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Range of Sizes of Peptide Products Generated during Degradation of Different Proteins by Archaeal Proteasomes

1998; Elsevier BV; Volume: 273; Issue: 4 Linguagem: Inglês

10.1074/jbc.273.4.1982

1083-351X

Alexei F. Kisselev, Tatos Akopian, Alfred L. Goldberg,

Protein Degradation and Inhibitors

The 20 S proteasome processively degrades cell proteins to peptides. Information on the sizes and nature of these products is essential for understanding the proteasome's degradative mechanism, the subsequent steps in protein turnover, and major histocompatibility complex class I antigen presentation. Using proteasomes from Thermoplasma acidophilum and four unfolded polypeptides as substrates (insulin-like growth factor, lactalbumin, casein, and alkaline phosphatase, whose lengths range from 71 to 471 residues), we demonstrate that the number of cuts made in a polypeptide and the time needed to degrade it increase with length. The average size of peptides generated from these four polypeptides was 8 ± 1 residues, but ranged from 6 to 10 residues, depending on the protein, as determined by two new independent methods.However, the individual peptide products ranged in length from approximately 3 to 30 residues, as demonstrated by mass spectrometry and size-exclusion chromatography. The sizes of individual peptides fit a log-normal distribution. No length was predominant, and more than half were shorter than 10 residues. Peptide abundance decreased with increasing length, and less than 10% exceeded 20 residues. These findings indicate that: 1) the proteasome does not generate peptides according to the "molecular ruler" hypothesis, and 2) other peptidases must function after the proteasome to complete the turnover of cell proteins to amino acids. The 20 S proteasome processively degrades cell proteins to peptides. Information on the sizes and nature of these products is essential for understanding the proteasome's degradative mechanism, the subsequent steps in protein turnover, and major histocompatibility complex class I antigen presentation. Using proteasomes from Thermoplasma acidophilum and four unfolded polypeptides as substrates (insulin-like growth factor, lactalbumin, casein, and alkaline phosphatase, whose lengths range from 71 to 471 residues), we demonstrate that the number of cuts made in a polypeptide and the time needed to degrade it increase with length. The average size of peptides generated from these four polypeptides was 8 ± 1 residues, but ranged from 6 to 10 residues, depending on the protein, as determined by two new independent methods. However, the individual peptide products ranged in length from approximately 3 to 30 residues, as demonstrated by mass spectrometry and size-exclusion chromatography. The sizes of individual peptides fit a log-normal distribution. No length was predominant, and more than half were shorter than 10 residues. Peptide abundance decreased with increasing length, and less than 10% exceeded 20 residues. These findings indicate that: 1) the proteasome does not generate peptides according to the "molecular ruler" hypothesis, and 2) other peptidases must function after the proteasome to complete the turnover of cell proteins to amino acids. The 20 S proteasome is a 700-kDa proteolytic complex that is present in all eukaryotic cells, archaea, and certain bacteria (1Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2215) Google Scholar, 2Tamura T. Nagy I. Lupas A. Lottspeich F. Cejka Z. Schoofs G. Tanaka K. Demot R. Baumeister W. Curr. Biol. 1995; 5: 766-774Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In eukaryotes, the proteasome is an essential component of the ATP-ubiquitin-dependent pathway for protein degradation (3Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1580) Google Scholar,4Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1444) Google Scholar). The 20 S particle functions as the proteolytic core of the 26 S proteasome complex, which catalyzes the rapid degradation of many short lived regulatory proteins and of proteins with abnormal conformation (1Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2215) Google Scholar, 4Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1444) Google Scholar, 5Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2172) Google Scholar, 6Lee D.H. Goldberg A.L. J. Biol. Chem. 1996; 271: 27280-27284Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). In mammals, the proteasome is also responsible for the breakdown of most long lived cell proteins and for the generation of most peptides presented to the immune system on MHC 1The abbreviations used are: MHC, major histocompatibility complex; Bis-tris propane, 1,3-bis[tris(hydroxymethyl)methylamino] propane; HPLC, high performance liquid chromatography; IGF, insulin-like growth factor.1The abbreviations used are: MHC, major histocompatibility complex; Bis-tris propane, 1,3-bis[tris(hydroxymethyl)methylamino] propane; HPLC, high performance liquid chromatography; IGF, insulin-like growth factor. class I molecules (5Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2172) Google Scholar,7Craiu A. Gaczynska M. Akopian T. Gramm C.F. Fenteany G. Goldberg A.L. Rock K.L. J. Biol. Chem. 1997; 272: 13437-13445Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). The 20 S particle is a barrel-shaped structure composed of four stacked rings. Each outer ring contains seven related α-subunits, and each of the inner rings seven related β-subunits, which catalyze peptide bond cleavage. The active sites are located within the central chamber of the 20 S particle, into which protein substrates must enter through a narrow openings in the α- and β-rings. Proteasomes cleave peptide bonds by a novel catalytic mechanism, in which the hydroxyl group of the threonine on the N terminus of the β-subunit serves as the active site nucleophile (8Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1363) Google Scholar, 9Seemüller E. Lupas A. Stock D. Lowe J. Huber R. Baumeister W. Science. 1995; 268: 579-582Crossref PubMed Scopus (581) Google Scholar, 10Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 11Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1492) Google Scholar, 12Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H. Huber R. Nature. 1997; 386: 463-471Crossref PubMed Scopus (1923) Google Scholar). Recently, we have shown that the 20 S proteasome degrades proteins by a highly processive mechanism to oligopeptides (10Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). A typical protease makes a single cleavage in a polypeptide substrate and then releases the fragments, which may be cleaved to smaller products in subsequent enzymatic rounds. In contrast, the proteasome appears to make many cleavages in the polypeptide and to digest it to small products without dissociation of the partially degraded substrates. This novel mode of degradation appears highly important for an intracellular proteolytic system, since the release of large protein fragments could interfere with cell function and regulation. However, definitive proof of this mode of degradation requires knowledge of the number of cleavages made in a protein substrate and the sizes of peptides generated. If the proteasome makes repeated cuts processively along the length of the polypeptide, one would predict that the enzyme should make a greater number of cleavages and take more time to digest longer polypeptides than shorter substrates. One goal of the present study was to test these predictions. Knowledge about the size distribution of peptides produced by the proteasome is important for understanding the subsequent steps in the protein degradative pathway and the process of MHC class I antigen presentation. In vivo, most of the peptides generated by the proteasome must be rapidly hydrolyzed to amino acids, which are utilized in synthesis of new proteins or in intermediary metabolism. In mammalian cells, some of these peptides are utilized in antigen presentation, possibly after further proteolytic processing to the final 8–9-residue peptides presented on the cell surface (13Goldberg A.L. Gaczynska M. Grant E. Michalek M. Rock K.L. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 479-490Crossref PubMed Scopus (46) Google Scholar). These latter steps are poorly understood, in part because of a lack of precise information on the sizes of the peptides released by the proteasome during protein breakdown. It is widely believed that the proteasome degrades polypeptides according to a "molecular ruler" to yield products of rather uniform size (8Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1363) Google Scholar, 12Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H. Huber R. Nature. 1997; 386: 463-471Crossref PubMed Scopus (1923) Google Scholar, 14Stuart D.I. Jones E.Y. Nature. 1997; 386: 437-438Crossref PubMed Scopus (10) Google Scholar, 15Baumeister W. Lupas A. Curr. Opin. Struct. Biol. 1997; 7: 273-278Crossref PubMed Scopus (65) Google Scholar), as first proposed by Wenzel et al. (16Wenzel T. Eckerskorn C. Lottspeich F. Baumeister W. FEBS Lett. 1994; 349: 205-209Crossref PubMed Scopus (147) Google Scholar). This group reported that a large fraction of the peptides generated during the breakdown of hemoglobin or insulin β-chains by archaeal proteasomes were between 7 and 9 residues long (16Wenzel T. Eckerskorn C. Lottspeich F. Baumeister W. FEBS Lett. 1994; 349: 205-209Crossref PubMed Scopus (147) Google Scholar). Since the distance between adjacent active sites corresponded to an octapeptide in an extended conformation (8Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1363) Google Scholar, 16Wenzel T. Eckerskorn C. Lottspeich F. Baumeister W. FEBS Lett. 1994; 349: 205-209Crossref PubMed Scopus (147) Google Scholar, 17Stock D. Ditzel L. Baumeister W. Huber R. Lowe J. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 525-532Crossref PubMed Scopus (30) Google Scholar), it was proposed that peptides of 7–9 residues were routinely generated as a result of coordinated cleavages by neighboring active sites. However, evidence for such a molecular ruler is quite limited. In the study by Wenzelet al. (16Wenzel T. Eckerskorn C. Lottspeich F. Baumeister W. FEBS Lett. 1994; 349: 205-209Crossref PubMed Scopus (147) Google Scholar) or in other studies of peptides generated by the proteasome (18Dick L.R. Moomaw C.R. DeMartino G.N. Slaughter C.A. Biochemistry. 1991; 30: 2725-2734Crossref PubMed Scopus (85) Google Scholar, 19Dick L.R. Aldrich C. Jameson S.C. Moomaw C.R. Pramanik B.C. Doyle C.K. DeMartino G.N. Bevan M.J. Forman J.M. Slaughter C.A. J. Immunol. 1994; 152: 3884-3894PubMed Google Scholar, 20Ehring B. Meyer T. Eckerskorn C. Lottspeich F. Tampe R. Eur. J. Biochem. 1996; 235: 404-415Crossref PubMed Scopus (74) Google Scholar, 21Groettrup M. Ruppert T. Kuehn L. Seeger M. Standera S. Koszinowski U. Kloetzel P.M. J. Biol. Chem. 1995; 270: 23808-23815Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 22Niedermann G. King G. Butz S. Birsner U. Grimm R. Shabanowitz J. Hunt D.F. Eichmann K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8572-8577Crossref PubMed Scopus (91) Google Scholar, 23Dick T.P. Ruppert T. Groettrup M. Kloetzel P.M. Kuehn L. Koszinowski U.H. Stevanovic S. Schild H. Rammensee H.G. Cell. 1996; 86: 253-262Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), the relative amounts of peptides of different sizes were not quantified. In addition, Wenzel et al. (16Wenzel T. Eckerskorn C. Lottspeich F. Baumeister W. FEBS Lett. 1994; 349: 205-209Crossref PubMed Scopus (147) Google Scholar) analyzed peptide products after prolonged incubations, during which the products were likely to undergo repetitive cleavage by the proteasome (20Ehring B. Meyer T. Eckerskorn C. Lottspeich F. Tampe R. Eur. J. Biochem. 1996; 235: 404-415Crossref PubMed Scopus (74) Google Scholar, 22Niedermann G. King G. Butz S. Birsner U. Grimm R. Shabanowitz J. Hunt D.F. Eichmann K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8572-8577Crossref PubMed Scopus (91) Google Scholar, 23Dick T.P. Ruppert T. Groettrup M. Kloetzel P.M. Kuehn L. Koszinowski U.H. Stevanovic S. Schild H. Rammensee H.G. Cell. 1996; 86: 253-262Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). The present studies were undertaken to determine the mean size of peptides generated from full-length proteins, to measure the relative amounts of peptides of different sizes, and thus to critically test the molecular ruler model. Most prior studies have focused on peptides ranging from 4 to 44 residues in length, which may be degraded differently from proteins. In contrast, in this study, we have investigated the digestion of proteins of different sizes ranging in length from 70 to 471 residues. We have introduced several new approaches to evaluate the mean number of cuts made in each protein substrate, the mean sizes of the peptides generated by proteasome, and the relative amounts of products of different lengths. Since only unfolded molecules can enter the 20 S particle and be degraded (24Wenzel T. Baumeister W. Nat. Struct. Biol. 1995; 2: 199-204Crossref PubMed Scopus (187) Google Scholar), the present studies utilized denatured polypeptides. Conditions were chosen where the protein substrate was present in large excess, such that the released peptides were not digested further. We have employed 20 S proteasomes from the archaeaThermoplasma acidophilum, because this particle is structurally simpler than the eukaryotic 20 S proteasome. It contains seven identical α- and β-subunits, and thus seven identical active sites, located at equal distances from each other (8Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1363) Google Scholar), unlike the eukaryotic particle which contains seven different α-subunits and seven different β-subunits, and has three active sites with different specificities (12Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H. Huber R. Nature. 1997; 386: 463-471Crossref PubMed Scopus (1923) Google Scholar). Therefore, the factors determining product size and rate of proteolysis should be easier to elucidate and the data easier to interpret with archaeal particle. The Thermoplasma proteasome was expressed in Escherichia coli and purified as described previously (10Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Recombinant human IGF was a kind gift of Dr. W. Prouty (Eli Lilly, Indianapolis, IN), bovine α-lactalbumin and β-casein, alkaline phosphatase from E. coli, Leu-enkephalin amide, substance P, and oxidized A- and B-chains of bovine insulin were purchased from Sigma. Synthetic peptides SIINFEKL, YPHPARIGL, TYQRTRALV, and YSDEDMQTM were kindly provided by Dr. K. Rock (University of Massachusetts Medical Center, Worcester, MA). All other peptides were from Bachem AG (Switzerland). For use as substrates, IGF and lactalbumin were unfolded by reduction of disulfide bonds and carboxymethylation as described previously (10Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The IGF, lactalbumin, and casein were exhaustively, reductively methylated (10Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) to prevent the extent of the reaction of the undegraded molecule with fluorescamine, and the modified polypeptide purified by HPLC prior to use. To denature alkaline phosphatase, it was oxidized with performic acid (25Hirs C.H.W. Methods Enzymol. 1967; 11: 197-198Crossref Scopus (878) Google Scholar). The concentrations of modified IGF, lactalbumin, and casein were determined by UV absorption at 280 nm. Calculations of the extinction coefficient for each protein were based on the contents of Tyr and Trp residues in each molecule (26Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5010) Google Scholar). The concentration of alkaline phosphatase was measured by the Lowry method, because Tyr and Trp are destroyed or modified during performic acid treatment. The protein substrates were incubated with proteasomes at 54 °C in 50 mm Bis-tris propane buffer, pH 7.5. Substrates incubated without proteasomes and proteasomes incubated without substrate served as controls. At different times, aliquots were taken from the reaction mixtures and mixed with an equal volume of 0.4% trifluoroacetic acid to stop the reaction. The concentration of the newly formed amino groups was measured by reaction with fluorescamine at pH 6.8 as described previously (10Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Another aliquot was run on the C18 HPLC column (Vydac peptide and protein, 0.46 × 25 cm, 10 μm) to separate products from the undegraded protein substrate. The column was equilibrated with 0.06% trifluoroacetic acid and eluted with a gradient of buffer B (80% acetonitrile, 0.05% trifluoroacetic acid) at a flow rate of 1.5 ml/min. A stepwise increase in buffer B concentration to 40–50% (depending on the substrate) was used in the region where peptide products were eluted, followed by isocratic elution for 2 min, and a gradient of 10% buffer B/min for 2 min was used in the region where the undegraded protein was eluted. Such a gradient allowed us to decrease the time required for analysis and the volume of pooled peptides. In some experiments (Table II and Fig. 6), the pooled peptides were collected for further analysis. (Precipitation with trichloracetic acid could not be used here because a significant fraction of peptides was found to precipitate together with the undegraded substrate.) The amount of undegraded protein was measured by integration of its HPLC UV absorbance peak at three different wavelengths (214, 230, and 280 nm).Table IIMean size of peptides generated by the proteasomeSubstrateMean sizeTwo rates methodAcid hydrolysis methodSize-exclusion chromatographyAlkaline phosphatase6.5 ± 0.59.3 ± 1.010.2Casein10.6 ± 2.19.0 ± 1.311.2Lactalbumin8.1 ± 0.57.7 ± 1.49.1IGF5.9 ± 0.86.7 ± 0.37.0 Average7.8 ± 1.88.2 ± 1.09.4 Open table in a new tab To obtain kinetic constants, the concentrations of amino groups and the area of the substrate peaks were plotted against the incubation time. The rates of substrate disappearance and of product accumulation were then determined from the slopes of these plots, which were linear under conditions used here. Less than half of the initial amount of the substrate was degraded at the end of incubation. To ensure this linearity, the initial substrate concentrations were at least 2-fold greater than the concentrations at which V maxwas reached (500 μm for IGF, 90 μm for lactalbumin, 25 μm for casein, and 12 μmfor alkaline phosphatase). The number of cuts in a polypeptide was calculated by dividing the rate of product accumulation by the rate of substrate consumption. The mean length of the products (in residues) was obtained by dividing the length of the protein by the number of cuts plus one. The substrates were incubated with proteasomes until 30–50% was degraded. The products were separated from the undegraded substrate by HPLC on the C18column, pooled, lyophilized, and redissolved in water. Free amino groups in the pool (i.e. the amount of peptides) were measured by the reaction with fluorescamine in 0.2 mphosphate buffer (pH 6.8) (10Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). These peptides were then hydrolyzed completely to amino acids with 6 m HCl in sealed ampoules for 24 h at 108 °C, and the amount of amino acids after hydrolysis was measured by the fluorescamine assay in 0.2 mborate buffer (pH 8.6). A standard mixture of amino acids, treated in the same fashion as the samples, was used for calibration of the amino acid assay. Then, the mean size was determined by dividing the molar amount of amino acids found after acid treatment by the molar amount of peptides before the treatment. Size-exclusion chromatography was performed on polyhydroxyethyl aspartamide column (0.46 × 20 cm, Poly LC, Columbia, MD), using a HP1090 chromatographer (Hewlett-Packard). The mobile phase was 0.2 m sodium sulfate, 25% acetonitrile, pH 3.0 (adjusted with phosphoric acid), and the flow rate was 0.125 ml/min. To determine the apparent molecular mass of the peptides eluted, the column was calibrated each time before use with 8–10 standard peptides in the 550–3500-Da range. The pool of proteasome's products (the same as in acid hydrolysis method) containing 5–10 nmol of peptides was dissolved in 50 μl of the mobile phase and loaded onto the column. Fractions (0.5 min) were collected, and the molar amount of peptides in each fraction was measured with the fluorescamine assay as described above. The corresponding fraction of the control mixture, in which the substrate was incubated without proteasome, was also run on the size-exclusion column. No fluorescamine-reactive material was found in the fractions of this run. To ensure rapid hydrolysis and to eliminate possible complications due to substrate folding, three substrates (alkaline phosphatase, IGF, and lactalbumin) were denatured prior to study, as described above. The other substrate studied, casein, had little or no tertiary structure and did not require denaturation to be degraded rapidly by the proteasome. To determine the number of cuts made by the proteasome in these different polypeptides, we measured the rate of disappearance of substrate molecules and the rate of appearance of the new amino groups during the same incubation. Denatured IGF, lactalbumin, casein, and alkaline phosphatase were incubated at 53 °C and pH 7.5 with highly purified recombinant Thermoplasma proteasomes. Initial substrate concentrations were high enough to ensure a constant rate of degradation during the entire incubation period. At different times, aliquots were removed, and one portion was analyzed by HPLC to determine the amount of substrate consumed (by measuring the amount of undegraded substrate by integration of its peak area), another portion was used to determine the amount of peptides produced by assaying them as the number of new primary amino groups generated that react with fluorescamine. As found previously (10Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), the rates of accumulation of peptide products and of the disappearance of the substrates paralleled each other. The ratio of the amount of new products generated to the amount of protein molecules degraded did not change with time, indicating a processive mechanism. Moreover, the products once released by the proteasome did not get cleaved again at later times under these incubation conditions, where there was a large molar excess of the substrate. If the proteasome makes n cuts in a protein molecule and the products generated do not undergo further cleavages, there should always be n-fold more new amino groups than substrate molecules consumed. Therefore, the number of cuts made per protein can be determined by dividing the rate of peptide product accumulation by the rate of substrate disappearance (Fig.1). This value increased with the length of the substrate, ranging from 11 cuts in IGF, which is 70-residues long, to 71 cuts in alkaline phosphatase, which contains 471 residues (Table I). The demonstration that a large number of cuts are made in a single substrate molecule, together with the previous finding that the proteasome does not release the substrate until all these cuts are made (10Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), is clear evidence of a highly processive mechanism for protein degradation (Fig.2).Table IKinetic parameters of protein degradation by the proteasomeSubstrateNo. of residuesProtein molecules degraded/minPeptide bonds cut/minCuts/ moleculeAlkaline phosphatase4710.221671Casein2091.11919Lactalbumin1233.24514IGF705.66111 Open table in a new tab Figure 2The number of cuts made by the 20 S proteasome in a protein depends on its length. The number of cuts was determined by the two rates method (open circles) and by the acid hydrolysis method (closed rectangles) (Fig. 1). The proteins are: 1, IGF; 2, lactalbumin;3, casein; and 4, alkaline phosphatase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This highly processive mechanism suggests that the proteasome should require more time to degrade a longer polypeptide than a short one, provided that it moves along the substrate at a relatively constant rate. These measurements of the rate of disappearance of the different substrates (Table I) allowed us also to determine the time needed to degrade a substrate molecule, because these experiments were performed atV max. The number of protein molecules degraded per min by a 20 S particle was calculated by dividing the rate of substrate disappearance by the molar concentration of the proteasome (Table I). The reciprocal therefore represents the time that the proteasome takes to degrade one substrate molecule, assuming it degrades only one substrate molecule at a time. The time required to degrade each of these polypeptides was a characteristic feature of the substrate. As shown in Fig.3, the time for degradation by the proteasome depended on the polypeptide's length. At 53 °C, the enzyme required 10 s to digest one IGF molecule (70 residues, 11 cuts) and 50 s to degrade one casein molecule (209 residues, about 20 cuts). When casein was modified by fluorescein isothiocyanate, the proteasome still took approximately 1 min to degrade it. Thus, with smaller polypeptides, there was almost a linear relationship between substrate length and duration of the degradative process. In contrast, almost 5 min were required to digest alkaline phosphatase (471 residues, 71 cuts). The disproportionately long time required for degradation of alkaline phosphatase suggests that there are additional rate-limiting steps in the digestive process aside from peptide bond cleavage, such as the unfolding of residual secondary structure or disassembly of substrate aggregates, which may slow substrate entry into the central chamber. However, this preparation of alkaline phosphatase appeared to contain another conformational form, which had a lower affinity for the proteasome but was degraded severalfold faster. It was impossible to measure the time required for degradation of this form because it was insoluble at high concentrations. Therefore, all measurements were done on the slowly degraded form of alkaline phosphatase, which remained soluble atV max. To study additional unfolded proteins as substrates with lengths between casein and alkaline phosphatase we tried to study rhodanese, glyceraldehyde-3-phosphate dehydrogenase and β-subunit of tryptophan synthase. However, at 53 °C (whereThermoplasma enzymes are quite active), these polypeptides were insoluble. Another polypeptide tested (ovalbumin) remained soluble, but was a poor substrate in vitro even after denaturation. The finding that the number of cuts made in a polypeptide is proportional to its length implies that the length of the peptides generated by the proteasome is similar for the different proteins. We have developed two simple methods to determine the mean size of these peptide products. In the first, which we term the two rates method, the mean size of the products was calculated by dividing the number of amino acids in the protein by the number of bonds cut plus one (Fig. 1). For example, if a protein containing 100 amino acid residues is cut at 9 sites to yield 10 pieces, their average length is 10 residues. The mean size of products found by this approach was similar with the four different substrates. The values obtained ranged from 6 for IGF to 11 for casein with an average length of 8 for the four proteins (TableII). These differences, although small, in the mean sizes of peptides generated from different proteins were found reproducibly (Table II). The standard errors on these values in Table II represent the range of the mean sizes obtained in independent experiments, rather than the variation in length about the mean (see below). To obtain an independent measure of the mean sizes of these peptide products, another approach was developed, which we call the acid hydrolysis method. The peptides generated by the proteasomes were separated from the undigested substrate by HPLC on a reverse-phase column. The amount of peptides produced was measured with fluorescamine, they were then hydrolyzed completely to amino acids by acid treatment, and the amount of amino acids was measured with fluorescamine. If an individual peptide c