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• W2041156762 abstract "Using the yeast two-hybrid system, we have identified a human ubiquitin-conjugating enzyme (hE2-25K) as a protein that interacts with the gene product for Huntington disease (HD) (Huntingtin). This protein has complete amino acid identity with the bovine E2-25K protein and has striking similarity to the UBC-1, −4 and −5 enzymes of Saccharomyces 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. We also demonstrate that huntingtin is ubiquitinated. These findings have implications for the regulated catabolism of the gene product for HD. Using the yeast two-hybrid system, we have identified a human ubiquitin-conjugating enzyme (hE2-25K) as a protein that interacts with the gene product for Huntington disease (HD) (Huntingtin). This protein has complete amino acid identity with the bovine E2-25K protein and has striking similarity to the UBC-1, −4 and −5 enzymes of Saccharomyces 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. We also demonstrate that huntingtin is ubiquitinated. These findings have implications for the regulated catabolism of the gene product for HD. Huntington disease (HD) 1The abbreviations used are: HDHuntington diseaseHIPhuntingtin interacting proteinHIP-2HIP consisting of amino acids 33-200 of hE2-25KE2ubiquitin-conjugating enzyme (where relevant, initial letter indicates species of origin: b, bovine, m, murine, h, human)GSHglutathioneGSTglutathione-S-transferaseRT-PCRreverse transcriptase-polymerase chain reactionPAGEpolyacrylamide gel electrophoresisFISHfluorescent in situ hybridizationFITCfluorescein isothiocyanatePVDFpolyvinylidine difluorideCAPS3-(cyclohexylamino)propanesulfonic acidDRPLAdentatorubropallidoluysian atrophyBDbinding domainADactivating domain is a member of a family of neurodegenerative disorders caused by CAG triplet expansion (1Huntington's Disease Collaborative Research Group Cell. 1993; 72: 971-983Google Scholar) which includes spinocerebellar ataxia types I and III, dentatorubropallidoluysian atrophy (DRPLA) and spinal bulbar muscular atrophy (SBMA) (2Willems P.J. Nat. Genet. 1994; 8: 213-215Google Scholar, 3Ross C.A. Neuron. 1995; 15: 493-496Google Scholar). Huntington disease huntingtin interacting protein HIP consisting of amino acids 33-200 of hE2-25K ubiquitin-conjugating enzyme (where relevant, initial letter indicates species of origin: b, bovine, m, murine, h, human) glutathione glutathione-S-transferase reverse transcriptase-polymerase chain reaction polyacrylamide gel electrophoresis fluorescent in situ hybridization fluorescein isothiocyanate polyvinylidine difluoride 3-(cyclohexylamino)propanesulfonic acid dentatorubropallidoluysian atrophy binding domain activating domain Each of these diseases is characterized by selective neuronal loss in discrete regions of the brain or spinal cord (3Ross C.A. Neuron. 1995; 15: 493-496Google Scholar). In HD the major site of neuropathology is the caudate nucleus and putamen (4Hayden M.R. Huntington's Chorea. Springer-Verlag, London1981Google Scholar). The cloning of the respective genes allowed testing of the hypothesis that the selective neuronal loss in these disorders was a reflection of restricted expression of the mRNA transcript or protein in those cells undergoing degeneration. However, the respective mRNA transcripts and the resulting proteins are widely expressed in the central nervous system and peripheral tissues (5Difiglia M. Sapp E. Chase K. Schwarz C. Meloni A. Young C. Martin E. Vonsattel J.P. Carraway R. Reeves S.A. Boyce F.M. Aronin N. Neuron. 1995; 14: 1075-1081Google Scholar, 6Sharp A. Loev S.J. Schilling G. Li S.H. Li X.J. Bao J. Wagster M.V. Kotszuk J.A. Steiner J.P. Lo A. Hedreen J. Sisodia S. Snyder S.H. Dawson T.M. Ryugo D.K. Ross C.A. Neuron. 1995; 14: 1065-1074Google Scholar, 7Jou Y-S. Myers R.M. Hum. Mol. Genet. 1995; 4: 465-469Google Scholar, 8Gutekunst C-A. Levey A.I. Heilman C.J. Whaley W.L. Yi H. Nash N.R. Rees H.D. Madden J.J. Hersch S.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8710-8714Google Scholar, 9Trottier Y. Devys D. Imbert G. Saudou F. An I. Lutz Y. Weber C. Agid Y. Hirsch E.C. Mandel J.L. Nat. Genet. 1995; 10: 104-110Google Scholar, 10Servadia A. Koshy B. Armstrong D. Antalffy B. Orr H.T. Zoghbi H.Y. Nat. Genet. 1995; 10: 94-98Google Scholar), and are not obviously altered in their abundance in affected tissues (8Gutekunst C-A. Levey A.I. Heilman C.J. Whaley W.L. Yi H. Nash N.R. Rees H.D. Madden J.J. Hersch S.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8710-8714Google Scholar, 9Trottier Y. Devys D. Imbert G. Saudou F. An I. Lutz Y. Weber C. Agid Y. Hirsch E.C. Mandel J.L. Nat. Genet. 1995; 10: 104-110Google Scholar, 10Servadia A. Koshy B. Armstrong D. Antalffy B. Orr H.T. Zoghbi H.Y. Nat. Genet. 1995; 10: 94-98Google Scholar, 11Yazawa I. Nukin N. Hashida H. Goto J. Yamada M. Kanazawa I. Nat. Genet. 1995; 10: 99-103Google Scholar, 12Aronin N. Chase K. Young C. Sapp E. Schwarz C. Matta N. Kornreich R. Landwehrmeyer B. Bird E. Beal F.M. Vonsattel J.P. Smith T. Carraway R. Boyce F.M. Young A.B. Penney J.B. DiFiglia M. Neuron. 1995; 15: 1193-1201Google Scholar, 13Schilling G. Sharp A.H. Loev S.J. Wagster M.V. Li S.H. Stine O.C. Ross C.A. Hum. Mol. Genet. 1995; 4: 1365-1371Google Scholar). Somatic mosaicism has also been invoked to account for the selective neuropathology. At both the DNA and protein level, significant somatic mosaicism of the CAG (polyglutamine) repeat was demonstrated in tissues from affected patients with HD, with the predominant levels of mosaicism in the caudate nucleus and the cortex, those regions most severely affected in HD (14Telenius H. Kremer B.J. Goldberg Y.P. Theilmann J. Andrew S.E. Adam S. Greenberg C. Ives E.J. Clarke L.A. Hayden M.R. Nat. Genet. 1994; 6: 409-414Google Scholar). Moreover, the cerebellum and brainstem show low levels of mosaicism and are infrequently involved in the neuropathology of HD (14Telenius H. Kremer B.J. Goldberg Y.P. Theilmann J. Andrew S.E. Adam S. Greenberg C. Ives E.J. Clarke L.A. Hayden M.R. Nat. Genet. 1994; 6: 409-414Google Scholar). However, the similar patterns of mosaicism are evident in SCA-I and DRPLA with limited mosaicism in the cerebellum even though this is one of the regions of the brain that is predominantly affected. This clearly indicates that somatic mosaicism was not likely to be a major factor in the genetically determined selective neuronal loss in HD or these other disorders (15Chong S.S. McCall A.E. Cota J. Subramony S.H. Orr H.T. Hughes M.R. Zoghbi H.Y. Nat. Genet. 1995; 10: 344-350Google Scholar, 16Ueno S. Kondah K. Kotani Y. Komure O. Kuno S. Kawai J. Hazama F. Sano A. Hum. Mol. Genet. 1995; 4: 663-666Google Scholar). It has recently been proposed that the selective neuropathology of these disorders could be related not only to expression of the gene containing CAG expansion, but to a protein with restricted expression with which the gene product normally associates (17Gusella J.F. MacDonald M.E. N. Engl. J. Med. 1994; 330: 1400-1451Google Scholar, 18Nasir J. Floresco S.B. O'Kusky J.R. Diewert V.M. Richman J.M. Zeisler J. Borowski A. Marth J.D. Phillips A.G. Hayden M.R. Cell. 1995; 81: 811-823Google Scholar). To identify proteins interacting with the HD gene product, we exploited the yeast two-hybrid system (19Fields S. Song O-K. Nature. 1989; 340: 245-246Google Scholar, 20Fields S. Sternglanz R. Trends Genet. 1994; 10: 286-292Google Scholar) and identified a human protein with an affinity for the amino terminus of the HD protein. This protein has complete amino acid identity with the previously described bovine ubiquitin-conjugating enzyme known as E2-25K (21Chen Z. Niles E.G. Pickart C.M. J. Biol. Chem. 1991; 266: 15698-15704Google Scholar). The E2-25K ubiquitin-conjugating enzyme belongs to a family of proteins that participate in the linking of COOH-terminal glycine residues of ubiquitin to specific lysine residues of target proteins (21Chen Z. Niles E.G. Pickart C.M. J. Biol. Chem. 1991; 266: 15698-15704Google Scholar). This ubiquitination leads to target protein degradation by the 26 S proteosome (22Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Google Scholar, 23Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Google Scholar, 24Deshaies R.J. Trends Cell Biol. 1995; 5: 428-434Google Scholar, 25Wefes I. Mastrandrea L.D. Haldeman M. Koury S.T. Tamburlin J. Pickart C.M. Finley D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4982-4986Google Scholar). We demonstrate that hE2-25K is highly expressed in brain and that cell-derived huntingtin interacts with purified hE2-25K in vitro. In addition, we clearly show that huntingtin is ubiquitinated in peripheral cells, which may have implications for the regulated catabolism of this protein. An HD cDNA construct (44EKpGBT9), with 44 glutamine repeats was generated containing amino acids 1-540 of the published HD cDNA (1Huntington's Disease Collaborative Research Group Cell. 1993; 72: 971-983Google Scholar, 26Goldberg Y.P. Kalchman M.A. Zeisler J. Graham R. Koide H.B. O'Kusky J. Sharp A.H. Ross C.A. Jirik F. Hayden M.R. Hum. Mol. Genet. 1996; 5 (177185): 2Google Scholar). This cDNA fragment was fused in-frame to the GAL4 DNA-binding domain (BD) of the yeast two-hybrid vector pGBT9 (Clontech). Another HD cDNA construct, 16EKpGBT9, was identical to 44EKpGBT9 but included only 16 glutamine repeats. Another clone (DMKΔBamHIpGBT9) containing the first 544 amino acids of the myotonic dystrophy gene (a gift from R. Korneluk) was fused in-frame with the GAL4-DNA BD of pGBT9 and was used as a negative control. Plasmids expressing the GAL4-BDRAD7 2D. Gietz, unpublished data. and SIR3 (27Paetkau D.W. Riese J.A. MacMorran W.S. Woods R.A. Gietz R.D. Genes Dev. 1994; 8: 2035-2045Google Scholar) were used as positive controls for the β-galactosidase filter assay. The yeast strain Y190 (MATa leu2-3, 112, ura3-52, trp1-901, his3-Δ200, ade2-101, gal4Δgal80Δ, URA3::GAL1-lacZ, Lys2::GAL-His3 cyh) (28Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Google Scholar) was used for all transformations and assays. Yeast transformations were performed using a modified lithium acetate transformation protocol (29Schiestl R.H. Manivasakum Woods R.A. Gietz R.D. Johnston M. Fields S. Methods: A Companion to Methods in Enzymology. Academic Press, Inc., New York1993: 79Google Scholar) and grown at 30°C using appropriate synthetic complete dropout media. The β-galactosidase chromogenic filter assays were performed by transferring the yeast colonies onto Whatman filters. The yeast cells were partially lysed by submerging the filters in liquid nitrogen for 15-20 s. Filters were allowed to dry at room temperature for at least 5 min and placed onto filter paper presoaked in Z-buffer (100 mM sodium phosphate (pH 7.0) 10 mM KCl, 1 mM MgSO4) supplemented with 50 mMβ-mercaptoethanol and 0.07 mg/ml 5-bromo-4-chloro-3-indolyl β-D-galactoside. Filters were placed at 37°C for up to 8 h. Liquid β-galactosidase assays were performed by inoculating a single yeast colony into appropriate synthetic complete dropout media and grown to OD600 0.6-1.5. Five milliliters of overnight culture was pelleted and washed once with 1 ml of Z-Buffer, then resuspended in 100 µl of Z-Buffer supplemented with 38 mMβ-mercaptoethanol and 0.05% SDS. Acid-washed glass beads (~100 µl) were added to each sample and vortexed for 4 min, by repeatedly alternating a 30-s vortex, with 30 s on ice. Each sample was pelleted and 10 µl of lysate was added to 500 µl of lysis buffer. The samples were incubated in a 30°C water bath for 30 s and then 100 µl of 4 mg/ml o-nitrophenyl β-D-galactopyranoside solution was added to each tube. The reaction was allowed to continue for 13 min at 30°C and stopped by the addition of 500 µl of 1 M Na2CO3 and placing the samples on ice. Subsequently, OD420 was taken in order to calculate the β-galactosidase activity with the equation 1000 × OD420/(t × V × OD600) (30Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar), where t is the elapsed time (minutes) and V is the amount of lysate used. A human adult brain Matchmaker™ cDNA library (Clontech) was transformed into the yeast strain Y190 already harboring the 44EKpGBT9 construct. The transformants were plated onto 100 150 × 15-mm circular culture dishes containing synthetic complete media deficient in Trp, Leu, and His. The herbicide 3-aminotriazole (25 mM) was utilized to limit the number of false His+ positives (31Durfee T. Becherer K. Chem P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Google Scholar). The yeast transformants were placed at 30°C for 5 days and β-galactosidase filter assays were performed on all colonies found after this time, as described above, to identify β-galactosidase+ clones. Primary His+/β-galactosidase+ clones were then orderly patched onto a grid on synthetic complete -Trp/-Leu/-His (25 mM 3-aminotriazole) 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 activating domain (AD) cDNA plasmids were isolated by growing the His+/β-galactosidase+ colony in synthetic complete -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-α for further manipulation. Oligonucleotide primers were synthesized on an ABI PCR-mate oligo-synthesizer. DNA sequencing was performed using an ABI 373 fluorescent automated DNA sequencer. The HIP cDNAs were confirmed to be in-frame with the GAL4-AD by sequencing across the AD-HIP-2 cloning junction using an AD oligonucleotide (5′-GAA GAT ACC CCA CCA AAC-3′). Subsequently, primer walking was used to determine the remaining sequences. 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 antisense oligonucleotide 5′-CCG TGC GGA GAG TCA TTG CAG CTG-3′ 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. The HIP-2 cDNA was released from the GAL4-AD library plasmid, pGAD10, by digestion with NotI, ligated into the NotI site of pGEX4T-2 (Pharmacia) and electroporated into DH5-α. A clone in the correct orientation was electroporated into the Escherichia coli host BL21 (Pharmacia) for expression of the GST protein. A single colony of both GST-HIP-2 and GST alone were inoculated into 5 ml of LB liquid media supplemented with 100 µg/ml ampicillin and grown overnight at 37°C with good aeration. The 5-ml culture was subcultured into a 30-ml LB culture supplemented with 100 µg/ml ampicillin and grown shaking overnight at 37°C. The 30-ml culture was poured into 500 ml of 2 × YT media supplemented with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside and 100 µg/ml ampicillin, and grown shaking overnight at 26°C. Two hundred and fifty-milliliter aliquots of culture were pelleted and resuspended in 12.5 ml of ice-cold 1 × phosphate-buffered saline. The bacterial suspension was sonicated with 30-s intervals for 10 min. The supernatant was passed through a glutathione-Sepharose (Pharmacia) column (500 µl). The column was washed 3 times with 10 ml of ice-cold 1 × phosphate-buffered saline. One milliliter 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 × phosphate-buffered saline to remove the glutathione. Clones containing either 44 or 16 glutamine repeats, from amino acid 1 through 540 were cloned (26Goldberg Y.P. Kalchman M.A. Zeisler J. Graham R. Koide H.B. O'Kusky J. Sharp A.H. Ross C.A. Jirik F. Hayden M.R. Hum. Mol. Genet. 1996; 5 (177185): 2Google Scholar) into the RcCMV vector (Invitrogen). The in vitro HD products were synthesized according to the manufacturers directions for the TnT-Rabbit Reticulocyte Lysate system (Promega) except 1 mM cold L-methionine was substituted for L-[35S]methionine. The in vitro products were aliquoted and stored at −70°C. To generate monoclonal antibodies against the HD protein, part of the HD cDNA from nucleotide 5345 in exon 39 to nucleotide 6257 in exon 44 was cloned in-frame into the bacterial pATH expression vector (32Koemer T.J. Hill J.E. Myers A.M. Tzagoloff A. Methods Enzymol. 1990; 194: 477-491Google Scholar). Approximately 20 µg of fusion protein was injected subcutaneously into BALB/c mice in the presence of Freund's adjuvant. Following three additional boosts, spleen cells from the immunized mice were fused with NS1 myeloma cells to generate antibody secreting hybridomas. Culture supernatants were screened for anti-HD antibodies by enzyme-linked immunosorbent assay, using bacterial TrpE as a negative control. Approximately 600 hybrid clones were generated, of which 35 recognized the fusion protein. Seven of these did not react with the trpE portion of the fusion protein and were used for further analysis. Western blot analysis revealed that one of the clones secreted antibodies (GHM1) that recognized the 350-kDa human HD protein but did not recognize the HD murine homologue. In order to generate an HD amino-terminal polyclonal antibody, a peptide corresponding to amino acids 3-16 (TLEKLMKAFESLKSC) was synthesized and coupled to keyhole limpet homocyanin using succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Two female New Zealand White rabbits were immunized with this peptide antigen with Freund's adjuvant. Antibodies were purified on an affinity column, made with synthesized peptide bound to thiol-activated Sepharose. Purified antibodies (BKP1) were used for Western blotting and detected a 350-kDa protein in human and mouse tissues and also in cell lines, including 293 cell and ES cell lines containing the HD gene, but not in ES cell lines lacking the HD gene. Five microliters of in vitro translated HD proteins (amino acids 1-540 with either 44 or 16 glutamine repeats) were incubated with GST-HIP-2 and GST (10 µg each) in 500 µl of reaction buffer (20 mM Tris-HCl (pH 7.5), 120 mM NaCl) for 2 h at 4°C. Glutathione-Sepharose beads (10 µl) were then added and incubated for an additional 2 h. The beads were pelleted for 5 min, and washed 3 times with reaction buffer containing 3% Nonidet P-40. Samples were mixed with Laemmli sample buffer, applied to 7.5% SDS-PAGE gel, and transferred to PVDF membrane (Millipore). Immunodetection was performed using one of two HD NH2-terminal polyclonal antibodies (AP78 or BKP1). For the experiments with 293 cell lysates, GST-HIP-2, GST-PTPase and GST were incubated with 300 µl of cell lysate (~500 µg of total protein) and 200 µl of reaction buffer. In order to confirm that HIP-2 is in fact encoded for the hE2-25K protein, an affinity-purified polyclonal anti-bE2-25K antibody (33Haldeman M. Finley D. Pickart C.M. J. Biol. Chem. 1995; 270: 9507-9516Google Scholar) was immunoreacted against the HIP-2 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. 3G. Xia and C. Pickart, unpublished experiments. The membranes were blocked in 5% skim milk powder in Tris-buffered saline (TBS) (pH 7.4) and immunoreacted in blocking buffer with the anti-bE2-25K polyclonal antibody (1:5000) for 1 h. After washing the membrane three times in TBS-T (Tris-buffered saline (pH 7.4), 0.05% Tween 20), a horseradish peroxidase-conjugated secondary antibody (1:10000) (Bio-Rad) was immunoreacted against the blots for 1 h 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 hE2-25K was mapped to chromosome 4p14 by fluorescent in situ hybridization (FISH) (34Lichter P. Tang C.J. Call K. Hermanson G. Evans G.A. Housman D. Ward D.C. Science. 1990; 247: 64-69Google Scholar) to normal human lymphocyte chromosomes counterstained with propidium iodide and DAPI. Biotinylated probe was detected with avidin-fluorescein isothiocyanate. Images of metaphase preparations were captured by a thermoelectrically cooled charge coupled camera (Photometrics). Separate images of DAPI banded chromosomes (35Heng H. Tsui L.C. Chromosoma (Berl.). 1993; 102: 325-332Google Scholar) and FITC targeted chromosomes were obtained. Hybridization signals were acquired and merged using image analysis software and pseudo colored blue (DAPI) and yellow (fluorescein isothiocyanate) as described (36Boyle A.L. Feltquite D.M. Dracopoli N.C. Housman D.E. Ward D.C. Genomics. 1992; 12: 106-115Google Scholar) and overlaid electronically. Northern blot analysis was performed as described previously (33Haldeman M. Finley D. Pickart C.M. J. Biol. Chem. 1995; 270: 9507-9516Google Scholar). Filters were hybridized with a PAGE purified antisense riboprobe as described previously (37MacDonald R.J. Smith G.H. Przybyla A.E. Chirgwin J.M. Methods Enzymol. 1987; 152: 219-227Google Scholar). The riboprobe was labeled to a specific activity of ~109 cpm/µg and approximately 2.0 × 106 cpm/ml of labeled probe was used to probe the filter. The human embryonic kidney cell line HEK293 was grown in Dulbecco's modified Eagle's medium F-12 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 Na3VO4, 20 mMβ-glycerophosphate: with 10 µg/ml each of leupeptin, aprotinin, antipain, soybean trypsin inhibitor, pepstatin, and 100 µM phenylmethylsulfonyl fluoride). Protein extracts (as specified in figure legends) were separated on 10% SDS-PAGE mini-gels and transferred to PVDF membrane. Filters were then probed with an affinity-purified anti-bE2-25K polyclonal antibody (33Haldeman M. Finley D. Pickart C.M. J. Biol. Chem. 1995; 270: 9507-9516Google Scholar), with detection by ECL. 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 Nonidet P-40, and supplemented with N-ethylmaleimide to inactivate endogenous de-ubiquitinating enzymes (38Haas A.L. Bright P.M. J. Biol. Chem. 1985; 260: 12464-12473Google Scholar). 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 µl. Five microliters of affinity-purified rabbit polyclonal antibodies against ubiquitin were added and the mixture rotated at 4°C for 3 h. Protein A-Sepharose (Sigma), 50 µl of a 1:1 slurry in dilution buffer, was then added, and the suspension was rotated at room temperature for 30 min. The resin was pelleted, then washed four times with 500 µl of dilution buffer. The beads were suspended in 40 µl of 2 × SDS-PAGE sample buffer and boiled for 1 min. The resin was pelleted and 20-µl 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. An HD cDNA spanning amino acids 1-540 and containing a polyglutamine tract of 44 residues was fused in-frame with the GAL4 DNA-binding domain and used in a yeast two-hybrid screen. Approximately 4.0 × 107 -Trp/-Leu transformants were screened to isolate a number of HIPs from an adult human brain Matchmaker™ cDNA library. A total of 14 positive clones passed through a second round of His+/β-galactosidase screening. These 14 clones could be divided into three classes. The single HIP-2 clone represented a 2048-base pair cDNA. In addition, other cDNAs coding for HIP-1 and HIP-3 were identified and are currently being characterized. The HIP-2-GAL4 activating domain fusion protein was shown to specifically interact with the GAL4BD-HD fusion protein, as yeast containing HIP-2 and the HD protein (amino acids 1-540) gave a His+ phenotype as well as showed β-galactosidase activity in a chromogenic filter assay (Fig. 1A). Specificity of interaction was shown by the finding that HIP-2 did not stimulate β-galactosidase activity with the DNA-binding domain, with vector alone (pGBT9), or with an unrelated fusion with myotonin kinase control (Fig. 1, A and B). We next sought to determine whether the size of the polyglutamine tract influenced the interaction of HIP-2 with the HD protein. We performed semi-quantitative analysis using liquid β-galactosidase assays (Fig. 1B). This revealed no difference in the strength of the interaction between HIP-2 and HD constructs (amino acids 1-540) containing either 44 or 16 glutamine repeats. Smaller fusion proteins containing either 44 or 16 glutamine repeats and the first 242 amino acids of the HD cDNA were also tested for interaction with HIP-2. There was no detectable interaction (Fig. 1B). 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 (data not shown). In order to assess the interaction between the HIP-2 protein and HD, in vitro binding assays were performed using GST fusion proteins. In vitro translated products corresponding to the first 540 amino acids containing either 44 or 16 glutamine repeats of the HD protein were incubated with GST-HIP-2 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. 2A). The interaction was not obviously influenced by repeat size, consistent with the results obtained in the quantitative interaction assays of fusion proteins bearing different length glutamine repeats (Fig. 1B). Co-affinity purification experiments were also performed using a human embryonic kidney cell line (HEK293) cell lysates to assess the interaction between HIP-2 and endogenous full-length HD protein. Incubation of HEK293 lysate with GST-HIP-2 linked to glutathione-Sepharose beads resulted in specific affinity purification of the 350-kDa HD protein on the beads (Fig. 2B). The HD protein failed to copurify with the GST-PTPase or GST protein alone. The detection of the HD protein on Western blots could be blocked by preincubation with peptide antigen (data not shown). Analysis of sequence data revealed that the HIP-2 protein had complete amino acid identity with a previously described bovine E2-25K (bE2-25K) ubiquitin-conjugating enzyme (21Chen Z. Niles E.G. Pickart C.M. J. Biol. Chem. 1991; 266: 15698-15704Google Scholar). Our original HIP-2 cDNA spanned all but the most 5′ 99 nucleotides of the published bovine sequence (21Chen Z. Niles E.G. Pickart C.M. J. Biol. Chem. 1991; 266: 15698-15704Google Scholar). Thus the N-terminal 33 residues of the E2 protein are not necessary for the interaction of E2-25K with huntingtin. The DNA 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 (“Experimental Procedures”). 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. 3A). Residue 23 in the hE2-25K amino sequence is a serine while the published bE2-25K has a threonine at this codon (21Chen Z. Niles E.G. Pickart C.M. J. Biol. Chem. 1991; 266: 15698-15704Google Scholar). However, resequencing of the bE2-25K cDNA revealed that the bovine enzyme also has a threonine at this codon. 4L. Mastrandrea and C. Pickart, unpublished experiments. There are a total of 19 conservative nucleotide changes in the coding region and nine nucleotide changes in the known 3′-untranslated region sequence between human and bovine E2-25K cDNA (Fig. 3A). The HIP-2 cDNA isolated from the HD yeast two-hybrid screen contains an additional 3′-untranslated region sequence relative to that published for the bovine gene (21Chen Z. Niles E.G. Pickart C.M. J. Biol. Chem. 1991; 266: 15698-15704Google Scholar). The complete identity between the bovine and human E2-25K enzymes places hE2-25K in the same class of conjugating enzymes as the E2- encoded by the UBC1, UBC4, and UBC5 genes of S. cerevisiae (21Chen Z. Niles E.G. Pickart C.M. J. Biol. Chem. 1991; 266: 15698-15704Google" @default.
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