Proteasomes Cleave at Multiple Sites within Polyglutamine Tracts

Abstract

Eukaryotic proteasomes have been reported to cleave only once within polyglutamine tracts and then only after the N-terminal glutamine (Venkatraman, P., Wetzel, R., Tanaka, M., Nukina, N., and Goldberg, A. L. (2004) Mol. Cell 14, 95–104). We have obtained results that directly conflict with that report. In the presence of the proteasome activator PA28γ(K188E) human red cell proteasomes progressively degraded fluorescein-GGQ10RR or fluorescein-HPHQ10RR into small fragments as shown by size exclusion chromatography and mass spectrometry. MALDI-TOF mass spectrometry revealed that proteolytic products arose from cleavage after every glutamine in fluorescein-HPHQ10RR, and mass accuracy rules out deamidation of glutamine to glutamic acid as an explanation for peptide degradation. Moreover, degradation cannot be attributed to a contaminating protease because peptide hydrolysis was completely blocked by the proteasome-specific inhibitors, lactacystin and epoxomicin. We conclude that proteasomes cleave repetitively anywhere within a stretch of ten glutamine residues. Thus our results cast doubt on the idea that mammalian proteasomes cannot degrade glutamine-expanded regions within pathogenic polyQ-expanded proteins, such as Huntingtin. Eukaryotic proteasomes have been reported to cleave only once within polyglutamine tracts and then only after the N-terminal glutamine (Venkatraman, P., Wetzel, R., Tanaka, M., Nukina, N., and Goldberg, A. L. (2004) Mol. Cell 14, 95–104). We have obtained results that directly conflict with that report. In the presence of the proteasome activator PA28γ(K188E) human red cell proteasomes progressively degraded fluorescein-GGQ10RR or fluorescein-HPHQ10RR into small fragments as shown by size exclusion chromatography and mass spectrometry. MALDI-TOF mass spectrometry revealed that proteolytic products arose from cleavage after every glutamine in fluorescein-HPHQ10RR, and mass accuracy rules out deamidation of glutamine to glutamic acid as an explanation for peptide degradation. Moreover, degradation cannot be attributed to a contaminating protease because peptide hydrolysis was completely blocked by the proteasome-specific inhibitors, lactacystin and epoxomicin. We conclude that proteasomes cleave repetitively anywhere within a stretch of ten glutamine residues. Thus our results cast doubt on the idea that mammalian proteasomes cannot degrade glutamine-expanded regions within pathogenic polyQ-expanded proteins, such as Huntingtin. Trinucleotide expansions, the cause of a number of human diseases, can occur in nontranscribed regions of the genome in transcribed but nontranslated sequences and in coding regions (1Orr H.T. Zoghbi H.Y. Annu. Rev. Neurosci. 2007; 30: 575-621Crossref PubMed Scopus (1063) Google Scholar). Expansions that result in altered protein structure involve two amino acids, an alanine encoded by GCG and glutamine (Q) encoded by CAG (2Albrecht A. Mundlos S. Curr. Opin. Genet. Dev. 2005; 15: 285-293Crossref PubMed Scopus (122) Google Scholar, 3Paulson H.L. Bonini N.M. Roth K.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12957-12958Crossref PubMed Scopus (100) Google Scholar). Both polyalanine and polyglutamine expansions lead to the formation of insoluble inclusions, usually nuclear, that often recruit chaperones, ubiquitin, proteasomes, and a variety of other proteins (4Ross C.A. Poirier M.A. Nat. Med. 2004; 10: S10-S17Crossref PubMed Scopus (2347) Google Scholar, 5Weydt P. La Spada A.R. Expert Opin. Therap. Targets. 2006; 10: 505-513Crossref PubMed Scopus (18) Google Scholar). Glutamine tract expansions occur in a disparate set of proteins with no common biochemical function, and the polyglutamine diseases, such as Huntington, Kennedy, and a number of ataxias, usually occur when the glutamine tract exceeds 35 residues in length (6Klein F.A. Pastore A. Masino L. Zeder-Lutz G. Nierengarten H. Oulad-Abdelghani M. Altschuh D. Mandel J.L. Trottier Y. J. Mol. Biol. 2007; 371: 235-244Crossref PubMed Scopus (76) Google Scholar). Whereas most polyQ-expanded proteins are expressed in numerous tissues, the degenerative process is restricted to a subset of neurons specific for each disease (7Brignull H.R. Moore F.E. Tang S.J. Morimoto R.I. J. Neurosci. 2006; 26: 7597-7606Crossref PubMed Scopus (139) Google Scholar). There are a number of hypotheses concerning the mechanism by which polyglutamine (polyQ) expansions cause disease. The most popular hypothesis is that polyQ-expanded regions interfere with transcription (8Schaffar G. Breuer P. Boteva R. Behrends C. Tzvetkov N. Strippel N. Sakahira H. Siegers K. Hayer-Hartl M. Hartl F.U. Mol. Cell. 2004; 15: 95-105Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 9Riley B.E. Orr H.T. Genes Dev. 2006; 20: 2183-2192Crossref PubMed Scopus (129) Google Scholar, 10Helmlinger D. Tora L. Devys D. Trends Genet. 2006; 22: 562-570Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). A number of findings support this hypothesis including the fact that changes in mRNA transcript levels are among the earliest manifestations of polyQ expression (11Luthi-Carter R. Strand A. Peters N.L. Solano S.M. Hollingsworth Z.R. Menon A.S. Frey A.S. Spektor B.S. Penney E.B. Schilling G. Ross C.A. Borchelt D.R. Tapscott S.J. Young A.B. Cha J.-H.J. Olson J.M. Hum. Mol. Genet. 2000; 9: 1259-1271Crossref PubMed Scopus (607) Google Scholar). Several articles have presented evidence that polyQ expansions interfere with axonal transport (12Gunawardena S. Her L.S. Brusch R.G. Laymon R.A. Niesman I.R. Gordesky-Gold B. Sintasath L. Bonini N.M. Goldstein L.S. Neuron. 2003; 40: 25-40Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 13Szebenyi G. Morfini G.A. Babcock A. Gould M. Selkoe K. Stenoien D.L. Young M. Faber P.W. MacDonald M.E. McPhaul M.J. Brady S.T. Neuron. 2003; 40: 41-52Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 14Morfini G. Pigino G. Brady S.T. Trends Mol. Med. 2005; 11: 64-70Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Several reports suggest that impairment of the ubiquitin-proteasome system may contribute to polyQ pathology (15Bence N. Sampat R. Kopito R. Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1783) Google Scholar, 16Bennett E.J. Bence N.F. Jayakumar R. Kopito R.R. Mol. Cell. 2005; 17: 351-365Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 17Bennett E.J. Shaler T.A. Woodman B. Ryu K.Y. Zaitseva T.S. Becker C.H. Bates G.P. Schulman H. Kopito R.R. Nature. 2007; 448: 704-708Crossref PubMed Scopus (422) Google Scholar, see Ref. 18Ciechanover A. Brundin P. Neuron. 2003; 40: 427-446Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar for review). In this context, we proposed that Huntington and the polyQ ataxias could be analogous to lysosomal storage diseases (19Goellner G.M. Rechsteiner M. Int. J. Biochem. Cell Biol. 2003; 35: 562-571Crossref PubMed Scopus (36) Google Scholar). Our proposal was based on the assumption that proteasomes would become less active upon filling with poorly degradable polyQ fragments. We also hypothesized that the proteasome activator PA28γ, which is highly expressed in neurons, could exacerbate polyQ pathology, because it suppresses the proteasome chymotrypsin-like active site (20Li J. Gao X. Ortega J. Nazif T. Joss L. Bogyo M. Steven A. Rechsteiner M. EMBO J. 2001; 20: 3359-3369Crossref PubMed Scopus (56) Google Scholar). The idea that PA28γ impacts polyQ disease was largely discredited by a recent collaborative study with the laboratory of Gillian Bates that examined proteasome activity in the brains of R6/2 mice, a well-studied model of Huntington disease (21Bett J.S. Goellner G.M. Woodman B. Pratt G. Rechsteiner M. Bates G.P. Hum. Mol. Genet. 2006; 15: 33-44Crossref PubMed Scopus (85) Google Scholar). Disease progression was unaffected by the presence or absence of PA28γ. Moreover, we did not observe any difference in proteasome activity in brain extracts from R6/2 and wild-type mice (21Bett J.S. Goellner G.M. Woodman B. Pratt G. Rechsteiner M. Bates G.P. Hum. Mol. Genet. 2006; 15: 33-44Crossref PubMed Scopus (85) Google Scholar). Similar results were obtained by Bowman et al. (22Bowman A.B. Yoo S.Y. Dantuma N.P. Zoghbi H.Y. Hum. Mol. Genet. 2005; 14: 679-691Crossref PubMed Scopus (176) Google Scholar) and by Diaz-Hernandez et al. (23Diaz-Hernandez M. Hernandez F. Martin-Aparicio E. Gomez-Ramos P. Moran M.A. Castano J.G. Ferrer I. Avila J. Lucas J.J. J. Neurosci. 2003; 23: 11653-11661Crossref PubMed Google Scholar) who found, respectively, that proteasome activity was unimpaired in the retinas of mice expressing polyQ-expanded ataxin or in brain extracts from the HD94 mouse model of Huntington disease. On the other hand, several investigators have reported that proteasome activity is inhibited in cells or tissues expressing polyQ-expanded proteins (24Seo H. Sonntag K.C. Isacson O. Ann. Neurol. 2004; 56: 319-328Crossref PubMed Scopus (145) Google Scholar, 25Nishitoh H. Matsuzawa A. Tobiume K. Saegusa K. Takeda K. Inoue K. Hori S. Kakizuka A. Ichijo H. Genes Dev. 2002; 16: 1345-1355Crossref PubMed Scopus (1114) Google Scholar). It has been reported that proteasomes do not cleave Gln-Gln bonds beyond the first two glutamines in polyQ tracts (26Venkatraman P. Wetzel R. Tanaka M. Nukina N. Goldberg A.L. Mol. Cell. 2004; 14: 95-104Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). This latter finding, if correct, would suggest that polyglutamine fragments might well accumulate within proteasomes. However, as shown by the results presented below, proteasomes are capable of cleaving Q-Q bonds anywhere within a tract of 10 contiguous glutamines. Thus, there is little reason to propose that proteasomes are inactivated by the buildup of polyQ fragments in their central chambers. Materials—Suc-LeuLeuValTyr-MCA and cbz-LeuLeuGlu-βNA were obtained from Sigma; boc-LeuArgArg-MCA was purchased from Peptides International (Louisville, KY). Lactacystin was purchased from CalBiochem (EMD Chemicals Darmstadt, Germany), and epoxomicin from A. G. Scientific (San Diego, CA). The peptide aldehydes, Ac-PGPH-al and Ac-EPFD-al, were gifts from Jennifer Harris of the Burnham Institute for Medical Research (La Jolla, CA). The peptides, Q5, Q3-R2, GG-Q7-RR, GG-Q10-RR, and HPH-Q10-RR, were synthesized at the University of Utah HSC Core Research Facilities. When employed, carboxyfluorescein was coupled to the N terminus of the peptide while still on the resin. Crude peptides were purified on a C18 column (Grace Vydac, Basel, Switzerland) with an acetonitrile gradient containing 0.1% trifluoroacetic acid. Purity was assessed by re-injection on reversed phase liquid chromatography and by mass spectrometry using either MALDI 2The abbreviations used are: MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; ESI, electrospray ionization; PGPH, postglutamyl-preferring; T, trypsin-like; CT, chymotrypsin-like; Fl, fluorescein; MCA, 7-amino-4-methylcoumarin; βNA, β-naphthylamide. or ESI ionization. Lyophilized samples of the peptides were dissolved by weight in either reticulocyte buffer (40 mm Tris, pH 7.8, 10 mm MgCl2, 20 mm KCl, 2 mm dithiothreitol) or in 10 mm ammonium phosphate, pH 7.8. The concentration of non-fluoresceinated peptides was determined by amino acid analysis, and the concentration of fluoresceinated peptides was assayed by comparison to a fluorescein standard curve. Degradation Assays—The 20 S proteasome was purified from outdated human red blood cells as previously described (27Hoffman L. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22362-22368Abstract Full Text PDF PubMed Google Scholar, 28Dubiel W. Pratt G. Ferrell K. Rechsteiner M. J. Biol. Chem. 1992; 267: 22369-22377Abstract Full Text PDF PubMed Google Scholar), and recombinant PA28γ and PA28γ(K188E) were purified as outlined in Realini et al. (29Realini C. Jensen C. Zhang Z. Johnston S. Knowlton R. Hill C.P. Rechsteiner M. J. Biol. Chem. 1997; 272: 25483-25492Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Assays of fluorogenic peptide substrates were carried out in 100-μl reactions in reticulocyte buffer (20 mm Tris, pH 7.8, 5 mm MgCl2, 10 mm KCl, and 1 mm dithiothreitol). Pre-mixes (50 μl) were prepared containing 0.3 μg of proteasome or 0.3 μg of proteasome plus 1 μg of PA28γ(K188E). Reactions were initiated by adding 50 μl of 200 μm fluorogenic peptides in reticulocyte buffer. After incubation, samples were quenched with 200 μl of ETOH, and fluorescence was measured in a PerkinElmer LS5 spectrofluorimeter with excitation/emission at for substrates and excitation/emission at for Assays fluoresceinated peptides were to described that the of fluorescein-GGQ10RR and fluorescein-HPHQ10RR peptide were μm and respectively, and the proteasome concentration was 1 to μg upon the reactions were by on to by or mass spectrometry. chromatography was on a column μl) were the column at in a buffer of mm Tris, pH containing mm After the 10 of were and their fluorescence was measured at and spectrometry was carried out in the Core of the University of of the mass shown were using on a mass samples were using the using a of acid in 0.1% trifluoroacetic acid. MALDI were in at a mass from to Peptides and by were by the from et al. (26Venkatraman P. Wetzel R. Tanaka M. Nukina N. Goldberg A.L. Mol. Cell. 2004; 14: 95-104Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) that proteasomes do not cleave beyond the first or two residues within a polyQ because a of proteasome that glutamines at the of fluorogenic cleavage after the amino acid J.L. J. Rechsteiner M. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of the degradation products upon hydrolysis of the substrates and of the to the that polyQ tracts are to proteasome hydrolysis (26Venkatraman P. Wetzel R. Tanaka M. Nukina N. Goldberg A.L. Mol. Cell. 2004; 14: 95-104Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). containing 20 S proteasomes and the proteasome activator were reported to cleave at a site and with the to and peptide products were in the proteasome products were to be However, the mass presented in of et al. (26Venkatraman P. Wetzel R. Tanaka M. Nukina N. Goldberg A.L. Mol. Cell. 2004; 14: 95-104Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) that numerous products might well be the at for products could result from of the residues from each of the glutamine tract in peptides containing only glutamine residues would be by mass spectrometry. We this by mass spectrometry of two peptides, and When the peptides were at and to mass the peptide containing was at in the Moreover, was as a the peptide was among and less mass spectrometry not a of products upon of polyQ the of to small peptides from cannot be as evidence for polyQ to proteasome degradation. within a of Glutamine mass spectrometry is not for we synthesized a fluorescein to degradation by size exclusion The was to fluorescein on the assumption that the residues would of N-terminal products by mass and the would proteasome cleavage to were as shown by the mass in When a of was assayed by mass spectrometry a number of cleavage products were and the Peptides from cleavage after each of the ten glutamines were in the mass and their was the accuracy of mass The absence of products containing only glutamine residues was in of their it was with the of of the products It that of the two residues is an in the proteolytic of of degradation in a The results in 2 and 1 are in conflict with the that proteasomes cleave only once within polyQ the presence of fluorescein at the N terminus of altered degradation by proteasomes such that in the polyQ tract this an peptide was synthesized with or fluorescein at N The two peptides were with proteasomes and a proteasome activator and samples were by mass spectrometry. of this analysis, presented in a of fluorescein on the of products from The presence of fluorescein in products residues. degradation of a of peptides of which or residues. the in 2 that hydrolysis after every glutamine in the non-fluoresceinated while an N-terminal fluorescein can by the the after glutamines within polyQ tracts in non-fluoresceinated Thus, in to the results of et al. (26Venkatraman P. Wetzel R. Tanaka M. Nukina N. Goldberg A.L. Mol. Cell. 2004; 14: 95-104Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar), we that human red cell proteasomes cleave anywhere within a stretch of or ten contiguous glutamine of degradation products from and in a Degradation of and by the of degradation could be by as well as mass the peptide was with 20 S proteasomes and the activator PA28γ(K188E). were after or for by MALDI-TOF or by for the proteolytic obtained from that shown in are in Whereas peptides containing or 10 glutamines were still at of incubation, were less at and by The of products was by an in peptides, and the of by of of the presented in with the mass at and in that there was a and to fluoresceinated in hydrolysis of in the we the column to the of and wild-type proteasome activator PA28γ on degradation of or for is and less mass spectrometry. our was on within the tract peptide cleavage products small to be well from on the peptide we were in of degradation of polyQ peptides by wild-type PA28γ, because polyQ degradation by proteasomes might Eukaryotic proteasomes of which are of the the chymotrypsin-like and Whereas by PA28γ only the in wild-type PA28γ suppresses the active site (29Realini C. Jensen C. Zhang Z. Johnston S. Knowlton R. Hill C.P. Rechsteiner M. J. Biol. Chem. 1997; 272: 25483-25492Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). we a site of recombinant PA28γ that (20Li J. Gao X. Ortega J. Nazif T. Joss L. Bogyo M. Steven A. Rechsteiner M. EMBO J. 2001; 20: 3359-3369Crossref PubMed Scopus (56) Google Scholar). degradation of fluorogenic with glutamine at the was by but only by PA28γ J.L. J. Rechsteiner M. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), we that PA28γ(K188E) would the of degradation by proteasomes. a of this we the peptide with proteasomes or proteasomes plus wild-type or PA28γ, and samples were by as described in shown by the results in degradation of was by PA28γ(K188E) by wild-type results were obtained with as degradation of substrates was by PA28γ, this may of the the proteasome L. Knowlton Y. Hill C.P. Nature. 2000; PubMed Scopus Google Scholar) of proteasome by PA28γ. In any PA28γ(K188E) cleavage within polyQ and this has for the of polyglutamine Degradation of and to a fact that the proteasome activator PA28γ(K188E) the of polyQ peptide is evidence that the proteasome is the PA28γ(K188E) is a recombinant protein that could be by an for a contaminating we examined the of two specific proteasome inhibitors, lactacystin and on peptide The in that degradation of was completely inhibited by and the in that in the presence of epoxomicin. mass that the after of with or proteasomes. fluorogenic or amino substrates were not degraded after of with proteasomes. results that our proteasome is of contaminating and evidence that the fluoresceinated peptides in were degraded by the Our of proteasome using a of fluorogenic in the presence of proteasomes cleave after glutamine but of cleaving or bonds J.L. J. Rechsteiner M. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). also to the of two peptide aldehydes, and which and active shown in inhibited degradation of that the active site of the proteasome is for cleavage within polyQ of and as proteasome in a The results presented evidence that proteasomes can cleave within polyQ This is from the mass spectrometry presented in 2 as well as 1 and degradation of and to peptides by hydrolysis after each glutamine in and of the glutamines in mass spectrometry and that degradation was to small peptides that only or two glutamines. We conclude that proteasomes can cleave at within a polyQ and can do The of peptide by lactacystin or epoxomicin A and the that degradation was to a contaminating glutamine residues in our peptide substrates were to to then peptide degradation might be attributed to the active site of the However, the difference and for each proteolytic was less This of mass accuracy rules out glutamine deamidation as a explanation for peptide degradation because of by would the mass of products by 1 if proteasome cleavage only after glutamic acid residues by the of the proteasome would the active would have to peptide but it did not Our results are in conflict with reported by et al. (26Venkatraman P. Wetzel R. Tanaka M. Nukina N. Goldberg A.L. Mol. Cell. 2004; 14: 95-104Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). we that of peptides containing only glutamine to the that proteasomes do not cleave within polyQ However, the our results and might in proteasome or we used mammalian proteasomes and because PA28γ(K188E) has to used in their we do not the idea that proteasomes cleave polyQ sequences only our of we it that proteasomes are capable of polyQ sequences In this we have that the of is the as in cells and that are degraded by proteasomes. G. G. C. and M. in the and PA28γ in their with proteasome while PA28γ only the and PA28γ also in their and M. Realini C. Biochem. J. 2000; PubMed Scopus Google Scholar). is is in and is in PA28γ is and is highly expressed in In PA28γ suppresses the proteasome that is for cleavage within polyQ tracts to that PA28γ might be to the of polyQ-expanded proteins, and of expression in brain could for polyglutamine to that (20Li J. Gao X. Ortega J. Nazif T. Joss L. Bogyo M. Steven A. Rechsteiner M. EMBO J. 2001; 20: 3359-3369Crossref PubMed Scopus (56) Google Scholar). Our recent that PA28γ did not disease progression in R6/2 mice (21Bett J.S. Goellner G.M. Woodman B. Pratt G. Rechsteiner M. Bates G.P. Hum. Mol. Genet. 2006; 15: 33-44Crossref PubMed Scopus (85) Google Scholar) this the of proteasomes to cleave within polyQ tracts and PA28γ(K188E) of polyQ peptide degradation the of a A number of have been proposed as for polyglutamine diseases Rev. 2005; Scopus (122) Google Scholar, M. D. S. F. Cell Mol. Sci. 2006; PubMed Scopus Google Scholar). proteins often and for the polyQ disease A has shown that of proteins the of cells expressing polyQ-expanded proteins Ref. J.L. Nat. Rev. Neurosci. 2005; PubMed Scopus Google Scholar for a review). polyQ-expanded proteins are as to be the T. A. A. S. Biochem. 2000; 272: PubMed Scopus Google Scholar, J.L. H. H. T. J. Hum. Mol. Genet. 2000; 9: Scopus Google Scholar), a in their levels also of human polyQ-expanded or in mouse of diseases in H. Orr H.T. H.L. L. Nat. Med. 2004; 10: PubMed Scopus Google Scholar, X. L. H.L. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). the degradation of polyQ-expanded proteins have the In this lysosomal of polyQ S. S. S. A. T.A. Nat. Chem. Biol. 2007; PubMed Scopus Google Scholar), and degradation of polyQ-expanded proteins have been E. A. Hernandez F. R. Avila J. Lucas J.J. J. Neurosci. 2001; PubMed Google Scholar). The presented that a PA28γ the degradation of peptides containing polyQ results to proteins, then a small to to wild-type PA28γ and proteasome to of PA28γ(K188E) might the of proteins in We and for the of and for in the of the We also for peptide and and for with the mass spectrometry.