVSGAaSEEELSA RJE^^JL^^TEESh PLI 661 -QL^3|AEEpKIRL|ELi 569 zk370.3 : HADifvEjpSniwas: Sla2p &lQTEV|HCVSD|3TSj 1QQLTLVPCA||AQY|FED| 694 HIPl dPEfflsGBlHSITIL. CLM|PSPA|S JBOYBHTLSIASI 751 zk370.3 "AG |L| IS IBS YE GpD CffiEv—L ASAKVAF 641 778 Sla2p : MS-^L||yGteE|l^v|HAB^QM]E|s^^P - - Id ^VKSB^^^EI^^^ 841 QA 731 Sla2p 866 HIPl 927 zk370.3 817 51a2p |GHIEDDH3 jjS QQQ Q PLD|_ 956 HIPl IsMojJEEHDH-- jjjgsj 1015 zk370.3 JQUlfflHDEGS- 90S 31a2p QDDD 968 HIPl !G|AE GBEE GTE ASPPTL QEWTEKE 1049 zk370.3 joJvAHKVSF 923 76 Chapter 3: Huntingtin-HIPl and the cytoskeleton Figure 3.6 Amino acid alignment of HIP1 (middle) with ZK370.3 (top) and Sla2p (bottom). Black shading represents identical or conservative amino acids between all three proteins. Grey shading represents identical or conservative amino acids between two of the pairs. Pairwise comparison reveals an overall level of 20 % identity and 40 % similarity between Sla2p and HIP1 and 26 % identity and 46 % similarity between HIP1 and ZK370.3. 77 Chapter 3: Huntingtin-HIPl and the cytoskeleton 3.2.3 The influence of polyglutamine length on the strength of the huntingtin-HIPl interaction In order to assess the influence of the polyglutamine tract on the interaction between HJP1 and huntingtin, liquid (3-galactosidase assays were performed. GAL4-BD-HD fusion proteins with 16, 44, 80 and 128 glutamine repeats were assayed for their strength of interaction with the GAL4-AD-HIP 1 fusion protein (Fig 3.7 a). The previously characterized 1T15-23Q-HAP1 and IT15-44Q-HAP1 interactions (Li et al., 1995) were used as positive controls and indicated somewhat increased interaction between huntingtin containing 44 polyglutamines with HAP1 compared to huntingtin with 23 polyglutamines as previously reported (Fig 3.7 a). The decreased interaction is not attributable to differences in efficiency of the yeast cells to translate the different GAL4-BD-huntington fusion proteins or increased instability of these proteins. Western blots were performed on yeast extracts, transferred with both the GAL4-HD and HIP1 constructs and immunoreacted with a GAL4-DNA Binding Domain monoclonal antibody (Fig 3.2). No difference in protein expression was observed between yeast cells with huntingtin with different sized polyglutamine tracts. HIP1 interacts most strongly with 16pGBT9 with a linear decrease in interaction with increasing polyglutamine length (p<0.0001; r2=0.481). Tukey analysis revealed that huntingtin with 80 or 128 polyglutamines interacted significantly less with HIP1 compared to huntingtin with 16 (p<0.0001) or 44 (p<0.0001) CAG repeats (not shown). Although the results presented above are reproducible and valid, the results of the liquid P-galactosidase assays with those original clones may not be a true assessment of the influence polyglutamine length has on the strength of the interaction between huntingtin and 78 Chapter 3: Huntingtin-HIPl and the cytoskeleton HIPl. It has been shown that reporter assays based on P-galactosidase induction in yeast may or may not be reflective of the true strength of the interaction observed between two proteins (Estojak et al., 1995). Any data observed via this method should be substantiated with accompanying biochemical analyses. This work is ongoing. 79 Chapter 3: Huntingtin-HIPl and the cytoskeleton Figure 3.7 Liquid P-galactosidase assays performed to assess the interaction strength between huntingtin and HIPl. The original huntingtin cDNA clones that were assessed for interaction with HIPl had an additional non-huntingtin 40 amino acids tail. In these assays the clones with 16 and 44 polyglutamines differed only at this region from the clones with 80 and 128 polyglutamines. Regardless of this tail, the interaction between huntingtin and HIPl demonstrated an influence in the interaction strength via the size of the polyglutamine length. In these assays n > 17 and a distinct trend that as the size of the polyglutamine tract increases, the interaction between huntingtin and HIPl had less affinity for each other. 81 Chapter 3: Huntingtin-HIPl and the cytoskeleton 3.2.4 Co-immunoprecipitation of huntingtin and HIP 1 Anti-HIPl antibodies directed toward amino acids 203-218 of the HIP1 protein detected a doublet at approximately 100 kDa in human brain lysate (Fig 3.8 first lane; lower panel). Huntingtin was immunoprecipitated from human frontal cortex lysate with GHM1 (Fig 3.8, lane 3) but not with Protein-A Sepharose alone (Fig. 3.8 lane 4) or with unrelated control antibodies (anti-synaptobrevin) (data not shown). Only GHM1 precipitated huntingtin (Fig 3.8 upper panel), as detected with the polyclonal anti-huntingtin antibody BKP1 (Kalchman et al., 1996). The anti-HJPl antibody recognized the approximately 100 kDa doublet in the same fraction (Fig 3.8 lower panel), showing that HIP1 was specifically co-immunoprecipitated with huntingtin. 3.2.5 HIP1 mRNA is enriched in the brain Analysis of HIPl mRNA by Northern blot analysis revealed an approximately 8 kb message present in all tissues assessed. However, the levels of mRNA were not uniform, with brain having the highest levels of expression (Fig 3.9) compared to peripheral tissues. In situ hybridization studies with anti-sense HIPl and Hdh RNA probes showed mouse HIPl mRNA to be ubiquitously expressed throughout the brain identical to that of Hdh (Fig 3.10). HIPl and Hdh mRNA was found to be ubiquitous in the cerebellum and cerebral cortex. HIPl was found in cerebellar Purkinje cells and caudate nucleus. Corresponding sense RNA probes provided negative controls. 82 Chapter 3: Huntingtin-HIPl and the cytoskeleton o o + 0> eg = in —• 5 P5 kDa Huntingtin -202 137 11 111 ^MVMIMHM) 83 Chapter 3: Huntingtin-HIPl and the cytoskeleton Figure 3.8 Coimmunoprecipitation of huntingtin and HIPl. The anti-huntingtin specific monoclonal antibody GHM1 was utilized to immunoprecipitate huntingtin and HIPl from human brain protein lysate (500 ag). The western blot was cut in half, and the upper portion immunoreacted with a polyclonal anti-huntingtin antibody (BKP1) and the lower half with the anti-HIPl pepl polyclonal antibody. Lane 1 is a control for the antibody, lane 2 is blank, lane 3 demonstrates huntingtin and HIPl coimmunoprecipitation and lane 4 is a negative control, where no lysate was added to antibody - Sepharose mixture was performed to confirm the interaction. 84 c 00 o 75 J* ca o —> >> o u -i—< T3 q X I .5 G — a 9 N3I\[Vlild Chapter 3: Huntingtin-HIPl and the cytoskeleton Figure 3.10 HIPl and Hdh mRNA distribution in horizontal mouse brain tissue sections. Autoradiography showing the identical ubiquitous expression of Hdh and HIPl mRNA in mouse cerebellum and brainstem (a and b respectively) and cerebral cortex (d and e respectively. Silver grains are shown for HIPl mRNA localized in cerebellar Purkinje cells (c) and caudate nucleus neurons (/). (Tissue was counterstained with Giemsa solution). ** Picture courtesy of Dr. K. Nishiyama. 87 Chapter 3: Huntingtin-HIPl and the cytoskeleton 3.2.6 HIPl protein is predominately found in the central nervous system Western blot analysis of peripheral and brain tissues revealed that the HIPl protein was only detected in the brain with no detectable protein observed outside the central nervous system. Within the brain, highest levels were seen in the cortex with slightly lower levels in the cerebellum, caudate and putamen (Fig 3.11 a) The fact that huntingtin and HIPl interact and that the expression levels of the HD gene are unchanged in the disease state, it was important to assess if the expression levels of the HIPl gene were altered. The expression level of an interacting protein involved with the HD gene product may be altered if plays a critical role in HD pathogenesis. However, the expression of HIPl appeared to be unaffected by mutant huntingtin. No obvious differences in HIPl mRNA expression were noted in brain samples from control samples and individuals affected with HD. Similarly, analysis of HIPl protein expression in the brain (Fig. 3.11b) revealed no remarkable differences between affected individuals and normal controls. 88 e ON o Cu i—H S -5 o § 53 c c ea CJ b/j b0 I G *3 c c lejuojj dn§ O 'ca 3 o 3 g '3 uinjpqojo^ — ca C c fm '53 "53 0 o 5 iun|[oqojo3 & ft o s u o jq 3 c c c ca J e < 0) T3 o o o g o •«* m 3 'C EQ c O inj # pnc3 o -C .2? .£> 'C CL> U iuri[[aqojo3 OH OX) Sonq c ri• u. ce; i jaAin[ kH oo C o o C ! Efl sa 05 o = >> TJ ft oo C X u I- c * .a 53 CD 13 m o ft C at 'C _o s- u 9 ft u T3 _>% DC 5 C o 3 c Chapter 3: Huntingtin-HIPl and the cytoskeleton The size difference observed with the different antibodies may be indicative of different HIPl family members. When the CMV-HIP1 construct is translated in vitro, a protein approximately 5-10 kDa smaller than that seen in brain lysate is detected by the anti- HIPl-FP but not anti-HIPl-pep 1 antibody (Fig 3.12 a). The difference in size between the in vitro synthesized protein and the endogenous protein detected with the anti HIP1-FP antibody, is a result that the original full-length HIPl cDNA construct lacked appropriate cDNA sequence to code for 176 amino acids at the amino terminal end. 90 Chapter 3: Huntingtin-HIPl and the cytoskeleton 91 Chapter 3: Huntingtin-HIPl and the cytoskeleton Figure 3.12 Assessment of CMV-HIP1 construct and comparative analysis of the two anti- HIP1 antibodies. a) Western blot of various sources of HIPl protein were assessed for their ability to immunoreact with anti-HIPl-FP. When the HIPl cDNA was expressed in an artificial translation system, under the control of the CMV promoter, the anti-HIPl-FP antibody can detect a protein product migrating at approximately 110 kDa. However, the immunoreactive band observed in the cortex samples (both mouse and human) is approximately 120 kDa. The anti-HIPl-FP antibody did not detect the mouse HIP la protein when expressed in vitro. The in vitro translated CMV-HD-1955 (44) construct and bacterially produced GST-HIP 1 proteins served as negative and positive controls, respectively. The size difference may be attributable to the lack of 5' sequence that is now being cloned to resolve this size difference. b) Autoradiogram of the radioactive in vitro translated proteins assessed in a). The lower panel is the same blot that was exposed to X-ray film before incubation with any components of the immunoblotting protocol. Therefore, the only samples that produced a signal on the X-ray film were those that incorporated the 35-S methionine as part of the in vitro translation procedure. The in vitro translated mouse HIP la protein is the same size as the HIPl protein detected by anti-HIPl-FP. The human HIPl protein differed from the mouse HIP la protein the same amount as it differs from endogenous HIPl protein, as detected in the upper panel. 92 Chapter 3: Huntingtin-HIPl and the cytoskeleton c) Western blot of in vitro synthesized proteins immunoreacted with anti-HIPl-pepl. Anti- HlPl-pepl did not detect the in vitro synthesized HIPl protein, but did detect the GST- HIP 1 protein. 93 Chapter 3: Huntingtin-HIPl and the cytoskeleton 3.2.7 Subcellular localization of HIPl protein in adult human and mouse brain To determine the subcellular localization of HIPl, normal tissue from human and mouse brain was fractionated by differential centrifugation (Fig 3.13). No immunoreactivity was observed by western blotting of the cytosolic fractions (Fig 3.13 a, lane 1). using the anti-HIPl polyclonal antibody. HIPl immunoreactivity was observed in all membrane fractions including cell debris and nuclei (PI), mitochondria and synaptosomes (P2), and microsomes and plasma membranes (P3) (Fig. 3.13 a, lanes 2-4 respectively). HIPl could be removed from membranes by high salt (0.5 M NaCl) buffers indicating it is not an integral membrane protein (Fig. 3.13 c, lane 1). However, since low salt (0.1- 0.25 M NaCl) was only able to partially remove HIPl from membranes, its membrane association is relatively strong (data not shown). The extraction of P3 membranes with the non-ionic detergent, Triton X-100 revealed HIPl to be a Triton X-100 insoluble protein (Fig. 3.13 d, lane 3). This characteristic is shared by many cytoskeletal and cytoskeletal-associated membrane proteins including actin, which was used as a control in this study (Fig. 3.13 e). HIPl co-localized with human huntingtin in all the membrane fractions (Fig. 3.13 a, lanes 2-4), including the high-salt membrane extractions, and in the Triton X-100 insoluble residue. 94 Chapter 3: Huntingtin-HIPl and the cytoskeleton Figure 3.13 Biochemical fractionation of huntingtin and HIPl from human cortex. One hundred micrograms of each protein fraction was analyzed on 7.5 % SDS-PAGE. a) The differential centrifugation of huntingtin (upper blots) and HIPl (lower blots) into soluble (lane 1) and PI, P2 and P3 membrane fractions (lanes 2- 4, respectively). The extraction of P3 membranes with Triton X-100 (b), 0.5M NaCl (c) and control buffer alone (d). In each condition (b, c, and d) P3 membranes were twice extracted (lanes 1 and 2) and a membrane pellet remains (lane 3). Actin is shown in soluble (lane 1) and membrane fractions (lanes 2, 3 and 6) as well as in the Triton X-100 insoluble residue (lane 6) but not in corresponding Triton X-100 soluble fractions (lanes 4 and 5). Huntingtin co-localizes with HIPl in all membrane fractions (a, lanes 2-4), in the Triton X-100 insoluble residues (b, lane 3), and both proteins can be largely removed from the membrane with high salt (c, lane 1), indicating similar subcellular localization. 96 Chapter 3: Huntingtin-HIPl and the cytoskeleton The subcellular localization of HIPl and huntingtin was further investigated by immunohistochemistry on normal adult mouse brain tissue or indirect immunofluorescence on HEK293-T cells (Fig 3.14,3-15) using huntingtin or HIPl specific anti-serum. Using adult mouse tissue (Figure 3.14) and anti-HIPl-pep 1, immunoreactivity was seen in a non• uniform, punctate pattern in the cytoplasm, appeared excluded from the nuclear cytoplasm and stained most intensely at the periphery of the cell. The HIPl immunoreactivity could be competed out using the HIPl-specific antigen. These results are consistent with the association of HIPl with intracellular membranes. Immunoreactivity occurred in all regions of the brain, including cortex, striatum, cerebellum and brainstem. The staining appeared more highly in neurons including the processes but was seen most intensely in the soma region. As described previously, huntingtin immunoreactivity was also seen exclusively and uniformly distributed in the cytosolic fraction (De Rooij et al., 1996; DiFiglia et al., 1995). HEK293T cells were transfected with CMV-HD promoter-constructs expressing the identical region used in the yeast two-hybrid experiments (amino acids 1-540) with 16, 44 or 128 polyglutamine repeats. Huntingtin demonstrated cytoplasmic staining with the 15Q construct, but displayed both cytoplasmic and perinuclear aggregates localization with constructs expressing either 44 or 128 repeats (Fig 3.15). Both perinuclear and intranuclear inclusions have been noted in cells expressing huntingtin (Martindale et al., 1998; DiFiglia et al., 1997; Sapp et al., 1997; Davies et al., 1997). When both huntingtin and HIPl were co-transfected into HEK293T cells, immunofluorescence indicated that the cytoplasmic form of huntingtin and HIPl appeared to co-localize (Fig 3.15). However, HIPl did not appear to be distinctly found within the huntingtin aggregates. 97 Chapter 3: Huntingtin-HIPl and the cytoskeleton 98 Chapter 3: Huntingtin-HIPl and the cytoskeleton Figure 3.14 Immunolocalization of HIPl and huntingtin in mouse brain. Sections from a normal mouse brain were stained with an avidin-biotin complex combined with diaminobenzidine (A,D,E), streptavidin-FITC (B) or streptavidin-Texas Red (C). Neurons in the brainstem were stained with the polyclonal anti-HIPl antibody (A,B) or the polyclonal anti-huntingtin BKP1 antibody at 1000 X magnification. The anti-HIPl antibody stained cells in the cytoplasm in a non-uniform, punctate pattern especially at the periphery of the cell. Huntingtin showed uniform staining in the cytoplasm of neurons. No staining was observed within the nucleus and all HIPl immunoreactivity could be competed out using a 10-fold molar excess of a HIPl specific peptide. Competition experiments were performed using cortex sections at 10 X magnification with (E) and without (D) peptide. 99 Chapter 3: Huntingtin-HIPl and the cytoskeleton Figure 3.15 Electronic overlays of immunofluorescence of huntingtin and HIPl in HEK293T cells. HD or HIPl cDNA constructs under control of the CMV promoter were transiently transfected and assess for co-localization. Huntingtin with 16 glutamine repeats was found to be cytoplasmic and co-localized with HIPl. Constructs expressing mutant forms of the huntingtin protein (44 or 128 glutamines) form perinuclear aggregates. HIPl is excluded from these aggregates (arrow). Huntingtin immunoreactivity was detected using the mAB2166 amino terminal antibody (Texas-red) and HIPl with anti-HIPl-FP (FITC). Neither antibody demonstrated immunoreaction when the CMV vector (pCI) was transfected into the HEK293T cells. 101 Chapter 3: Huntingtin-HIPl and the cytoskeleton 3.2.8 HIPl maps to human chromosome 7q 11.23 Fluorescent in situ hybridization (FISH) revealed that HIPl maps to a single genomic locus at 7ql 1.2 (Fig 3.16). 15- • \2 -11 1 M.2- H- -35 36- Figure 3.16 Genomic mapping of HIPl locus. A single genomic locus for HIPl was identified at 7ql 1.23 using the original cDNA probe isolated from the two-hybrid screen as the probe. 102 Chapter 3: Huntingtin-HIPl and the cytoskeleton 3.3 DISCUSSION The identification of HIPl from the yeast two-hybrid screen resulted in the identification of a novel human gene. HIPl was isolated by another research group at the same time as the HIPl described here (Wanker, et al., 1997). The HIPl cDNA isolated by Wanker et al. (1997) is identical to the HIPlpGADlO cDNA shown in Fig 3.3. Although no interaction with the first 242 amino acids of huntingtin could be detected in the system used by myself, Wanker et al. (1997) demonstrated that they could see a weak interaction with the first 171 amino acids of huntingtin. They speculated that it is the HEAT repeat (huntingtin, elongation factor 3 (EF3), the regulatory A subunit of protein phosphatase 2A (PP2A) and TORI, a target of rapamycin that is intricate in the cell cycle pathway) of huntingtin and the putative HIPl leucine zipper (coiled-coil domain) of HIPl (Andrade and Bork, 1995) that are mandatory domains required for a strong interaction (Wanker et al., 1997). Intriguingly, the HEAT repeat is found in proteins that are important in vesicle mediated transport (VP 15) and protein secretion (Andrade and Bork, 1995), functions speculated for huntingtin. Furthermore, Wanker, et al. (1997) managed to deduce that amino acids 588-609 of HIPl, the region that includes the coiled-coil domain (leucine zipper), was necessary for an interaction with huntingtin to occur. The lack of an interaction using the first 242 amino acids (APstl pGBT9 constructs) of huntingtin with HIPl in my system could be a result of the GAL4 vectors used. The pGBT9 vectors are known for their low levels of expression (Clontech, Inc.) and have been described to sometimes not produce results identical to those seen with higher expression vectors (Clontech, Inc.). 103 Chapter 3: Huntingtin-HIPl and the cytoskeleton HIPl was found to share significant sequence homology and biochemical characteristics with the known membrane cytoskeletal-associated protein Sla2p in S. cerevisiae. This suggests that HIPl is the human homologue of Sla2p (Holtzman et al., 1993). HIPl, SLA2/END4, and the C. elegans ZK370.3 gene products are similar in molecular weight and share significant homology in their carboxy terminal domains with the mammalian membrane cytoskeletal-associated protein, talin (Rees et al., 1990). The biological role HIPl plays in mammalian cells may be predicted from studies of its yeast homologue SLA2/END4. SLA2 was first identified as a null mutation causing temperature-sensitive growth defects related to a general disorganization of the membrane cytoskeleton (Li et al., 1995; Holtzman et al., 1993). This mutation is lethal when combined with actin mutants deficient in binding ABP1 and fimbrin (actin binding proteins) or when mated with abpl deletion mutants (Holtzman et al., 1993). A second mutant, initially described as end4 (Raths et al., 1993), was observed to be required for normal endocytosis in yeast. The third mutation, also later assigned to the SLA2 gene, was reported as Mop2p (modifier of plasma membrane-associated protein) (Na et al., 1995). Mop2p was described as a protein regulating the abundance of the yeast H+-ATPase in the plasma membranes and is known to be structurally and functionally analogous to the mammalian cation translocating P-type ATPases including Na+/K+, H+/K+ and Ca+-ATPases (MacLennan et al., 1987; Brandl et al., 1986; Shull et al., 1985). Therefore, Sla2p and by analogy, HIPl, appear to function in the regulation of membrane events through interactions with the underlying cytoskeleton. Recent biochemical studies reveal that sldl null mutants accumulate polarized vesicles and are defective in exocytosis at the site of growing yeast bud (Mulholland et al., 104 Chapter 3: Huntingtin-HJPl and the cytoskeleton 1997). The role of Sla2p as an important vesicle associated protein in yeast is consistent with the localization of HIPl as a protein that interacts with huntingtin. Several researchers have speculated about huntingtin's involvement with vesicle trafficking (Blockgalarza et al., 1997; Wood et al., 1996; DiFiglia et al., 1995). The huntingtin-HIPl interaction is compatible with these reports and provides the first molecular link between huntingtin and the membrane cytoskeleton. Furthermore, the fact that HIPl is located both cytoplasmic and bound to cellular membranes, a site where huntingtin is also found, suggests that the interaction between huntingtin and HIPl is occurring at the membrane. Both huntingtin and HIPl were also found in the cytosolic compartments of the cell, suggesting that there may be a dynamic interaction between huntingtin and HIPl occurring at the membrane. In order for a protein that interacts with huntingtin to play a biologically significant role in HD, the HIP must be expressed in the same region of the brain affected in HD patients as where huntingtin is expressed. HIPl is predominately found in the central nervous system as is huntingtin. In situ hybridization data demonstrate that both HIPl and huntingtin messages are found in the brainstem, cerebellar Purkinje cells and caudate nucleus neurons (Fig 3.10). Within the brain, both huntingtin and HIPl are found in the highest levels in the cortical regions, with lower levels seen in the cerebellum. Wanker et al. (1997) observed expression of HIPl outside the CNS, however at much lower levels than within it. Even though HIPl RNA could be detected outside the CNS, no HIPl protein expression outside the CNS could be detected. The two different antibodies generated against HIPl, anti-HIPl-pep 1 and anti-HIPl - FP, detected different sized gene products. Anti-HIPl-pep 1 detected a doublet at approximately 100 kDa, whereas the anti-HIPl-FP detected a single protein at approximately 105 Chapter 3: Huntingtin-HIPl and the cytoskeleton 115 kDa. Both antibodies detected the GST-HIP 1 fusion protein. The size difference may be accounted for by the fact that it appears as though HIPl belongs to a family of proteins. The family of HIPl proteins have been designated HIP la (or the murine equivalent mHIPl and mHIPla) based upon sequence similarity to either cDNA or EST clones isolated in Michael Hayden's lab or that available through the EST database at NCBI. Recent cloning of HIPl cDNAs from both human and mouse libraries show that HIPl belongs to a family of proteins. The different sized bands detected with the different anti- HIPl antibodies may be indicative of the HIPl family of proteins. The different sized gene products observed between the in vitro synthesized HIPl and the endogenous HIPl is due to the fact that the HIPl cDNA cloned into the expression vector lacks the most 5' coding region of the HIPl gene. The homologues of HIPl in both human and mouse have genomic locations different than HIPl itself (Vik Chopra, personal communication). Perhaps through evolution duplication, mutation and recombination events involving the HJPl gene other genes with varying degrees of identity to HIPl arose. Alternatively, the HIPl gene may be the result of similar events, resulting in the location and properties that exist. Transient transfection experiments of HD and HIPl cDNA constructs into HEK293T cells demonstrated that the cytoplasmic forms of huntingtin and HIPl did co-localize. However, HIPl appeared to be excluded from the perinuclear aggregates (Fig 3.17). If the pathogenesis of HD is related to the association of huntingtin with HIPl, their interaction in some way be crucial for normal cellular function. One possibility is that increased polyglutamine tracts result in huntingtin aggregate formation, both peri and intranuclear, disturbing the normal interaction of huntingtin with HIPl which, in turn, could lead to an 106 Chapter 3: Huntingtin-HIPl and the cytoskeleton alteration of biochemical events at the membrane causing premature cell death and ultimately the clinical manifestations of HD. 107 Chapter 3: Huntingtin-HIPl and the cytoskeleton 3.4 REFERENCE LIST Andrade, M.A. and Bork, P. (1995). HEAT repeats in the Huntington's disease protein. Nat. Genet. 11, 115-116. Blockgalarza, J., Chase, K.O., Sapp, E., Vaughn, K.T., Vallee, R.B., DiFiglia, M., and Aronin, N. (1997). Fast transport and retrograde movement of huntingtin and hap 1 in axons. NeuroReport 5, 2247-2251. Brandl, C.J., Green, N.M., Korczak, B., and MacLennan, D.H. (1986). Two Ca2+ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell 44(4), 597-607. Burke, J.R., Enghild, J.J., Martin, M.E., Jou, Y.S., Myers, R.M., Roses, A.D., Vance, JM, and Strittmatter, W.J. (1996). Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH. Nat. Med. 2, 347-350. Davies, S.W., Turmaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L., and Bates, G.P. (1997). Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90, 537-548. De Rooij, K.E., Dorsman, J.C., Smoor, M.A., Den Dunnen, J.T., and van Ommen, G.J. (1996). Subcellular localization of the Huntington's disease gene product in cell lines by immunofluorescence and biochemical subcellular fractionation. Hum. Mol. Genet. 5, 1093- 1099. DiFiglia, M., Sapp, E., Chase, K., Schwarz, C, Meloni, A., Young, C, Martin, E., Vonsattel, J.P., Carraway, R., and Reeves, S.A. (1995). Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron 14, 1075-1081. DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Vonsattel, J.P., and Aronin, N. (1997). Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277, 1990-1993. Estojak, J., Brent, R., and Golemis, E.A. (1995). Correlation of two-hybrid affinity data with in vitro measurements. Mol. and Cell. Biol. 75, 5820-5829. Holtzman, D.A., Yang, S., and Drubin, D.G. (1993). Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae. J. Cell Biol. 122(3), 635-644. 108 Chapter 3: Huntingtin-HIPl and the cytoskeleton John, M., Briand, J.P., Granger-Schnarr, M., and Schnarr, M. (1994). Two pairs of oppositely charged amino acids from Jun and Fos confer heterodimerization to GCN4 leucine zipper. J. Biol. Chem. 269(23), 16247-16253. Kalchman, M.A., Graham, R.K., Xia, G., Koide, H.B., Hodgson, J.G., Graham, K.C., Goldberg, Y.P., Gietz, R.D., Pickart, CM., and Hayden, M.R. (1996). Huntingtin is ubiquitinated and interacts with a specific ubiquitin-conjugating enzyme. J. Biol. Chem. 271, 19385-19394. Kozak, M. (1995). Adherence to the first-AUG rule when a second AUG codon follows closely upon the first. Proc. Natl. Acad. Sci.USA 92, 7134 Kozak, M. (1996). Interpreting cDNA sequences: some insights from studies on translation. Mam. Gen. 7, 563-574. Li, R., Zheng, Y., and Drubin, D.G. (1995). Regulation of cortical actin cytoskeleton assembly during polarized cell growth in budding yeast. J. Cell Biol. 128(4), 599-615. Li, X.J., Li, S.H., Sharp, A.H., Nucifora, F.C.J., Schilling, G., Lanahan, A., Worley, P., Snyder, S.H., and Ross, CA. (1995). A huntingtin-associated protein enriched in brain with implications for pathology. Nature 378, 398-402. Lupas, A., Van Dyke, M., and Stock, J. (1991). Predicting coiled coils from protein sequences. Science 252, 1162-1164. MacLennan, D.H., Brandl, C.J., Korczak, B., and Green, N.M. (1987). Calcium ATPases: contribution of molecular genetics to our understanding of structure and function. Society of General Physiologists Series 41, 287-300. Martindale, D., Hackam, A.S., Wieczorek, A., Ellerby, L., Wellington, C.L., McCutcheon, K., Singaraja, R., Kazemi-Esfarjani, P., Devon, R., Bredesen, D.E., Tufaro, F., and Hayden, M.R. (1998). Length of the protein and polyglutamine tract influence localization and frequency of intracellular aggregates of huntingtin. Nat. Genet. 18 (2), 150-154. Mulholland, J., Wesp, A., Riezman, H., and Botstein, D. (1997). Yeast actin cytoskeleton mutants accumulate a new class of golgi-derived secretory vesicle. Molecular Biology of the Cell 8, 1481-1499. Na, S., Hincapie, M., McCusker, J.H., and Haber, J.E. (1995). MOP2 (SLA2) affects the abundance of the plasma membrane H+-ATPase of Saccharomyces cerevisiae. J. Biol. Chem. 270(12), 6815-6823. Pearlman, J.A., Powaser, P.A., Elledge, S.J., and Caskey, CT. (1994). Troponin T is capable of binding dystrophin via a leucine zipper. FEBS Letters 354, 183-186. 109 Chapter 3: Huntingtin-HIPl and the cytoskeleton Raths, S., Rohrer, J., Crausaz, F., and Riezman, H. (1993). end3 and end4: Two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cervisiae. J. Cell Biol. 120(1), 55-65. Rees, D.J., Ades, S.E., Singer, S.J., and Hynes, R.O. (1990). Sequence and domain structure of talin. Nature 347, 685-689. Sapp, E., Schwarz, C, Chase, K., Bhide, P.G., Young, A.B., Penney, J., Vonsattel, J.P., Aronin, N., and DiFiglia, M. (1997). Huntingtin localization in brains of normal and huntingtons-disease patients. Ann. Neurol. 42, 604-612. Shull, G.E., Schwartz, A., and Lingrel, J.B. (1985). Amino-acid sequence of the catalytic subunit of the (Na+ + K+)ATPase deduced from a complementary DNA. Nature 316(6030), 691-695. Wanker, E.E., Rovira, C, Scherzinger, E., Hasenbank, R., Walter, S., Tait, D., Colicelli, J., and Lehrach, H. (1997). HJP-I: a huntingtin interacting protein isolated by the yeast two- hybrid system. Hum. Mol. Genet. 6, 487-495. Wood, J.D., MacMillan, J.C., Harper, P.S., Lowenstein, P.R., and Jones, A.L. (1996). Partial characterisation of murine huntingtin and apparent variations in the subcellular localisation of huntingtin in human, mouse and rat brain. Hum. Mol. Genet. 5, 481-487. 110 Chapter 4: HJP2: Ubiquitination of Huntingtin CHAPTER 4 - HUNTINGTIN INTERACTING PROTEIN 2 The data presented in this chapter contributed to the following manuscript: Kalchman MA, Graham RK, Koide HB, Xia G, Hodgson JG, Graham KC, Goldberg YP, Gietz RD, Pickart CM and Hayden MR. Huntingtin interacts with a ubiquitin conjugating enzyme which is highly expressed in brain. Journal of Biological Chemistry (1996). 271, 19385-19394. Ill Chapter 4: H1P2: Ubiquitination of Huntingtin 4.1 HUNTINGTIN AND UBIQUITIN HIP2 was isolated as a single clone from the same yeast two-hybrid screen as HIPl. HIP2 has complete amino acid identity with the bovine E2-25K ubiquitin conjugating enzyme and has striking similarity to the UBC-1, -4 and -5 enzymes of 5. cerevisiae. This protein is highly expressed in brain and a slightly larger protein recognized by an anti-E2- 25K polyclonal antibody is selectively expressed in brain regions affected in HD. The huntingtin-E2-25K interaction is not obviously modulated by CAG length. The data presented in this chapter also represents the first reporting of direct ubiquitination of a protein containing a disease causing polyglutamine repeat. 112 Chapter 4: HIP2: Ubiquitination of Huntingtin 4.2 RESULTS 4.2.1 Isolation of Huntingtin Interacting Protein 2 (HIP") The HIP2 cDNA was isolated as a single cDNA from the Matchmaker cDNA two- hybrid library (Clontech) in the screen for HIPs. The HJP2-GAL4 activating domain (AD) fusion protein was shown to specifically interact with the GAL4BD-HD fusion protein, as yeast containing HIP2 and the HD protein (amino acids 1-540) gave a His+ phenotype as well as showed P-galactosidase activity in a chromogenic filter assay (Fig 4.1). Specificity of the interaction was demonstrated by the fact that HIP2 did not stimulate P-galactosidase activity with the DNA binding domain, with vector alone (pGBT9), or with an unrelated fusion with myotonin kinase control (Fig 4.1). We next sought to determine whether the size of the polyglutamine tract influenced the interaction of HIP2 with the HD protein. Semi-quantitative analysis using liquid p- galactosidase assays (Fig 4.2) were performed. This revealed no difference in the strength of the interaction between HIP2 and HD constructs (amino acids 1-540) containing either 16, 44 or 128 glutamine repeats. Smaller fusion proteins containing either 16, 44 or 128 glutamine repeats and the first 242 amino acids of the HD cDNA were also tested for interaction with HIP2, with negative results. Furthermore, assessment of a fusion protein containing residues 125 to 540 alone did not reveal any interaction suggesting that an intact amino-terminal region encompassing the entire first 540 residues is essential for this interaction. 113 so * c a X OS <+- o H a o % d M '3 — + GO OS H + o OS H pa I O 5 QO 9 1 Chapter 4: HIP2: Ubiquitination of Huntingtin Figure 4.1 Specific interaction of HJP2 with the 5' region of the HD gene. The specificity of the huntingtin-HJP2 interaction is shown by the activation of the LacZ reporter gene only when the GAL4-BD fusion expressing amino acids 1-540 of huntingtin are co-transformed with H1P2. Co-transformation of HJP2 with other control constructs (pGBT9 and DMKpGBT9) failed to activate the yeast LacZ reporter gene. HIP2 did not produce (3-galactosidase activity when assessed for interaction at either the amino or carboxyl terminus of the fragment used to isolate it from the two-hybrid screen. Unlike HIPl, however, when the vector backbone is switched huntingtin and HIP2 fail to interact. The huntingtin-HAPl interaction was used as a positive control. 115 Chapter 4: HJP2: Ubiquitination of Huntingtin p-Galactosidase Acitivity 350-i 300 A 1504 1004 50 A 16pGBT9+HIP2 44pGBT9+HIP2 128pGBT9+HIP2 IT15-23Q+HAP1 U15-44Q+HAP1 Clone Figure 4.2 Liquid p-galactosidase assays showing the interaction between huntingtin and HIP2. No striking difference in activity is apparent when constructs with different sized polyglutamine tracts are assessed for interaction strength. 116 Chapter 4: JTJP2: Ubiquitination of Huntingtin 4.2.2 HJP2 is the human E2-25K ubiquitin conjugating enzyme Analysis of sequence data revealed that the HIP2 protein had complete amino acid identity with a previously described bovine E2-25K (bE2-25K) ubiquitin conjugating enzyme gene (Chen et al., 1991). The original HJP2 cDNA spanned all but the most 5' 99 nucleotides of the published bovine sequence (Chen et al., 1991). Thus the N-terminal 33 residues of the E2-protein are not necessary for the interaction of E2-25K with huntingtin. The cDNA sequence spanning the coding region for the first 33 amino acids was generated by RT-PCR using a 5' primer based on the published E2-25k sequence. There is 95% nucleotide identity and 100% amino acid identity between the bE2-25K and this human E2-25K (hE2-25K) protein, both of which comprise 200 amino acids (Fig 4.3). Residue 23 in the hE2-25K amino sequence is a serine while the published bE2-25K has a threonine at this codon (Chen et al., 1991). However, resequencing of the bE2-25K cDNA revealed that the bovine enzyme also has a threonine at this codon (C. Pickart, unpublished data). There are a total of nineteen conservative nucleotide changes in the coding region and nine nucleotide changes in the known 3' UTR sequence between human and bovine E2-25K cDNA (Fig 4.3). The HIP2 cDNA isolated from the HD yeast two-hybrid screen contains additional 3' UTR sequence relative to that published for the bovine gene (Chen et al., 1991). The complete identity between the bovine and human E2-25K enzymes places hE2- 25K in the same class of conjugating enzymes as the E2s encoded by the UBC1, UBC4 and UBC5 genes of S. cerevisiae (Chen et al., 1991). The latter three E2 proteins have essential, and partially overlapping, functions in ubiquitin-mediated protein turnover (Varshavsky, 1997; Jentsch and Schlenker, 1995; Seufert et al., 1990b; Seufert and Jentsch, 1990a). Cys- 117 Chapter 4: HJP2: Ubiquitination of Huntingtin 92 is the active site Cys of hE2-25K, based both on extensive homology of the surrounding sequence to the active site sequences of other E2s (Chen et al., 1991), and on the inability of Cys92A-bE2-25K to form a thiol ester with ubiquitin. As expected based on its identity to the corresponding portions of bE2-25K, the purified GST-H1P2 fusion protein reacted strongly with affinity-purified antibodies raised against bE2-25K (Chen et al., 1991), while GST exhibited no reaction (Fig 4.4). 118 Chapter 4: HJP2: Ubiquitination of Huntingtin BOVINE HUMAN GACATCGCCAACATCGCGGTCCAGCGAATCAAGCGG 36 -26 GGTACGAATCAGCTGCGGGCGGA. - i HAHIAVQRIKR GAGTTCAAGGAGGTGCTGAAGAGCGAGGAGACGAGCAAAAATCAAAf TAAAGTAGATCTTGTAGAT 102 34 12 1FKEVJ.KS8KTSKHQIKV0LVD GAGAATTT7ACAGAATTAAGAGGAGAAATAGCAGGACCTCCAGACACACCATATGAAGGAGGAAGA 168 100 34 EHFTELRGEIAOPFBTFYEOOR TACCAACTAGAGATAAAAATACCAGAAACATACCCATTTAATCCCCCTAAGGTCCGGTTTATCACT 234 166 . ,T .T T 56 YQLXXKXPETYPFNPFKVRFXT AMATATGGCATOCTAATAttAGftCCGfCACAGGGGCf^ATCTGWWJATAfCCTOAAAGATC^ 3 0 0 232 _ 78 KIWHPNISSVTGAI [c] L D I L K D Q TGGGCAGCTGCAATGACTCTCCGCACGGTATTATTGTCATT CCAGftTGATCCACAGGATGCTGTAGTAGCAAATCAGTACAAACAAAATCCCGAAATGTTCAAACAG 432 364 ..G A A T 122 PDDPQDAVVANQYKQNPEMFKQ ACAGCTCGACTTTGGGCACATGTGTATGCTGGAGCACCAGTTTCTAGTCCAGAATACACC AAAAAA 498 430 G 144 TARLWAHVYAGAPVSSPEYTKK ATAGAAAACCTATGTGCTATGGGCTTTGATAGGAA7GCAGTAATAGTGGCCTTGTCTTCAAAATCA 564 496 G 166 ZENtiCAMGFORNAVZVALSSICS IXWGATGT^AGACTGCAACAGAATTGCTTCTGAGTAACTGAGGCATAGAGAGC- -TGCTGATATA 628 562 . AA A....AGA 188 WDVETATELLLSN* GTCAAGCTTGCCTCTTCTT-GAGGAGCACCAACATCTGTTATTTTTAGGATTCTGCA 693 ITTAATCTGGCATTCTCGCCTAATGATGTTATCTAGGCACCATTGGAGACTGAAAAAAAAAAATCC 759 694 A T ICC CTGCTCTGTAAATAAAGCTAATTAAACGTCTGTGTAAATTTAAAAAGGGGAAATACTTTAATTTTT 825 756 800 TTTCI^AATAGTGTAAAAATTCCCTGAGCTAAGCTAAAACCATGGAAGAAACAIGCTACTTTAGTG 891 TTTAGCAGTGTACCAAGACTAGCAAGAGTTTGCTTCAGGATTrGGTrcAATAATTAAGA 957 TGGAGTGTGTCAGGGCCATTCAAATTCTTGGTGTTGCATCACAGC^ 1023 GGATCCTCTGTGCCTGTGAATTTACTTGC ATGCTTGTACTTGACTTCTTAGGATGGGTAGCTGAAA 1089 AGACCACCATTTTAAGCATTTGAGAATTCTTAAATATGAAATTTATTCAGAATTGAAGATGGTGAC 1155 CTATTCAGAGCCTTTTTGTCCTTGTCAACAGACTGGGACAGTGTCTGATTCCCCCTTCACCCCCCC 1221 CCACCCCCGCCTTGGC ACACACAGCTAATATTCTAATGGTAAATTTCTCTGTATCAGGTGGGGAAA 1287 TCTGCTGAAGGACAGTATGTATCCCTTGCTTCATTTTTAGGTC 1353 AGTTCTTCAAACACTCTTAAATTrT-TCTT^ 1419 TTGCAAAAATAGTAAATACTTGATGTTACATTATTCCCAGGTTTAATCAAAGAACCCAACTTAGTT 1485 TTTCAGTGAArTTGACACCTATTTTTTAGT^ 1551 TCAGC TCTTTGCAGTTTTTAGCCTCATTTTGGGGTCTATA 1617 TCATTCTTGCTTGCACTTCCCCTATTGACACATGAAAGCTGTGT^ 1683 CAGATGCACATAGGAATAGAAGTGTGTTATAAATCTAGCTTTCTTTATGATGTTTCTGATAAfACG 1749 AGAATTGAAAACTTT ACCTTCTCTrGTACATAGTCAGACTArrTGTATTAAATTTACATTTCArTC 1815 TAAGTTCC AAAAGTTTGAAAATT ATT AGTTTTGCAAGATC ACAC ACTAATGTAACCATTTTATGAA 1881 GGTTGAAGT^SGATTTATGCAGGCAGTTCTATATATAGAAATOCAATTOT 1947 CAATACAAAATAACACAAATGTAATGGAATCAGACTGAATTAAAGTAAGGCTGTATATTGAAAGTC 2013 ATATTATAAAAGGTTTGCTTTCTTTAAGTGTT 2079 AGATAATTTTTGAATCATAACGTCAGCATAACTTCATTTGACTTCTCAATAATCTTGTCGACGCGG 2145 CCGC 2149 Chapter 4: HIP2: Ubiquitination of Huntingtin Figure 4.3 DNA and amino acid sequences of the H1P2 (hE2-25K). The bovine E2-25K sequence is aligned and shows 95% nucleotide (left) and 100% amino acid (right) identity. The arrow indicates the first amino acid that was part of the cDNA isolated from the yeast two-hybrid screen. The active site cysteine is at residue 92 enclosed with a box. The sequence 5' to the arrow was generated from RT-PCR from human frontal cortex RNA. The amino acid residues corresponding to both the bovine and human are under the bovine nucleotide position on the left side of the figure. 120 Chapter 4: HJP2: Ubiquitination of Huntingtin kDa -82 -32 Figure 4.4 GST-HIP2 fusion protein is detected with the anti-bE2-25K antibody. The purified GST or GST-HIP2 fusion protein was purified over GSH-agarose beads and loaded on an 10 % SDS-PAGE system along side a sample of purified bE2-25k protein. An antibody specific for the bE2-25k protein was used as the primary antibody on the western blot. The GST-HJP2 protein and bE2-25k purified protein was detected, whereas the purified GST protein was not. 121 Chapter 4: HJP2: Ubiquitination of Huntingtin 4.2.3 Interaction between GST-HIP2 and the HD protein In order to assess the interaction between the HIP2 protein and HD, in vitro binding assays were performed using Glutathione-S-transferase (GST) fusion proteins. In vitro translated products corresponding to the first 540 amino acids containing either 16 or 44 glutamine repeats of the HD protein were incubated with GST-HJP2 protein linked to Glutathione-Sepharose beads. The HD protein was retained on the beads, whereas no significant interaction was observed with the GST protein alone (Fig 4.5 a). Co-affinity purification experiments were also performed using human embryonic kidney cell line (HEK293) lysates to assess the interaction between HIP2 and endogenous full length HD protein. Incubation of HEK293 lysate with GST-HIP2 linked to Glutathione- Sepharose beads resulted in specific affinity purification of the 350 kDa HD protein on the beads (Fig 4.5 b). The HD protein failed to co-purify with the GST protein alone or with GST-PTPase (protein tyrosine phosphatase). The detection of the HD protein on western blots could be blocked by preincubation with peptide antigen (data not shown). 122 Chapter 4: HIP2: Ubiquitination of Huntingtin o o b a) g I a a > ^7 *T so Tt 5/3 mi IH? H H Cfi B9 cw Cfwrij \#CAJ v#j Ov«rN\ rk. rt> rK OOOOkDa -83 -205 -144 -85 Figure 4.5 Interaction of HIP2 with the HD protein (western blots). a) In vitro translation products of nucleotides 314 1955 of the HD gene were incubated with GST-HIP2 or GST protein alone bound to Glutathione-Sepharose beads. Huntingtin was retained on beads with GST-H1P2 but not with the GST protein alone. b) Incubation of GST-HIP2 with cell extracts from HEK-293 cells resulted in specific purification of the endogenous HD protein whereas the GST protein alone failed to co- purify with the HD protein. Two independent experiments demonstrate the specificity of the interaction of hE2-25K protein with the gene product for HD. 123 Chapter 4: H1P2: Ubiquitination of Huntingtin 4.2.4 The hE2-25K ubiquitin conjugating enzyme is highly expressed in brain Northern blots revealed that the 1.2 and 2.4 kb transcripts previously detected in bovine thymus and murine erythroleukemic cells are also present in all human tissues studied (C. Pickart, data not shown) (Chen et al., 1991), and a 25 kDa immunoreactive band was present in virtually all murine, human, and rat tissues examined (Fig 4.6). However, this 25 kDa protein was most highly expressed in brain, where three immunoreactive bands, of 25 kDa, approximately 28 kDa and 45 kDa were detected, with the 28 kDa band predominating (Fig 4.6 a). All three bands were eliminated when the E2-25K antibody was preincubated with purified GST-HIP2 protein (data not shown). The precise nature of the two higher-molecular weight immunoreactive proteins remains to be determined. They could represent modified forms of E2-25K, other members of the E2 protein family, or possibly cross-reacting proteins. Anion exchange analysis performed on the mE2-25k protein revealed that the various forms share charge properties, consistent with the 25 and 28 kDa proteins representing native and modified forms, respectively, of mE2-25K (C. Pickart, personal communication). It is noteworthy that the 28 kDa band, but not the 45 kDa band, shows a striking selectivity of expression in the central nervous system, where the highest levels of expression are seen in the cortex and striatum, with lower levels of expression in the cerebellum and brain stem (Fig 4.6 b). This expression pattern is consistent with the regional neuropathology in HD and suggests that it is the 28 kDa band that interacts with huntingtin. The mutation underlying HD is an expansion of a polyglutamine stretch at the amino terminus of the gene. Even though the site of interaction of the hE2-25K protein with HD is close to the amino terminus between amino acids 1 and 540, the data do not show any 124 Chapter 4: HIP2: Ubiquitination of Huntingtin obvious influence of CAG length on the interaction between huntingtin and HIP2. Furthermore, examination of patterns of expression of the hE2-25K in the frontal cortex from affected and unaffected individuals reveals no obvious differences (Fig 4.6 d). 125 uin||3q3J33 | i snureieqx j j ureag \ j §unq O) u^ja) OS ldH 9£ ioj^uo3 6liaH PZI0JJU03 8SdH WW J3Aiq Chapter 4: HJP2: Ubiquitination of Huntingtin Figure 4.6 Tissue and regional specificity of E2-25K expression, (western blots using anti- bovine E2-25K polyclonal antibody). a) Protein extract (20 iig/lane) from various rat tissues. Both the cytosolic (C) and membrane (M) fractions from the whole rat brain are shown. The antibody detects a 25 kDa band in peripheral tissue and a slightly higher band (-18 kDa) in brain. In addition the antibody cross reacts with a 45 kDa band seen in brain and testis. b) Protein extracts from various mouse brain regions (10 pig/lane). The highest levels are in the frontal cortex and striatum, with much lower levels of expression in the cerebellum and brain stem. c) Protein extracts from human peripheral and brain (40 p:g/lane). Higher levels of expression are seen in brain tissue compared to peripheral tissues. d) Protein extracts from normal and HD affected individuals. (20 p:g/lane of frontal cortex protein extract). 128 Chapter 4: HJP2: Ubiquitination of Huntingtin 4.2.5 The HD gene product is ubiquitinated Members of the family of ubiquitin conjugating enzymes participate in the conjugation of ubiquitin to cellular proteins. The interaction of E2-25K with HD thus suggested that HD might be a substrate for ubiquitination within the cell. To address this possibility, ubiquitin conjugates were immunoprecipitated from lysates of transformed lymphoblasts derived from an individual heterozygous for HD. As seen in Fig 4.7 a, the immunoprecipitate obtained with the affinity-purified anti-ubiquitin antibodies faithfully reproduced the spectrum of conjugates present in the starting lysate (compare lanes 4 and 5 to lane 1). As expected, no conjugates were observed in precipitates from control incubations lacking either antibodies or lysate (Fig 4.7 a, lanes 2 and 3, respectively). When the same immunoprecipitates were probed with an huntingtin-specific antibody (GHM-1), precipitates from complete incubations (Fig 4.7 b, lanes 4 and 5), but not precipitates from control incubations (lanes 2 and 3), were seen to contain an HD immunoreactive band running slightly above the major band seen in the starting lysate (lane 1). An even higher molecular-weight immunoreactive "smear" was also evident in the immunoprecipitates (lanes 4 and 5, Fig 4.7 b). Similar results were obtained with a different anti-huntingtin antibody (BKP-1 data not shown). The enhanced molecular weight of these immunoreactive proteins, and their detection with anti-huntingtin antibodies suggest that these proteins are ubiquitinated forms of huntingtin. Immunoprecipitates derived from complete incubations also contained anti- huntingtin-immunoreactive proteins that were smaller than intact HD (lanes 4 and 5, Fig 4.7 b). It remains to be determined whether these could represent ubiquitinated forms of processed or partially degraded forms of the HD protein. 129 Chapter 4: HJP2: Ubiquitination of Huntingtin o x X u C X X x o P s P p + + CD o o +41 el 5* «5 S? cw an J J J J hJ + ' + + B Ub-HD Co-IP © x b < XXX © g p 5 5 81 OS A + 1 + + HD Protein 130 Chapter 4: HIP2: Ubiquitination of Huntingtin Figure 4.7 The HD protein co-immunoprecipitates with ubiquitin. The HD protein is ubiquitinated (western analysis of immunoprecipitated ubiquitin conjugates). Protein extracts (50 fig) from an Epstein-Barr virus (EBV) transformed cell line were immunoprecipitated using a polyclonal anti-ubiquitin antibody (38). The immunoprecipitates were divided in half and run either on 10.0% (a) or 5.5% (b) SDS-PAGE gels and transferred to PVDF membranes. The results shown are representative of those obtained in three independent experiments. a) Immunodetection with anti-ubiquitin antibodies. Free ubiquitin (Ub) is indicated by an arrow. The intense band near the middle of the gel in lanes 3-5 is the heavy chain of the immunoprecipitating antibody. In b), only the region of the gel above this band is shown. The negative controls for a) and b) were as indicated: (+) indicates addition of a reagent, whereas (-) indicates the omission of a reagent. The immunoprecipitates analyzed in lanes 4 and 5 differed in that the lysate in lane 5 was incubated at 37 °C for 45 minutes prior to addition of anti-ubiquitin antibodies. The similar results seen in lanes 4 and 5 indicate that the method used for preparation of the extract successfully inactivated endogenous de-ubiquitinating enzymes. b) Immunodetection with anti-HD monoclonal antibody GHM1. An aliquot of the starting extract was run in lane 1 to provide a migration standard for the HD protein. ** The above figure is courtesy of Dr. Cecile Pickart. 131 Chapter 4: HIP2: Ubiquitination of Huntingtin 4.2.6 hE2-25K Maps to Chromosome 4pl4 The cytogenetic location of the hE2-25K gene was determined using fluorescent in situ hybridization and revealed a single locus suggesting a single hE2-25K gene. The hE2- 25K locus is contained within chromosomal band 4pl4, centromeric to the HD locus within chromosomal band 4pl6 (Fig 4.8). HIP 2 * Figure 4.8 Fluorescent in-situ hybridization localized the hE2-25K protein to cytogenetic band 4pl4. 132 Chapter 4: HIP2: Ubiquitination of Huntingtin 4.3 DISCUSSION The HJP2 cDNA was identified from the same screen of the human brain Matchmaker cDNA library as HIPl. However, unlike HIPl, HIP2 was isolated as a single clone and has complete amino acid identity with a previously described bovine ubiquitin conjugating enzyme bE2-25K. The mouse E2-25K protein was isolated from a yeast two- hybrid screen using a mouse GAL4 activating domain cDNA library (M. McDonald, personal communication). This provides further support that the interaction between human huntingtin and the hE2-25K protein is indeed a true interaction. The human E2-25K (hE2-25K) also shares significant homology with other members of the large family of E2 proteins with especially striking similarity to the UBC-1, -4 and -5 enzymes of S. cerevisiae. These three E2 enzymes in yeast play an essential role in the catabolism of abnormal proteins and have partially overlapping functions (Persichetti et al., 1996). Substrates of the ubiquitination pathway include the tumor suppressor protein P53 (Scheffner et al., 1990), other oncoproteins, transcription factors (Orian et al., 1995) and cell cycle regulatory proteins (Pagano et al., 1995). In addition, even proteins apart from huntingtin are demonstrating a link between disease and ubiquitination for example, it has been demonstrated that the gene product for cystic fibrosis is also ubiquitinated and degraded by the ubiquitin dependent pathway (Ward et al., 1995). The ubiquitin pathway functions in many processes that occur broadly in many cell types, for example cell cycle progression (Ward et al., 1995). However, the pathway can also function in processes that are tissue- or cell type-specific, for example terminal erythroid differentiation (Wefes et al., 1995) and programmed cell death (Haas et al., 1995). In the 133 Chapter 4: JTJP2: Ubiquitination of Huntingtin latter two examples, the role of the pathway is mediated in part through the induction of enzymes responsible for the conjugation of ubiquitin to target proteins, including several ubiquitin conjugating enzymes (Wefes et al., 1995; Haas et al., 1995). It has also recently been suggested that altered patterns of cellular ubiquitination could play a role in neurodegenerative disorders. Elevated levels of free ubiquitin pools are seen in Alzheimer's disease (Taddei et al., 1993), Parkinson's disease (Sugiyama et al., 1994) and amyotrophic lateral sclerosis (Schiffer et al., 1994) although the precise relationship of these findings to the respective disease phenotypes is not yet clear. An increase in the number of ubiquitin reactive neurites has also been reported in HD brains compared to controls (Cammarata et al., 1993). Ubiquitin staining of aggregates has been described in four diseases and one model system expressing a polyglutamine expansion. The proteins required for spinocerebellar ataxia type 1 (ataxinl), Machado Joseph disease (ataxin3), DRPLA (atrophinl) and huntingtin, as well as a polyglutamine stretch fused into the HPRT protein, all appear to have ubiquitinated aggregates present only in cells expressing a mutant sized polyglutamine allele (Becher et al., 1998; Butler, 1998; DiFiglia et al., 1997; Davies et al., 1997; Sapp et al., 1997; Ordway et al., 1998; Paulson et al., 1997; Matilla et al., 1997). The 167 amino acids encoded by the original HIP2 cDNA were completely identical to residues 33 through 200 of the previously-described bovine ubiquitin conjugating enzyme bE2-25K, and shown that hE2-25K and bE2-25K are identical over their entire respective 200 amino acid sequences (Fig 4.3). The two-hybrid results indicate that the interaction between E2-25K and HD protein minimally requires residues 33 through 200 of E2-25K, and the first 540 residues of the HD protein (Fig 4.1). Although this region of the HD protein 134 Chapter 4: HIP2: Ubiquitination of Huntingtin contains the polyglutamine tract that is amplified in Huntington disease, the interaction between HD and hE2-25K was not sensitive to the length of this polyglutamine tract. This may be inferred as the results of quantitative two-hybrid assays (Fig 4.2) and qualitative in vitro interaction assays (Fig 4.1). More biochemical and/or in vitro experiments that assess the influence the length of both huntingtin and the size of the polyglutamine tract within huntingtin play in modulating the interaction between huntingtin and HIP2 and ubiquitin should be performed. As detected by binding of the HD protein to a GST-HIP2 fusion protein, E2-25K forms a complex with a heterologously expressed HD protein derivative in rabbit reticulocyte lysate (Fig 4.5 a), and with the endogenous HD protein in human embryonic kidney cells (Fig 4.5 b). A similar complex can be formed in yeast cells, as shown by the two-hybrid results (Fig 4.1). Within cells, both E2-25K (Seufert and Jentsch, 1990a) and the HD protein (DiFiglia et al., 1995; Aronin et al., 1995) are localized in the same (cytosolic) compartment, supporting the potential in vivo relevance of the complex revealed by the data. Furthermore, the identification of ubiquitinated forms of huntingtin (Sapp et al., 1997; Davies et al., 1997; DiFiglia et al., 1997) reveal a biologically significant role the interaction between a protein involved in the ubiquitin degradative pathway and huntingtin. While in the simplest case this is a binary complex, the results do not exclude a model in which complex formation requires an additional, unidentified protein to be present in all three types of cells. A requirement for such a component, specifically a ubiquitin-protein ligase (E3), might be expected based on an emerging model for specificity in ubiquitination. In this model, the E3 interacts directly with the target protein, while E2 specificity arises at the level of the E2-E3 interaction (Scheffner et al., 1995). So far, this model based on the 135 Chapter 4: HIP2: Ubiquitination of Huntingtin analysis of the interactions between specific ubiquitinating enzymes and substrate-based ubiquitination signals in only a few substrates, notably p53 (Huibregtse et al., 1993), N-end rule substrates (Varshavsky, 1992), and mitotic cyclins (Pagano et al., 1995). Thus, it is not excluded that an E2 protein can make a substantial direct contribution to substrate recognition in selected cases. However, even if a third component is required for the formation of a complex between E2-25K and the HD protein, the involvement of E2-25K appears to be specific. A high degree of specificity in the interactions of E2-25K with either substrates or enzymatic cofactors (if any) had been apparent from the results of prior biochemical analyses of this E2 protein (Pickart et al., 1992). Prior to the present work, the only substrate known to be recognized by E2-25K was ubiquitin itself. This latter reaction is E3-independent in vitro, and results in the formation of long multiubiquitin chains such as are known to efficiently target proteins for degradation by the 26S proteasome (Chen and Pickart, 1990). It remains to be determined whether E2-25K can generate such ubiquitin chains on the HD protein. It is possible that the hE2-25K protein serves an intricate role in the formation of the ubiquitin conjugates seen not only in HD, but with the other polyglutamine associated diseases as well. E2-25K is highly related to yeast UBC4 and 5. These E2s function, probably with an unidentified E3, in the turnover of a large body of short-lived proteins in yeast (Chen et al., 1991). There are numerous homologues of yeast UBC4 in mammals, many of which appear to be broadly expressed (Wing and Jain, 1995). None of these homologues was detected in the two-hybrid screen, providing an indication of specificity in the huntingtin-E2-25K interaction. Moreover, in a converse two-hybrid screen of a Matchmaker cDNA library from 136 Chapter 4: HIP2: Ubiquitination of Huntingtin murine brain using bE2-25K as the bait, huntingtin was not identified and only a single positive clone was isolated (C. Pickart, personal communication). The failure to detect huntingtin is not surprising, since E2-25K interacts with the amino terminal region of the 350 kDa HD protein (Fig 4.5), and the relevant region of the cDNA is likely to be under- represented in the library. However, the results of this converse screen provide a strong indication that E2-25K does not engage in a broad, non-specific set of protein-protein interactions. The finding of an interaction between the HD protein and E2-25K immediately suggested that the HD protein might be a substrate for ubiquitination within the cell. Immunoprecipated ubiquitin conjugates indeed contained protein species that reacted with monoclonal and polyclonal anti-HD antibodies (Fig 4.8). These results contrast with those obtained in a prior study (Aronin et al., 1995), where no ubiquitination of huntingtin was observed. It is likely that the failure to detect ubiquitinated huntingtin in this earlier work reflected the use of post-mortem material. The use of immunohistochemistry on transgenic mice and transfected cells lines has circumvented some of the post-mortem issue, and revealed that, indeed, huntingtin is ubiquitinated. It is important to remember that the steady-state level of conjugated forms of a given substrate depends on the relative rates of substrate ubiquitination and de-ubiquitination (Haas and Bright, 1985). Since ubiquitination but not de-ubiquitination is ATP-dependent, ubiquitin conjugates will rapidly decay post-mortem (Riley et al., 1988). Consistent with this expectation, in simple western analysis of extracts prepared from post-mortem human brain (with anti-ubiquitin antibodies) ubiquitin conjugates were not observed at a detectable level (data not shown). On the other hand, inclusion of a thiol-alkylating agent during 137 Chapter 4: JTJP2: Ubiquitination of Huntingtin lymphoblast lysis quantitatively inhibited endogenous de-ubiquitinating enzymes, and enabled us to detect ubiquitinated HD in these cells (Fig 4.7). While the results on ubiquitination (Fig 4.7) were necessarily obtained in peripheral cells rather than in brain tissue, it is expected that these events occur in brain as well, since E2-25K and HD protein are co-expressed in those cells (e.g. Fig 4.6). On the other hand, these results do not resolve an unanswered enigma for HD and other disorders associated with CAG expansion, which is that degeneration is specifically observed in neurons, even though the genes harboring these mutations are widely expressed. Similar questions must be raised for the presenilin 1 and 2 genes which contain mutations associated with some forms of familial Alzheimer's disease (Rogaev et al., 1995; Sherrington et al., 1995). While E2- 25K is broadly expressed (Fig. 4.6), it is intriguing that a slightly larger protein recognized by the polyclonal anti-E2 antibody is predominantly expressed in the central nervous system (Fig 4.6 c), with a pattern of expression that appears to parallel the neuropathology of HD. This expression pattern might be expected for an interactive protein with potential to explain the selective neuronal loss in this disease. 138 Chapter 4: JTIP2: Ubiquitination of Huntingtin 4.4 REFERENCE LIST Aronin, N., Chase, K., Young, C, Sapp, E., Schwarz, C, Matta, N., Kornreich, R., Landwehrmeyer, B., Bird, E., and Beal, M.F. (1995). CAG expansion affects the expression of mutant Huntingtin in the Huntington's disease brain. Neuron 15, 1193-1201. Becher, M., Kotzuk, J.A., Sharp, A.H., Davies, S.W., Bates, G.P., Price, D.L., and Ross, CA. (1998). Intranuclear neuronal inclusions in Huntington's disease and Dentatorubral Pallidoluysian Atrophy: correlation between the density of inclusions and IT15 triplet repeat length. Neuro. Biol. Dis. (in press) Cammarata, S., Caponnetto, C, and Tabaton, M. (1993). Ubiquitin-reactive neurites in cerebral cortex of subjects with Huntington's chorea: a pathological correlate of dementia? Neuro. Lett. 156, 96-98. Chen, Z., Niles, E.G., and Pickart, CM. (1991). Isolation of a cDNA encoding a mammalian multiubiquitinating enzyme (E2-25K) and overexpression of the functional enzyme in Escherichia coli. J. Biol. Chem. 266, 15698-15704. Chen, Z. and Pickart, CM. (1990). A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin. J. Biol. Chem. 265, 21835-21842. Davies, S.W., Turmaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L., and Bates, G.P. (1997). Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90, 537-548. DiFiglia, M., Sapp, E., Chase, K., Schwarz, C, Meloni, A., Young, C, Martin, E., Vonsattel, J.P., Carraway, R., and Reeves, S.A. (1995). Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron 14, 1075-1081. DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Vonsattel, J.P., and Aronin, N. (1997). Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277, 1990-1993. Haas, A.L., Baboshina, O., Williams, B., and Schwartz, L.M. (1995). Coordinated induction of the ubiquitin conjugation pathway accompanies the developmentally programmed death of insect skeletal muscle. J. Biol. Chem. 270, 9407-9412. Haas, A.L. and Bright, P.M. (1985). The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. J. Biol. Chem. 260, 12464-12473. 139 Chapter 4: HJP2: Ubiquitination of Huntingtin Huibregtse, J.M., Scheffner, M., and Howley, P.M. (1993). Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins. Mol. and Cell. Biol. 13, 4918-4927. Jentsch, S. and Schlenker, S. (1995). Selective protein degradation: a journey's end within the proteasome. Cell 82, 881-884. Matilla, A., Koshy, B.T., Cummings, C, Isobe, T., Orr, H.T., and Zoghbi, H.Y. (1997). The cerebellar leucine-rich acidic nuclear protein interacts with ataxin-1. Nature 389, 974-978. Ordway, J.M., Tallaksen-Greene, S., Gutekunst, C.A., Bernstein, E.M., Cearley, J.A., Wiener, H.W., Dure TV, L.S., Lindsey, R., Hersch, S.M., Jope, R.S., Albin, R.L., and Detloff, P.J. (1998). Ectopically expressed CAG repeats cause intranuclear inclusions and a progressive late onset neurological phenotype in the mouse. Cell 91, 753-763. Orian, A., Whiteside, S., Israel, A., Stancovski, I., Schwartz, A.L., and Ciechanover, A. (1995). Ubiquitin-mediated processing of NF-kappa B transcriptional activator precursor pl05. Reconstitution of a cell-free system and identification of the ubiquitin-carrier protein, E2, and a novel ubiquitin-protein ligase, E3, involved in conjugation. J. Biol. Chem. 270, 21707-21714. Pagano, M., Tarn, S.W., Theodoras, A.M., Beer-Romero, P., Del Sal, G., Chau, V., Yew, P.R., Draetta, G.F., and Rolfe, M. (1995). Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269, 682-685. Paulson, H. L., Perez, M. K., Trottier, Y., Trojanowski, J. Q., Subramony, S. H., Das, S. S., Vig, P., Mandel, J.-L., Fischbeck, K. H., and Pittman, R. N. Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3. (1997). Neuron 19, 333- 334. Persichetti, F., Carlee, L., Faber, P.W., McNeil, S.M., Ambrose, CM., Srinidhi, J., Anderson, M., Barnes, G.T., Gusella, J.F., and MacDonald, M.E. (1996). Differential expression of normal and mutant huntingtons disease gene alleles. Neurobiol. Dis. 3, 183-190. Pickart, CM., Haldeman, M.T., Kasperek, E.M., and Chen, Z. (1992). Iodination of tyrosine 59 of ubiquitin selectively blocks ubiquitin's acceptor activity in diubiquitin synthesis catalyzed by E2(25K). J. Biol. Chem. 267, 14418-14423. Butler, R Leigh PN. Mcphaul MJ. Gallo JM. (1998). Truncated forms of the androgen receptor are associated with polyglutamine expansion in X-linked spinal and bulbar muscular atrophy. Hum. Mol. Genet. 7, 121-127. Riley, D.A., Bain, J.L., Ellis, S., and Haas, A.L. (1988). Quantitation and immunocytochemical localization of ubiquitin conjugates within rat red and white skeletal muscles. J. Histo. and Cyto. 36, 621-632. 140 Chapter 4: HIP2: Ubiquitination of Huntingtin Rogaev, E.I., Sherrington, R., Rogaeva, E.A., Levesque, G., JJceda, M., Liang, Y., Chi, H., Lin, C, Holman, K., and Tsuda, T. (1995). Familial Alzheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer's disease type 3 gene. Nature 376, 775-778. Sapp, E., Schwarz, C, Chase, K., Bhide, P.G., Young, A.B., Penney, J., Vonsattel, J.P., Aronin, N., and DiFiglia, M. (1997). Huntingtin localization in brains of normal and huntingtons-disease patients. Ann. Neurol. 42, 604-612. Scheffner, M., Nuber, U., and Huibregtse, J.M. (1995). Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 373, 81-83. Scheffner, M., Werness, B.A., Huibregtse, J.M., Levine, A.J., and Howley, P.M. (1990). The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation ofp53. Cell 63, 1129-1136. Schiffer, D., Attanasino, A., Chio, A., Migheli, A., and Pezzulo, T. (1994). Ubiquitinated dystrophic neurites suggest corticospinal derangement in patients with amyotrophic lateral sclerosis. Neuro. Lett. 180, 21-24. Seufert, W. and Jentsch, S. (1990a). Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins. EMBO J. 9, 543-550. Seufert, W., McGrath, J.P., and Jentsch, S. (1990b). UBC1 encodes a novel member of an essential subfamily of yeast ubiquitin-conjugating enzymes involved in protein degradation. EMBO J. 9, 4535-4541. Sherrington, R., Rogaev, E.I., Liang, Y., Rogaeva, E.A., Levesque, G., Ikeda, M., Chi, H., Lin, C, Li, G., and Holman, K. (1995). Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease. Nature 375, 754-760. Sugiyama, H., Hainfellner, J.A., Yoshimura, M., and Budka, H. (1994). Neocortical changes in Parkinson's disease, revisited. Clin. Neuropath. 13, 55-59. Taddei, N., Liguri, G., Sorbi, S., Amaducci, L., Camici, G., Cecchi, C, and Ramponi, G. (1993). Cerebral soluble ubiquitin is increased in patients with Alzheimer's disease. Neuro. Lett. 151, 158-161. Varshavsky, A. (1992). The N-end rule. Cell 69, 725-735. Varshavsky, A. (1997). The ubiquitin system. Trends Biochem Sci 22, 383-387. Ward, C.L., Omura, S., and Kopito, R.R. (1995). Degradation of CFTR by the ubiquitin- proteasome pathway. Cell S3, 121-127. 141 Chapter 4: HIP2: Ubiquitination of Huntingtin Wefes, I., Mastrandrea, L.D., Haldeman, M., Koury, S.T., Tamburlin, J., Pickart, CM, and Finley, D. (1995). Induction of ubiquitin-conjugating enzymes during terminal erythroid differentiation Proc. Natl. Acad. Sci. USA 92, 4982-4986. Wing, S.S. and Jain, P. (1995). Molecular cloning, expression and characterization of a ubiquitin conjugation enzyme (E2(17)kB) highly expressed in rat testis. Biochem. J. 305, 125-132. 142 Chapter 5: HJP3 CHAPTER 5 - HUNTINGTIN INTERACTING PROTEIN 3 143 Chapter 5: HIP3 5.1 HUNTINGTIN AND HIP3 HJP3, like HJP2, was isolated as a single positive from the yeast two-hybrid screen. Sequence analysis of the available cDNA demonstrates that HIP3 shares identity with Akrlp, a protein from S. cerevisiae. Akrlp is a protein that is intimately involved in receptor mediated endocytosis of particular yeast pheromones. Interestingly, a western blot of HIP3 shows its expression to be highest in the caudate nucleus and putamen, two regions highly prone to premature neuronal death in HD patients. The HIP3 amino acid sequence, similar to that of Akrlp, has an ankyrin repeat. Ankyrin repeats are known to be sequences involved in associating or complexing to the cellular membrane. Therefore, H1P3 may play a role in a complex of huntingtin - HIPl and HIP3 at the cellular membrane, possibly influencing protein trafficking or signaling. 144 Chapter 5: HIP3 5.2 RESULTS 5.2.1 Isolation and sequencing of JTIP3 The HJP3 cDNA was isolated as a single clone from the same screen of the GAL4 activating domain human brain cDNA library (Clontech, Inc). The interaction data generated using HIP3 was limited to that the GAL4AD-HIP3 16pGBT9 and 44pGBT9 GAL4 DNA binding domain clones (amino acids 1-540 of huntingtin). The interaction between huntingtin and HIP3 is as specific, in the yeast two-hybrid system, as both HIPl and HIP2, as no spurious interaction with the pGBT9 vector or the DMKpGBT9 construct was observed (Fig 5.1). 5.2.2 HTP3 shares identity with the yeast Akrlp protein Preliminary analysis of the 489 base pair HJP3 cDNA sequence (Fig 5.2) shows no identity with known cloned human genes. However, similarities were observed with amino acid sequence from D2021.B, a gene from C. elegans with unknown function. HIP3 also shares a high degree of identity (7.6e-21) with a gene cloned from S. cerevisiae, Akrlp (Fig 5.3) (Givan and Sprague, Jr., 1997; Pryciak and Hartwell, 1996; Kao et al., 1996). The identity between HIP3 and Akrlp is high not only within the ankyrin repeat but also high outside the ankyrin repeat. However, it should be noted from the alignment that the position of the HIP3 homology within Akrl may slide along the protein length if more ankyrin repeats are found which may share a higher degree of identity throughout the region selected by the algorithim used by the ClustalW program. Furthermore, the ankyrin repeat found 145 Chapter 5: HJP3 within the HJP3 polypeptide shares identity with the 33 residue ankyrin repeat found in other proteins containing an ankyrin repeat (Fig 5.4). The Akrlp protein has been shown to be a critical protein involved in the endocytotic pathway of yeast pheromone receptors (Givan and Sprague, Jr., 1997). 146 Chapter 5: HJP3 LJ 16pGBT9 + fflP3 E3 44pGBT9 + fflP3 D3 16pGBT9 + HIPl • pGBT9 + fflP3 • DMKpGBT9 + HIP3 Figure 5.1 The interaction of huntingtin with HIP3. The yeast two-hybrid (3- galactosidase filter assay show the specificity of huntingtin with HIP3. No activity was seen when the vector alone (pGJ3T9) or an unrelated binding domain construct (DMKpGJ3T9) assessed for an interaction with HJP3. 147 Chapter 5: HJP3 When the entire stretch of the HIP3 sequence was assessed for identity with Akrlp, the 163 amino acids share 41.7 % identity in the region of identity, suggesting that HJP3 is the human homologue of the yeast Akrlp protein (Fig 5.4). 148 Chapter 5: JTJP3 1 CGATACCGAAGCGGGCTGTGTGCCCCTTCTCCACCCAGAGGAAATCAAAC 5 0 CAAAGCCATTATAACCATGGATATGGTGAACCTCTTGGACGGAAAACTCA 100 TATTGATGATTACAGCACATGGGACATAGTCAAGGCTACACAATATGGAA 15 0 TATATGAACGCTGTCGAGAATTGGTGGAAGCAGGTTATGATGTACGGCAA 2 00 CCGGACAAAGAAAATGTTACCCTCCTCCATTGGGCTGCCATCAATAACAG 2 5 0 AATAGATTTAGTCAAATACTATATTTCGAAAGGTGCTATTGTGGATCAAC 3 00 TTGGAGGGGACCTGAATTCAACTCCATTGCACTGGGACACAAGACAAGGC 3 50 CATCTATCCATGGTTGTGCAACTAATGAAATATGGTGCAGATCCTTCATT 40 0 AATTGATGGAGAAGGATGTAGCTGTATTCATCTGGCTGCTCAGTTCGGAC 450 ATAC C TCAATTGTTGCTTATC TCATAGCAAAAGGACAG 489 Figure 5.2 Partial cDNA sequence of HIP3. The 489 bp of JTIP3 cDNA shares no significant identity with any DNA submitted to the nr database at Genbank. 149 Chapter 5: HIP3 HIP3 : DTEAGCVPfflLHPEE^PQSHYNHEYGHpLGRKTHIDBYSTKIDIVK 45 Akrlp : MVNELENVPRASTLTNEEQTVDPSNNDSQEDISLGDSNEITS|JASLKA|ESGNEEESENEBVNHNDEAEESPLLTRYHT : 79 HIP3 0THYElYERC^^KEAGYDVRQPffl KHN^LTOHA^^^DfflSKHYroKrai^QLGEDfflNS^^MDTSQEHfflsffl 121 Akrlp BcMRfiDLATVlBafflHGKLLEWNlaGDSTHHfflGlffi 15 8 HIP3 BvQ^gYSUjl^y igG^C S C^gAAQF GHT S HKA^HAKGQ 163 Akrlp BDF{^H^gTOTBD™FNLl^gWSSNIMlffiLHBlFHVVgKGLLDIDCRDPKGRTSLL18IAAY0GDSLTVAELT,KFG 237 HIP3 : : Akrlp : ASIKIADTEGFTPLHWGTVKGQPHVLKYLIQDGADFFQKTDTGKDCFAIAQEMNTVYSLREALTHSGFDYHGYPIKKWF : 316 HIP3 : : Akrlp : KKSQHAKLVTFITPFLFLGIAFALFSHINPLFVIIVLFLLAIATNKGLNKFVLPSYGRMGVHNVTLLRSPLLSGVFFGT : 395 HIP3 : : Akrlp : LLWTIWFFKVMPRTFSDEQYTNILMLVILVSVFYLFGQLVIMDPGCLPEETDHENVRQTISNLLEIGKFDTKNFCIE : 474 HIP3 : : Akrlp : TWIRKPLRSKFSPLNNAWARFDHYCPWIFNDVGLKNHKAFIFFITLMESGIFTFLALCLEYFDELEDAHEDTSQKNGK : 553 HIP3 : : Akrlp : CFILGASDLCSGLIYDRFVFLILLWALLQSIWVASLIFVQAFQICKGMTNTEFNVLMKESKSIGPDGLSFNENFNTTPE : 632 HIP3 : : Akrlp : GFAPSIDPGEE SNDTVLAPVPGSTIRKPRTCFGVCYAVTGMDQWLAVIKETIGIKDSTGHNVYSITSRIPTNYGWKRNV : 711 HIP3 : : Akrlp : KDFWLTSDINAPLlflRRILYPPSGSKALLNGIEVDYFKLYKLPNKDVEQGNDMV- : 764 Figure 5.3 Alignment of HIP3 with Akrlp. The HIP3 ankyrin repeat is found between 108- 140 of HJP3. It is apparent that the majority of the HIP3 cDNA is missing if in fact the Akrlp is the yeast homologue of the protein. The screening of cDNA libraries is underway in the laboratory of Dr. Hayden. 150 Chapter 5: HJP3 CONSENSUS - G - T p L h - A A ------V - - L L - - G A - - - - - D HIP3 L N S T P L H D T R Q G H L S IV V V Q L M K Y G A. D P S L I D Akrlp L H A T P H w A A R Y G Y V Y I HI D F •L L K H G A D P T M 1 D HHBi AnkyririB G Y T 'T P L •H I A A K K N Q M Q I S T L L N Y G A E T N I V T Rat Ankyrin G L T T p '4: H Q A A Q Q G H T H I - N V L L Q H G A K P N A 1 T Human Ankyrin G L T P :l H V A S F M G H L P I V K N L L Q R G A S P N V s N Figure 5.4 JTIP3 and other ankyrin repeats. The ankyrin repeat of HJP3 was aligned with ankyrin repeats from the proteins indicated. The consensus for the ankyrin repeat is shown at the top. 151 Chapter 5: HJP3 5.2.3 JTIP3 protein is highly expressed in the brain Western blots show that JTJP3 is approximately 184 kDa and is highly expressed in brain, with limited expression outside the CNS. High levels of expression was observed within the caudate and putamen, with expression also seen in areas of the brain not affected by HD (Fig 5.5). Once more information about the open reading frame, and production of other antibodies is completed more definitive conclusions can be drawn from the expression data. 152 Chapter 5: HJP3 EH 3 Figure 5.5 Western blot showing expression of the human HJP3 protein. An immunoreactive band of approximately 185 kDa was observed using anti-HIP3-pepl. Approximately 75 (ig of protein was loaded in each lane. Expression of HJP3 is high in the central nervous system, with highest levels of protein seen in the caudate and putamen (left panel). 153 Chapter 5: HIP3 5.2.4 HJP3 maps to a single genomic locus in humans HJP3 was seen as a single genomic locus by FISH at 12ql2-14 (Fig 5.6). The size of the genomic region is still unresolved as no larger genomic clones have been identified as of yet. « r 11.2- 11.1- 13- 15- 1-21 23 24. 1- -24.2 24.3- Figure 5.6 Fluorescence in situ Hybridization of H1P3 shows a single genomic locus for the HIP3 gene at 12ql2-14. 154 Chapter 5: HIP3 5.3 DISCUSSION The JTJP3 cDNA clone from the same yeast two-hybrid screen that identified HIPl and HIP2 presented as an unknown human gene, sharing a significant similarity with the yeast Akrl protein (Pryciak and Hartwell, 1996; Givan and Sprague, Jr., 1997; Kao et al., 1996). Two independent groups identified Akrlp from yeast two-hybrid screens using two different GAL4-DNA binding domain constructs. One of the groups isolated the AKR1 gene from a two-hybrid screen using the cytoplasmic tail of a-factor receptor (Ste3p) (Givan and Sprague, Jr., 1997). Subsequently, Akrlp was shown to be critical in the constitutive endocytosis of the a-factor receptor. Deletion mutants of akrl could not internalize either the a- or a- factor receptors. The authors here (Givan and Sprague, Jr., 1997) emphasize the important role the organization of the actin cytoskeleton plays in endocytosis, and implicate the SLA2/END4 gene products, as well as calmodulin (CAM) in the proper organization of endocytotic vesicles. Primary structure analysis revealed that Akrlp has six ankyrin repeats (Pryciak and Hartwell, 1996), suggesting that once the full-length HIP3 cDNA is isolated more that just a single ankyrin repeat will be noted. It is possible that each ankyrin repeat in HIP3 may serve as a macromolecular assembly focus, resulting in neuronal specific interactions. The high degree of similarity (41.7 %) observed between Akrlp and the limited amount of HIP3 sequence does suggest that a similar function in humans may occur. A genetic screen in yeast for mutations that show synthetic lethality with a mutant form of the bud emergence gene (BEM1) (Kao et al., 1996) was performed. Akrlp was one 155 Chapter 5: HIP3 of the proteins isolated from this screen and the phenotype of these cells are reminiscent of cells that show a synthetic lethality when the SLA2 gene is deleted in abpl in yeast (Holtzman et al, 1993). The results found in the genetic screen for critical proteins in the BEM1 pathway suggest that the SLA2 gene product is involved in the organization of the underlying cytoskeletal components required for bud site emergence (Li et al., 1995; Kao et al., 1996). Interestingly, the data that Bemlp binds directly to Cdc24p (Kao et al., 1996), and that Cdc24p and Cdc42p are required for the process of pheromone signaling and bud site progression may be quite informative. There is evidence that Cdc42 is critical in actin assembly and bud site formation, in cooperation with Sla2p, in cdc42 mutant yeast strains (Li et al., 1995). Therefore, a cohesive model placing Sla2p and Arklp in the same compartment of the living yeast provides for these proteins to possibly interact. By extending this model to humans, a similar complex may be formed between HIPl and HIP3. Since HIPl and HIP3 share similar biochemical properties it may be reasonable to extrapolate a model to involve HD. The endocytotic deficient yeast strains show that end4 mutants are deficient in endocytosis, accumulate vesicles and have a disorganized actin cytoskeleton (Mulholland et al., 1997; Raths et al., 1993). Similarly akrl yeast are defective for endocytosis of yeast pheromone receptors, and also have a gross abnormal cytoskeletal phenotype. It is quite feasible that HIPl, H1P3 and huntingtin are associated with vesicles, synaptic or otherwise, that are responsible for neurotransmitter transport, pre or post-synaptic membrane recycling or endocytosis of a receptor that binds directly to huntingtin. 156 Chapter 5: HJP3 5.4 REFERENCE LIST Givan, S.A. and Sprague, G.F., Jr. (1997). The ankyrin repeat-containing protein Akrlp is required for the endocytosis of yeast pheromone receptors. Molecular Biology of the Cell 8, 1317-1327. Holtzman, D.A., Yang, S., and Drubin, D.G. (1993). Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae. J. Cell Biol. 122(3), 635-644. Kao, L.R., Peterson, J., Ji, R., Bender, L., and Bender, A. (1996). Interactions between the ankyrin repeat-containing protein Akrlp and the pheromone response pathway in Saccharomyces cerevisiae. Mol. and Cell. Biol. 16, 168-178. Li, R., Zheng, Y., and Drubin, D.G. (1995). Regulation of cortical actin cytoskeleton assembly during polarized cell growth in budding yeast. J. Cell Biol. 128(4), 599-615. Mulholland, J., Wesp, A., Riezman, H., and Botstein, D. (1997). Yeast actin cytoskeleton mutants accumulate a new class of golgi-derived secretory vesicle. Molecular Biology of the Cell 8, 1481-1499. Pryciak, P.M. and Hartwell, L.H. (1996). AKR1 encodes a candidate effector of the G beta gamma complex in the Saccharomyces cerevisiae pheromone response pathway and contributes to control of both cell shape and signal transduction. Mol. and Cell. Biol. 16, 2614-2626. Raths, S., Rohrer, J., Crausaz, F., and Riezman, H. (1993). end3 and end4: Two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cervisiae. J. Cell Biol. 120(1), 55-65. 157 Chapter 6: Discussion and future work CHAPTER 6 - SUMMARY. FUTURE WORK AND CONCLUSIONS 158 Chapter 6: Discussion and future work 6.1 SUMMARY The molecular mechanism underlying diseases that demonstrate genetic anticipation began to unravel with the cloning of the mutation causing spinal and bulbar muscular atrophy (SBMA) (La Spada et al., 1991). The fact that an expanded trinucleotide repeat could cause disease represented a novel type of disease-causing mutation. Since the description of the SBMA mutation, other diseases, such as fragile X and myotonic dystrophy were discovered to have a similar molecular etiology. This trinucleotide expansion presented an explanation for the genetic anticipation seen in some families. The larger the size the trinucleotide repeat, the earlier age of onset. The identification of the SBMA mutation, an expansion of a CAG trinucleotide repeat encoding polyglutamine, set the stage for the cloning of 7 other diseases that can now be attributed to the same molecular mutation (see Table 1.1). The position of the polyglutamine tract within the protein does not seem to adhere to a strict rule, for example the polyQ tract in huntingtin is at the amino terminal, whereas in atrophinl the polyQ tract is in the middle of the protein. The function of the Huntington disease gene product (huntingtin) remains unknown. It would be rare for a protein, especially the size of huntingtin (348 kDa) to exist without interacting with other proteins within the cell. It is possible that the presence of an expanded polyglutamine tract influences the interaction between huntingtin and its interactors, or in fact it may cause interactions with a protein that it does not normally associate with. In addition to the basic interaction between huntingtin and the HIPs, it is possible that increased 159 Chapter 6: Discussion and future work ubiquitination and/or precipitation of huntingtin within the intranuclear inclusions or peri• nuclear aggregates could contribute to HD pathology. The mutation in each of the diseases caused by the expression of an expanded polyglutamine tract is fundamentally identical. The specificity for each disease may be a result of unique protein-protein interactions occurring between the disease causing proteins and other cellular proteins. To date, four of the eight diseases caused by the presence of an expanded polyglutamine tract have been shown to be associated with ubiquitinated inclusions of the mutant protein. The ubiquitination may be the final step before specific cell loss, after protein partners specify which regions of the brain become affected in each disease. Various groups have embarked on discovering the protein targets for each of these relatively novel proteins. Different approaches have been employed to identify proteins that interact with proteins with a polyglutamine tract, however, the yeast two-hybrid (Chien et al., 1991; Fields and Song, 1989) has been the most productive system (Table 6.1). In order for an interacting protein to be considered a candidate for a biologically significant interaction in HD, it must fulfill some basic requirements. Firstly, it must be expressed in regions of the brain affected in HD. Secondly, the biological significance of the interaction should be consistent with the subcellular localization of the protein. A huntingtin interacting protein may also shed light onto the normal function of the HD gene product. 160 NO 00 ON r- ON ON ON ON ON NO ON ON ^ ON ON ON OsoN & s ^ >—i ON ON ON ON ON °2 ON ON ON ON r—I ON ea —; r ON ca -4—» ea 4_> ca (V) ! c • ,2 CD O TO3 ca ea 5? 43 3 T3 T3 CD CD 43 43 43 bO •c •c u bO 43 ca >^ CD ca 43 43 bO ea 43 43 43 43 >. 43 •4—» > 43 ca > o O 43 > ca O O O o ea s I CD o CD O c3 43 O ea In i o CD 60 42 ca T3 bO (U 13 e C o '•4—t "-4—» N bD t« G o o ca 3> G o k. CD c c t3 •4-* ^« c -I 00 <0 c Crt Cfl O -H SH 6 o 3 a, O P o O R § 8.2 1) 1 1 o o S g o O 8 I 0 N o 43 CD "S -*-» > o 3 c "bb bO > OX) > O 4 U > T3 1 (U s C ca <44 G CO bO _ o o o in a O O CO < T3 G "•4—> OH -co OH c ll 43 T3 C b0 G '53 _G 3 ^5 EG ca SC o T3 Q Q Q Q c^ 00 Ul ca ca Sol U J a. s -4-t la CA c O ca ca '-4-t U ea 43 O ca u U It has been speculated that an expansion of a polyglutamine tract yields a "gain of function". This hypothesis is supported by the fact that mice heterozygous for a functional mouse Hdh gene demonstrate no HD-like phenotype, and mice lacking Hdh in a homozygous state die in early embyrogenesis (Nasir et al., 1995; Duyao et al., 1995; Zeitlin et al., 1995). In order to address the issue of huntingtin interacting proteins, I performed a yeast two-hybrid screen using the first 540 amino acids of huntingtin (with 44 glutamines). Three populations of cDNAs were isolated and assigned the names HIPl, HIP2 and HIP3 (Chapters 3-5, respectively). Calmodulin Calmodulin (CAM) is a member of a protein family required for calcium binding and cell signaling. CAM directly modulates the activity of protein kinases and phosphatases, ion channels, and nitric oxide synthetases, and is generally involved in such diverse processes as cell proliferation, endocytosis, cellular adhesion, protein turnover, smooth muscle contraction and nuclear import (Niki, et al., 1996). To date two independent groups have found evidence for huntingtin to have an indirect association with calmodulin (Tukamoto et al., 1997; Bao et al., 1996). Immunoprecipitation data suggest that the expanded form of huntingtin interacts with CAM in the absence of calcium, and with a stronger affinity than that of the normal sized HD allele (Bao et al., 1996). The significance of the huntingtin-CAM interaction lies in the fact that many processes in the brain, such as vesicle function and recycling are calcium dependent. 162 Chapter 6: Discussion and future work HAP1 Huntingtin was shown to interact with HAP1 in a polyglutamine dependent manner (Li et al., 1995). Subsequent yeast two-hybrid screening for proteins that interact with HAP1 isolated a protein called Duo (Colomer et al., 1997). Biochemical analysis of both HAP 1 and Duo demonstrated that they are both enriched in subcellular cytoskeletal fractions (Colomer et al., 1997; Li et al., 1996). Duo has a GEF domain that may regulate the activity of racl. Racl is a protein essential in the organization of the actin cytoskeleton, endocytosis, exocytosis, and free radical production (Colomer et al., 1997). Furthermore, racl is a member of the Rho family of GTPase proteins and is implicated in neuritogenesis (Colomer et al., 1997). The possibility that huntingtin may be involved in the ras-related signaling pathway should be investigated. Caspases Caspases define a family of enzymes related to the C. elegans protein CED3, a protein required for programmed cell death (Villa et al., 1997). To date, 11 mammalian caspases have been cloned and catagorized based on criteria such as sequence homology, function and target specificity (Villa et al., 1997). The of ability caspases to cleave proteins responsible for the neurodegenerative diseases caused by an expanded polyglutamine tract provides insight into the particular manner in which neurons may be vulnerable to death. The generation of mice that are transgenic for a highly expanded form of exon 1 of the human HD cDNA demonstrate neurological and cellular phenotypes reminiscent of that seen in affected HD patients (Mangiarini et al., 1996; Davies et al., 1997; DiFiglia et al., 163 Chapter 6: Discussion and future work 1997; Sapp et al., 1997). This suggests that the minimal amount of the huntingtin protein required for manifestation of the phenotype lies within exon 1, and specific neuronal loss may be attributable to the interacting proteins and remaining sequence of the gene. Four of the disease proteins, HD (huntingtin), SBMA (androgen receptor), SCA1 (ataxinl) and MJD/SCA3 (ataxin3) have been generated in transgenic mice (Burright et al., 1997). Mice expressing mutant forms of huntingtin, ataxinl and ataxin3 all have a particular phenotype, that includes but not limited to, neuronal loss, ataxia, and motor deficits. The transgenic SBMA (androgen receptor) mice presented with no striking phenotype according to the authors, attributable to low levels of transgene expression or a lack of a response to the human protein (Bingham et al., 1995). Although both the androgen receptor and ataxinl are normally found within the nucleus, in the mutant form ataxinl can form the intranuclear inclusions as can huntingtin and ataxin3, both of which have been demonstrated to be predominately cytosolic proteins. Coincidentally, the recent description of atrophinl forming intranuclear aggregates coupled with its ability to be cleaved by the caspases now places huntingtin, atrophinl, ataxin3, and ataxinl within the nucleus, a place where it is not normally located, possibly a result of cleavage by the active caspases. 6.2 HUNTINGTIN INTERACTING PROTEINS The yeast two-hybrid screen that I performed to identify HJPs resulted in the isolation of 14 positive clones. Twelve of the 14 clones represented identical cDNAs, and were 164 Chapter 6: Discussion and future work assigned the nomenclature HIPl. HJP2 and HJP3 were isolated as individual positive colonies. With only cDNA sequence data available, each of the clones represented an ideal candidate to interact with huntingtin. First, HIPl provided a molecular link to the cytoskeleton, a connection that had only been shown indirectly through biochemical techniques (DiFiglia et al., 1995; Aronin et al., 1995; Wood et al., 1996b; Wood et al., 1996a). Secondly, HIP2 represented the human homologue of an enzyme involved in the ubiquitin proteolytic pathway. Previously, data suggested that HD patients had increased ubiquitin immunoreactive neurites, compared to those of controls (Cammarata et al., 1993). And as a result of this discovery, the ubiquitination status of huntingtin was investigated, and indeed, we were the first to demonstrate that huntingtin is ubiquitinated (see Chapter 4) (Kalchman et al., 1996). The speculation that huntingtin is associated with membranes and possibly synaptic vesicles provides a cohesive model for the huntingtin interacting proteins (HIPs) described in this thesis. It appears that HIPl and HIP3 may not be mutually exclusive in their interaction with huntingtin, and assessing the role the polyglutamine length plays in their interaction with huntingtin becomes a critical line of research in the future. The death of neurons can come about by different mechanisms. There has been suggestion of a glutamate toxicity model for neuronal death in HD, known as the excitotoxicity model (Hannan, 1996). In this model, as a result of the expanded polyglutamine tract, an increase in intracellular glutamine results in glutamate receptors allowing an influx of Ca2+ into the cell via both NMDA and non-NMDA receptors (Chen et al., 1995). 165 Chapter 6: Discussion and future work The cellular function of HIPl and HIP3, although unknown in human cells, may be extrapolated from the function of their homologues in lower eukaryotes, such as the budding yeast S. cerevisiae. The implication of these two novel human proteins in the disease pathway for HD may be intercalated with the glutamate toxicity model for neuronal death. Various groups, including ours (see Chapter 3) (DiFiglia et al., 1995; Kalchman et al., 1997; Bao et al., 1996) have demonstrated that huntingtin is associated with membranes, including the membranes of vesicles, specifically synaptic vesicles (DiFiglia et al., 1995). By taking the data available on HIPl, HIP3, in conjunction with knowledge of the yeast homologues of the latter two proteins, the huntingtin protein may be part of a complex that is responsible for the transport of some type of neurotransmitter(s). In short, HIPl, HIP3 (as well as other interacting proteins) and huntingtin may form a complex at the membrane of the synaptic vesicle and bind to an unidentified receptor and mediate the movement of the bound protein. The yeast homologues of both HIPl and H1P3 have been implicated in the receptor mediated endocytotic pathway, and are critical for the formation of the endocytotic vesicle (Mulholland et al., 1997; Raths et al., 1993; Givan and Sprague, Jr., 1997). The phenotype of yeast strains deleted for the yeast homologue of HIPl, SLA2/END4, is that of a defective pathway for the internalization of receptor-borne and fluid-phase markers (Raths et al., 1993). Other defects in the same pathway have been suggested in sla2/end4 mutants where there is an indication of the accumulation of vesicles, suggesting a role of SLA2/END4 in the exocytosis pathway (Givan and Sprague, Jr., 1997). The role that Sla2p plays in the formation of the actin cytoskeleton and the formation of vesicles appears to be separable and may be explained by unique protein-protein interactions (Givan and Sprague, Jr., 1997). 166 Chapter 6: Discussion and future work In establishing a link between the size of the polyglutamine tract within huntingtin and its influence on its ability to interact with protein partners is relevant to establish a role the polyglutamine tract plays in disease. However, the results obtained using the different constructs with HIPl emphasizes the limitations of the yeast two-hybrid system for quantitative assessment of interactions. Is it a coincidence that yeast cells deleted for AKR1 also demonstrate a defect in receptor mediated endocytosis (Givan and Sprague, Jr., 1997) of Ste3p in S. cerevisiae! The yeast homologue of HIP3, Akrlp has 6 ankyrin repeats, any of which may serve as a site to anchor it to the membrane of a vesicle or as a bridge for its interaction with other proteins. Furthermore, Ste3p has a C-terminal tail that mediates its endocytosis. However, the pathway of endocytosis and vesicle formation is complex. Akrlp was identified during a genetic screen for mutations that influenced survival of yeast with a mutant form of the bud emergence gene BEM1 (Kao et al., 1996). Subsequently, it was also found that Akrlp is an essential protein that interacts with the Ga subunit of the pheromone receptor-coupled G protein, a protein involved in signal transduction (Kao et al., 1996). Further two-hybrid screens with Akrlp as the bait isolated other proteins such as Ste2p and Ste4p, demonstrating that Akrlp is involved in the regulation of pheromone uptake via an endocytotic pathway (Givan and Sprague, Jr., 1997; Kao et al., 1996). As part of the communication between neurons, synaptic vesicles are delivered to nerve termini, their contents released and picked up at the post-synaptic cleft, and the vesicle remnants recycled. The process of recycling the vesicle and other endocytotic events that occur at nerve termini are analogous to receptor-mediated endocytosis (Sudhof, 1995). The finding that huntingtin and HIPl are found in the same biochemical fraction with synaptic 167 Chapter 6: Discussion and future work vesicles (see Chapter 3) (DiFiglia et al., 1995; Kalchman et al., 1997; Wanker et al., 1997) coupled with the HIP3 data suggest that huntingtin and the HIPs may have a conserved function in humans to assist in receptor-mediated endocytosis or vesicle transport. Regardless of the degree of interaction between huntingtin and the HIPs, as of yet there has been no report of any of the interacting proteins being found within the intra or perinuclear huntingtin aggregates. However, it has been shown that a construct similar to that used in the yeast two-hybrid experiments here demonstrates a proteolytic cleavage via the proapoptotic enzyme apopain (caspase3) (Goldberg et al., 1996). The cleavage of huntingtin within the first 540 amino acids by.caspase3 has been speculated to generate a toxic fragment that can form huntingtin aggregates and initiate apoptosis in the striatal neurons (Davies et al., 1997; DiFiglia et al., 1997; Martindale et al., 1998; Wellington et al., 1997). It is important to state that although aggregates do contain huntingtin, it has not been determined how much of the cellular form of huntingtin gets targeted for these aggregates. Interestingly, it has been shown that caspase3 is required for glutamate-induced apoptosis in cultured neurons (Du et al., 1997). It is also known that movement of synaptic vesicles into and out of the synaptic termini is influenced by the concentration of Ca2+ (von Gersdoff and Matthews, 1994). It appears that elevated levels of calcium inhibit endocytosis at the synaptic terminal (von Gersdoff and Matthews, 1994). Therefore, if the glutamate receptors are allowing an influx of calcium into the HD affected neuron as a result of elevated internal levels of glutamine, an abnormal interaction between the mutant form of huntingtin and one of its interacting proteins within the neuron may render it vulnerable to premature apoptotic cell death. 168 Chapter 6: Discussion and future work All the interacting proteins isolated to date have contributed to furthering the understanding the role huntingtin plays within the cell. However, none of the evidence presented to date can easily explain the selective neuronal death observed not only in HD but in any of the diseases caused by an expanded polyglutamine tract. 6.3 WHAT DOES THE IDENTIFICATION OF INTERACTING PROTEINS TEACH US ABOUT THE PATHOGENESIS OF HUNTINGTON DISEASE? One model for the complex relationships between the expanded polyglutamine tract within the HD gene, aggregate formation, interacting proteins and apoptotic cell death is outlined in Figure 6.1. It has been shown that huntingtin, in both the normal and mutant form, is a target for cleavage via caspases 1 and 3 (Wellington et al., 1998; Goldberg et al., 1996), and for degradation through the ubiquitin proteolytic pathway (Chapter 4). The cleavage of huntingtin via the caspases and/or other proteolytic systems produces a truncated N-terminal fragment that has been shown in vitro to be associated with increased aggregate formation and toxicity (Hackam et al., 1998; Martindale et al., 1998). In vivo, truncated huntingtin fragments are also associated with increased aggregate formation (Davies et al., 1998; Mangarini et al., 1997). 169 Chapter 6: Discussion and future work Huntingtin (PolyQ >35) Stress activation of caspases/proteases Altered Association with HIPl / HIP3 Toxic Fragment I ! Increased Susceptibility to Cell Death t Altered Proteosome Function HIP2/Ubiquitination 170 Chapter 6: Discussion and future work Figure 6.1 Model of potential pathway leading to increased susceptibility to cell death as a result of an expanded polyglutamine tract in huntingtin. The open arrows represent postulated associations yet to be proven, whereas the black arrows represent established relationships. The influence the interacting proteins play on the production of the toxic fragment is undetermined. 171 Chapter 6: Discussion and future work Ubiquitination of huntingtin is likely to occur through the El - E2 - E3 pathway of ubiquitin degradation. HIP2, an essential protein in this pathway, may serve to link ubiquitin to huntingtin, contributing to the ubiquitination status of the aggregates. It is still unclear, however, if the ubiquitination happens before or after the formation of the insoluble aggregates or inclusions. Huntingtin aggregates that stain positive with ubiquitin are seen in vitro (Cooper et al., 1998) and in vivo (Becher et al., 1998). Mice transgenic for exonl of the HD gene also possess ubiquitinated intranuclear inclusion (Mangarini et al., 1997) providing further evidence of the involvement of the ubiquitin dependent proteolytic pathway for huntingtin. This would predict further catabolism of these proteins through the proteosome. Aggregates found in SCA1 affected cells stain positive for the 20S component of the proteosome (Cummings et al., 1998) as well as for molecular chaperones including heat shock protein (HSP) 70. These findings suggest that mutant ataxinl forms aggregates which are directed to components of the proteosome. As a result, the proteosome is trapped in the aggregate together with the molecular chaperones. This trapping may make the cells more susceptible to cell death. It is possible that a similar mechanism is occurring within affected neurons in HD. The isolation of Huntingtin Interacting Proteins has provided new insights into potential pathways which may compromise cell viability. The decreased strength of interaction between mutant huntingtin and HIPl may liberate HIPl from mutant huntingtin and allow for HIPl to serve a neurotoxic function by activating the caspase or other proapoptotic pathways. In addition, the decreased interaction between mutant huntingtin and 172 Chapter 6: Discussion and future work HIPl may liberate huntingtin allowing it to freely aggregate in either the perinuclear or intranuclear regions. This model of a decreased interaction between two proteins promoting a toxic effect was noted with tau and its ability to bind microtubules (Spillantini et al., 1998). Here a mutation in the tau protein ultimately results in a decreased ability of tau to bind microtubules, such that it is free to aggregate and disrupt normal cell function. Even though no neurological disease is associated with mutations in the tau protein, hypotheses regarding tau's involvement with the P amyloid protein in Alzheimer's disease has been speculated (Vogel, C, 1998). HIP3, similar to HIPl, is a membrane associated protein that also associates with huntingtin. Preliminary evidence suggests the presence of the expanded polyglutamine tract may also decrease the huntingtin-HIP3 (data not shown) interaction in such a way as to directly increase the neurons susceptibility to cell death. The huntingtin-HIPl and HIP3 interactions occurs at membranes and the loss of huntingtin's affinity for both these interacting proteins may be crucial in activating the cell death pathway. This activation of cell death may be a result of an excess of unbound HIPl and HIP3 now free to perform a novel apoptotic function within the cell (Figure 6.1). None of the proteins shown to associate with huntingtin have expression exclusive to the regions of the central nervous system affected in HD patients. A possible explanation for the selective neuronal loss may lie in a mechanism similar to that of the NMDA subunits. If the HIPs influence receptors that are selectively expressed within affected regions of the brain that particular neuron population of neurons may die. A particular environment within particular cells may render them highly susceptible to the toxic effects of mutant huntingtin 173 Chapter 6: Discussion and future work or excess of the interacting protein. However, identical factors in a different cell population may not result in an increased susceptibility to cell death. This difference in sensitivity may be the result of the expression of particular receptors or neurotransmitters in particular regions. For example, NMDA receptors are found throughout the brain, however not all subunits are expressed in all subregions of the brain. The NMDA receptor NR2B is found at low levels in the cerebellum, a region spared in HD. However, it is highly expressed in the caudate and putamen, regions highly affected in HD (Wenzel et al., 1997). The NR2C subunit of the NMDA receptor has the highest level of expression in the cerebellum compared to other regions of the brain (Wenzel et al., 1997). There may be other receptors with regulated expression similar to that of the NMDA family and it is these yet unidentified proteins which will provide further clues to the specific regions of neuronal loss see in in HD. The identification of proteins that interact with the other polyglutamine containing proteins will provide further insight into the function of the respective normal and mutant proteins. The specific regions of the brain affected in each of these unique neurological diseases should be the primary source for investigating interacting proteins. A further understanding of HD pathogenesis, progression and neuronal specificity will be unveiled as more research uncovers the essential proteins and mechanisms responsible for the increased susceptibility to cell death. 174 Chapter 6: Discussion and future work 6.4 FUTURE EXPERIMENTS Although the cloning of the HIPs has provided a critical step in furthering the understanding of HD and the role huntingtin plays in cells, more experiments using the resources generated throughout my PhD work are warranted. Below I suggest further research to be performed as an extension of the data presented in this thesis. For example: 1. An in-depth biochemical approach to investigate the nature of the interaction between huntingtin and the HIPs should be pursued. For example, biochemical analyses such as co-affinity purifications of the HIPs and huntingtin, using an in vitro translation system or a bacterially based GST-expression system could be employed. The availability of various huntingtin constructs with different sized polyglutamine tracts will also be extremely useful in assessing the influence that the mutation has on the interaction strength. Coimmunoprecipitations using antibodies specific for huntingtin or the HIPs would also be extremely useful in providing further evidence of a biologically significant interaction. Each of the interacting proteins can be compared for their relative affinity for huntingtin. Those proteins that demonstrate a higher affinity for huntingtin may serve a more critical role in any huntingtin "complex" that may be formed. 2. The hypothesis that the HIPs may play a role in promoting an increased susceptibility to cell death should be investigated. The finding that as the length of the polyglutamine tract increases the binding affinity for HIPl decreases provides clues that excess HIPl may be found within the cell. The affect that excess HIPl has in cells expressing 175 Chapter 6: Discussion and future work expanded polyglutamne tracts should be investigated for cell survival, i.e. the putative toxic effect of excess HIPl within a cell. 3. Recently, more 5' HIPl sequence has been generated demonstrating that the CMV-HIP1 construct that was pieced together is incomplete. As a result, additional transfection experiments to more accurately assess the subcellular localization of HIPl within cells should be performed. This data will also provide evidence as to the function that the most amino terminal residues of HIPl have in the targeting of HIPl to its subcellular compartment. 4. It is critical to further refine the site of interaction between huntingtin and the HIPs. Therefore, more two-hybrid constructs that can identify the minimal region of huntingtin required for the interaction should be generated. Subsequently, more biochemical studies can be performed in parallel that can help delineate a minimal region of the amino terminal of the huntingtin protein that is responsible for the interaction. In addition to delineating the minimal region required for the interaction, investigation into the influence larger huntingtin fragments have on the interaction kinetics of huntingtin with HIPs should be performed. 5. If the HIPs and huntingtin are involved in the formation of a complex, it may be reasonable to assume that HIPl and HIP3 may interact as well. Two-hybrid and co- purification experiments may show such a complex. 6. Interestingly, the aggregates seen in cells expressing mutant huntingtin may provide a novel way to assess the interacting proteins. It might be possible to biochemically purify the aggregates and immunodetect specific interacting proteins that are integral 176 Chapter 6: Discussion and future work components of these aggregates. If the aggregates contain a specific structural organization that requires membranous structures to be formed, the HIPl and HIP3 proteins may be vital in the maintenance of the structures. 7. Even though the intranuclear inclusions have been shown to stain positive for ubiquitin, the perinuclear aggregates have not been investigated for the same property. The collaboration with Cecile Pickart (John's Hopkins) provides an excellent foundation for this type of research. The HEK293T cells, when transfected with a construct expressing an expanded CAG tract, definitely form perinuclear aggregates. By using various anti- ubiquitin antibodies available, immunofluorescence can be performed using the 293T cells transfected with HD cDNA constructs in order to investigate possible ubiquitination of the perinuclear aggregates. 8. The limited data available on HJP3 reveals that it shares identity with a yeast protein involved in receptor mediated endocytosis and is highly expressed in the caudate nucleus and putamen. The first thing that must be done is the isolation of a full-length HIP3 cDNA clone. As a result, the full-length open reading frame can be determined and further structural analyses may be performed. Experiments that provide further support of the huntingtin - HIP3 interaction should be pursued. For example, coimmunoprecipitations can provide an additional method of demonstrating the interaction would be highly informative. Furthermore, the influence the polyglutamine tract length has on the interaction with HIP3 is a crucial set of experiments to be done. Cellular, subcellular and biochemical analyses of H1P3 and huntingtin will be insightful 177 Chapter 6: Discussion and future work and informative to determine if the two proteins are found within the same cellular compartments. 6.5 CONCLUSIONS The identification of HIPs generated novel data that has contributed significantly to understanding the molecular biochemistry of the huntingtin protein. HIPl and HIP3 have high levels of expression in the appropriate tissues and place huntingtin at the cytoskeleton, possibly engaging in a modified type of receptor mediated endocytosis. HIP2, the human E2-25K ubiquitin conjugating enzyme, is not only expressed at high levels in the appropriate tissue, but unveiled a molecular link between huntingtin and ubiquitin. Ubiquitinated inclusions have been noted in five of the eight diseases associated with polyglutamine expansion, and could be the result of cells destined for death. The role proteins that interact with huntingtin play in HD will be a critical avenue of research to further the understanding of this devastating disease. 178 Chapter 6: Discussion and future work 6.6 REFERENCE LIST Aronin, N., Chase, K., Young, C, Sapp, E., Schwarz, C, Matta, N., Kornreich, R., Landwehrmeyer, B., Bird, E., and Beal, M.F. (1995). CAG expansion affects the expression of mutant Huntingtin in the Huntington's disease brain. Neuron 15, 1193-1201. Becher, M.W., Kotzuk, J.A., Sharp, A.H., Davies, S.W., Bates, G.P., Price, D.L., and Ross, CA. (1997) Intranuclear neuronal inclusions in Huntington's disease and dentatorubral and palidoluysian atrophy: correlation between the density of inclusions and IT 15 CAG triplet repeat length. Neurobiol. Dis. (in press). Bao, J., Sharp, A.H., Wagster, M.V., Becher, M., Schilling, G., Ross, C.A., Dawson, VL, and Dawson, T.M. (1996). Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin. Proc. Natl. Acad. Sci. USA 93, 5037-5042. Bingham, P. M., Scott, M. O., Wang, S., McPhaul, M. J., Wilson, E. M., Garbern, J. Y., Merry, D. E., and Fischbeck, K. H. Stability of an expanded trinucleotide repeat in the androgen receptor gene in transgenic mice. (1995). Nat. Genet. 9, 191-196. Burke, J.R., Enghild, J.J., Martin, M.E., Jou, Y.S., Myers, R.M., Roses, A.D., Vance, JM, and Strittmatter, W.J. (1996). Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH. Nat. Med. 2, 347-350. Burright, E.N., Orr, H.T., and Clark, H.B. (1997). Mouse models of human CAG repeat disorders. Brain Path. 7, 965-977. Cammarata, S., Caponnetto, C, and Tabaton, M. (1993). Ubiquitin-reactive neurites in cerebral cortex of subjects with Huntington's chorea: a pathological correlate of dementia? Neuro. Lett. 156, 96-98. Chen, Q., Harris, C, Brown, C.S., Howe, A., Surmeier, D.J., and Reiner, A. (1995). Glutamate-mediated excitotoxic death of cultured striatal neurons is mediated by non-NMDA receptors. Exp. Neur. 136, 212-224. Chien, C.-T., Barrel, P.L., Sternglanz, R., and Fields, S. (1991). The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci.USA 55, 9578-9582. Colomer, V., Engelender, S., Sharp, A.H., Duan, K., Cooper, J.K., Lanahan, A., Lyford, G., Worley, P., and Ross, CA. (1997). Huntingtin-associated protein 1 (Hapl) Binds to a trio• like polypeptide, with a rad guanine nucleotide exchange factor domain. Hum. Mol. Genet. 6, 1519-1525. 179 Chapter 6: Discussion and future work Cooper, J.K., Schilling, G., Peters, M.F., Herring, W.J., Sharp, A.H., Kaminsky, Z., Masone, J., Khan, F.A., Delanoy, M., Borchelt, D.R., Dawson, V., Dawson, T.M., and Ross., CA. (1998) Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture. Hum. Mol. Genet. 7, 783-790. Davies, S.W., Turmaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L., and Bates, G.P. (1997). Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90, 537-548. Davies SW. Beardsall K. Turmaine M. DiFiglia M. Aronin N. Bates GP. (1998). Are neuronal intranuclear inclusions the common neuropathology of triplet-repeat disorders with polyglutamine-repeat expansions? The Lancet. 351, 131-133. DiFiglia, M., Sapp, E., Chase, K., Schwarz, C, Meloni, A., Young, C, Martin, E., Vonsattel, J.P., Carraway, R., and Reeves, S.A. (1995). Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron 14, 1075-1081. DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Vonsattel, J.P., and Aronin, N. (1997). Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277, 1990-1993. Du, Y., Bales, K.R., Dodel, R., Hamilton-Byrd, E., Horn, J.W., Czilli, D.L., Simmons, L.K., Ni, B., and Paul, S.M. (1997). Proc. Natl. Acad. Sci. USA 94, 11657-11662. Duyao, M.P., Auerbach, A.B., Ryan, A., Persichetti, F., Barnes, G.T., McNeil, S.M., Ge, P., Vonsattel, J.P., Gusella, J.F., and Joyner, A.L. (1995). Inactivation of the mouse Huntington's disease gene homologue Hdh. Science 269, 407-410. Fields, S. and Song, O. (1989). A novel genetic system to detect protein-protein interacts. Nature 340, 245-246. Givan, S.A. and Sprague, G.F., Jr. (1997). The ankyrin repeat-containing protein Akrlp is required for the endocytosis of yeast pheromone receptors. Mol. Biol. Cell 8, 1317-1327. Goldberg, Y.P., Nicholson, D.W., Rasper, D.M., Kalchman, M.A., Koide, H.B., Graham, RK, Bromm, M., Kazemi-Esfarjani, P., Thornberry, N.A., Vaillancourt, J.P., and Hayden, M.R. (1996). Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract. Nat.Genet. 13, 442-449. Hackam, A.S., Singaraja, R., Wellington, C.W., Metzler, M., McCutcheon, K., Zhang, T., Kalchman, M., and Hayden, M.R. The influence of huntingtin protein size on nuclear localization and cellular toxicity. J. Cell Bio. 141, 1097-1105. 180 Chapter 6: Discussion and future work Hannan, A.J. (1996). Trinucleotide-repeat expansions and neurodegenerative disease: a mechanism of pathogenesis. Clin. Exp. Phar. & Phys. 23, 1015-1020. Kalchman, M.A., Graham, R.K., Xia, G., Koide, H.B., Hodgson, J.G., Graham, K.C., Goldberg, Y.P., Gietz, R.D., Pickart, CM., and Hayden, M.R. (1996). Huntingtin is ubiquitinated and interacts with a specific ubiquitin-conjugating enzyme. J. Biol. Chem. 271, 19385-19394. Kalchman, M.A., Koide, H.B., McCutcheon, K., Graham, R.K., Nichol, K., Nishiyama, Kazemi-Esfarjani, P., Lynn, F.C, Wellington, C, Metzler, M., Goldberg, Y.P., Kanazawa, I., Gietz, R.D., and Hayden, M.R. (1997). HIPl, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nat.Genet. 16, 44-53. Kao, L.R., Peterson, J., Ji, R., Bender, L., and Bender, A. (1996). Interactions between the ankyrin repeat-containing protein Akrlp and the pheromone response pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 168-178. Koshy, B., Matilla, T., Burright, E.N., Merry, D.E., Fischbeck, K.H., Orr, H.T., and Zoghbi, H.Y. (1996). Spinocerebellar ataxia type-1 and spinobulbar muscular atrophy gene products interact with glyceraldehyde-3-phosphate dehydrogenase. Hum. Mol. Genet. 5, 1311-1318. La Spada, A.R., Wilson, E.M., Lubahn, D.B., Harding, A.E., and Fishbeck, K.H. (1991). Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 352, 77-79. Li, X.J., Li, S.H., Sharp, A.H., Nucifora, F.C.J., Schilling, G., Lanahan, A., Worley, P., Snyder, S.H., and Ross, CA. (1995). A huntingtin-associated protein enriched in brain with implications for pathology. Nature 378, 398-402. Li, X.J., Sharp, A.H., Li, S.H., Dawson, T.M., Snyder, S.H., and Ross, CA. (1996). Huntingtin-associated protein (HAP1): discrete neuronal localizations in the brain resemble those of neuronal nitric oxide synthase. Proc. Natl. Acad. Sci. USA 93, 4839-4844. Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., hetherington, C, Lawton, M., Trottier, Y., Lehrach, H., Davies, S.W., and Bates, G.P. (1996). Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87, 493-506. Martindale, D., Hackam, A.S., Wieczorek, A., Ellerby, L., Wellington, C.L., McCutcheon, K., Singaraja, R., Kazemi-Esfarjani, P., Devon, R., Bredesen, D.E., Tufaro, F., and Hayden, M.R. (1998). Length of the protein and polyglutamine tract influence localization and frequency of intracellular aggregates of huntingtin. Nat.Genet. 18 (2), 150-154. 181 Chapter 6: Discussion and future work Miyashita, T., Okamura-Oho, Y., Mito, Y., Nagafuchi, S., and Yamada, M. (1997). Dentatorubral pallidoluysian atrophy (DRPLA) protein is cleaved by caspase-3 during apoptosis. J. Biol. Chem. 272, 29238-29242. Mulholland, J., Wesp, A., Riezman, H., and Botstein, D. (1997). Yeast actin cytoskeleton mutants accumulate a new class of golgi-derived secretory vesicle. Molecular Biology of the Cell 8, 1481-1499. Nasir, J., Floresco, S.B., O'Kusky, J.R., Diewert, V.M., Richman, J.M., Zeisler, J., Borowski, A., Marth, J.D., Phillips, A.G., and Hayden, M.R. (1995). Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell 81, 811-823. Nikil. YokokuraH. SudoT. KatoM. Hidaka H. (1996). Ca2+signaling and intracellular Ca2+ binding proteins. J. Biochem. 128, 685-697. Raths, S., Rohrer, J., Crausaz, F., and Riezman, H. (1993). end3 and end4: Two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cervisiae. J.Cell Biol. 120(1), 55-65. Sapp, E., Schwarz, C, Chase, K., Bhide, P.G., Young, A.B., Penney, J., Vonsattel, J.P., Aronin, N., and DiFiglia, M. (1997). Huntingtin localization in brains of normal and huntingtons-disease patients. Ann. Neurol. 42, 604-612. Spillantini, M.G., Murrell, J.R., Goedert, M., Farlow, M.R., Klug, A., and Ghetti, B. (1998). Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc. Natl. Acad. Sci. USA. 95: 7737-7741. Sudhof, T.C. (1995). The synaptic vesicle: a cascade of protein-protein interactions. Nature 375, 645-653. Tukamoto, T., Nukina, N., Ide, K., and Kanazawa, I. (1997). Huntingtons disease gene product, huntingtin, associates with microtubules in vitro. Mol. Brain Res. 51, 8-14. Villa, P., Kaufmann, S.H., and Earnshaw, W.C. (1997). Caspases and caspase inhibitors. Trends Biochem Sci 22, 388-393. von Gersdoff, H. and Matthews, G. (1994). Inhibition of endocytosis by elevated internal calcium in a synaptic terminal. Nature 370, 652-655. Wanker, E.E., Rovira, C, Scherzinger, E., Hasenbank, R., Walter, S., Tait, D., Colicelli, J., and Lehrach, H. (1997). HJP-I: a huntingtin interacting protein isolated by the yeast two- hybrid system. Hum. Mol. Genet. 6, 487-495. 182 Chapter 6: Discussion and future work Wellington, C.L., Ellerby, L.M., Hackam, A.S., Margolis, R.L., Trifiro, M.A., Singaraja, R., McCutcheon, K., Salvesen, G.S., Propp, S.S., Bromm, M., Rowland, K.J., Zhang, T., Rasper, D., Roy, S., Thornberry, N., Pinsky, L., Kakizuka, A., Ross, C.A., Nicholson, D.W., Bredesen, D.E., and Hayden, M.R. (1998). Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract. J.Biol.Chem. 273, 9159-9167. Wenzel A. Fritschy JM. MohlerH. Benke D. (1997). NMDA receptor heterogeneity during postnatal development of the rat brain differential expression of the NR2A, NR2B, and NR2C subunit proteins. 68, 469-78. Wood, J.D., Harper, P.S., and Jones, A.L. (1996a). Characteristics of cytosolic and membrane bound forms of huntingtin. Am. J. Hum. Genet. 59(4), A295 Wood, J.D., MacMillan, J.C., Harper, P.S., Lowenstein, P.R., and Jones, A.L. (1996b). Partial characterisation of murine huntingtin and apparent variations in the subcellular localisation of huntingtin in human, mouse and rat brain. Hum. Mol. Genet. 5, 481-487. Zeitlin, S., Liu, J.P., Chapman, D.L., Papaioannou, V.E., and Efstratiadis, A. (1995). Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue. Nat. Genet. 11, 155-163. 183