Advertisement

Binding of Hydrophobic Peptides to Several Non-catalytic Sites Promotes Peptide Hydrolysis by All Active Sites of 20 S Proteasomes

EVIDENCE FOR PEPTIDE-INDUCED CHANNEL OPENING IN THE α-RINGS*
  • Alexei F. Kisselev
    Footnotes
    Affiliations
    From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Daniel Kaganovich
    Affiliations
    From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Alfred L. Goldberg
    Correspondence
    To whom correspondence should be addressed: Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1855; Fax: 617-232-0173;
    Affiliations
    From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Author Footnotes
    * This work was supported by NIGMS grants from the National Institutes of Health (to A. L. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Fellow of the Medical Foundation and a Special Fellow of the Leukemia and Lymphoma Society during this work.
Open AccessPublished:April 01, 2002DOI:https://doi.org/10.1074/jbc.M112360200
      The eukaryotic 20 S proteasome contains the following 6 active sites: 2 chymotrypsin-like, 2 trypsin-like, and 2 caspase-like. We previously showed that hydrophobic peptide substrates of the chymotrypsin-like sites allosterically stimulate peptide hydrolysis by the caspase-like sites and their own cleavage. More thorough analysis revealed that these peptides also stimulate peptide hydrolysis by the trypsin-like site. This general activation by hydrophobic peptides occurred even if the chymotrypsin-like sites were occupied by a covalent inhibitor and was highly cooperative, with an average Hill coefficient of 7. Therefore, this stimulation of peptide hydrolysis at all active sites occurs upon binding of hydrophobic peptides to several non-catalytic sites. The stimulation by hydrophobic peptides was not observed in the yeast ΔNα3 mutant 20 S proteasomes, in 20 S-PA26 complexes, or SDS-activated proteasomes and was significantly lower in 26 S proteasomes, all of which appear to have the gated channel in the α-rings in an open conformation and hydrolyze peptides at much faster rates than 20 S proteasomes. Also the hydrophobic peptides alteredK m , V max of active sites in a similar fashion as PA26 and the ΔNα3 mutation. The activation by hydrophobic peptides was decreased in K+-containing buffer, which favors the closed state of the channels. Therefore, hydrophobic peptides stimulate peptide hydrolysis most likely by promoting the opening of the channels in the α-rings. During protein breakdown, this peptide-induced channel opening may function to facilitate the release of products from the proteasome.
      Ac
      acetyl
      amc
      7-amido-4-methylcoumarin
      Boc
      tert-butyloxycarbonyl
      DTT
      dithiothreitol, IEF, isoelectrofocusing
      mna
      4-methoxy-2-naphthylamide
      na
      2-naphthylamide
      nL
      norleucyl
      NLVS
      4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Leu-vinyl sulfone
      pna
      4-nitroanilide
      Suc
      succinyl
      VS
      vinylsulfone
      Z
      carbobenzoxy
      Bistris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
      wt
      wild type
      The majority of proteins in mammalian cells is degraded by 26 S proteasomes (
      • Rock K.L.
      • Gramm C.
      • Rothstein L.
      • Clark K.
      • Stein R.
      • Dick L.
      • Hwang D.
      • Goldberg A.L.
      ). This 2.4-MDa proteolytic enzyme consists of the 20 S proteasome and one or two 19 S regulatory complexes (
      • Voges D.
      • Zwickl P.
      • Baumeister W.
      ,
      • Baumeister W.
      • Walz J.
      • Zühl F.
      • Seemüller E.
      ). The 20 S proteasome, which also exists in mammalian cells as a free 700-kDa particle, is a hollow cylinder composed of two outer α- and two inner β-rings (
      • Baumeister W.
      • Walz J.
      • Zühl F.
      • Seemüller E.
      ). Each ring contains seven different subunits, and each β-ring contains three proteolytic sites, which differ in their substrate specificities. The “chymotrypsin-like” (β5) site cleaves peptide bonds preferentially after hydrophobic residues; the “trypsin-like” (β2) site cuts mainly after basic residues, and the third site (β1) cuts preferentially after acidic residues (
      • Chen P.
      • Hochstrasser M.
      ,
      • Arendt C.S.
      • Hochstrasser M.
      ,
      • Dick T.P.
      • Nussbaum A.K.
      • Deeg M.
      • Heinemeyer W.
      • Groll M.
      • Schirle M.
      • Keilholz W.
      • Stevanovic S.
      • Wolf D.H.
      • Huber R.
      • Rammensee H.G.
      • Schild H.
      ,
      • Heinemeyer W.
      • Fischer M.
      • Krimmer T.
      • Stachon U.
      • Wolf D.H.
      ). This latter site has been traditionally termed “post-glutamyl peptide hydrolase” site. However, because it hydrolyzes standard fluorogenic substrates of caspases and cleaves after aspartate residues better than after glutamates, we prefer the more accurate and simpler term “caspase-like” site (
      • Kisselev A.F.
      • Akopian T.N.
      • Castillo V.
      • Goldberg A.L.
      ).
      When isolated under gentle conditions (e.g. in the presence of glycerol), 20 S proteasomes are in a latent state (
      • Coux O.
      • Tanaka K.
      • Goldberg A.L.
      ) in which they are unable to degrade proteins and hydrolyze model peptide substrates only at low rates. This low peptidase activity is suppressed further by physiological concentrations of potassium ions (
      • Köhler A.
      • Cascio P.
      • Leggett D.S.
      • Woo K.M.
      • Goldberg A.L.
      • Finley D.
      ), but the activity of such preparations increases dramatically upon a variety of treatments, such as heating, removal of glycerol, or addition of low concentrations of SDS (
      • Coux O.
      • Tanaka K.
      • Goldberg A.L.
      ). The explanation for the low basal activity (latency) of the 20 S proteasome is that all of its proteolytic sites are located within this cylindrical particle (
      • Löwe J.
      • Stock D.
      • Jap B.
      • Zwickl P.
      • Baumeister W.
      • Huber R.
      ,
      • Groll M.
      • Ditzel L.
      • Löwe J.
      • Stock D.
      • Bochtler M.
      • Bartunik H.
      • Huber R.
      ), and access of substrates to these sites is restricted by two gated axial channels in the α-rings (
      • Löwe J.
      • Stock D.
      • Jap B.
      • Zwickl P.
      • Baumeister W.
      • Huber R.
      ). These channels allow entry or exit of small peptides, but even in their most open state they can be traversed only by unfolded polypeptides (
      • Wenzel T.
      • Baumeister W.
      ). In the crystal structure of the yeast 20 S particle these channels were found to be completely sealed by the N-terminal portions of 7 α-subunits (
      • Groll M.
      • Ditzel L.
      • Löwe J.
      • Stock D.
      • Bochtler M.
      • Bartunik H.
      • Huber R.
      ). However, when the nine N-terminal residues of the α3 subunit were deleted, an open channel was found (
      • Groll M.
      • Bajorek M.
      • Kohler A.
      • Moroder L.
      • Rubin D.M.
      • Huber R.
      • Glickman M.H.
      • Finley D.
      ). This ΔNα3 mutant also showed greatly increased rates of peptide hydrolysis, which were not further enhanced by SDS (
      • Groll M.
      • Bajorek M.
      • Kohler A.
      • Moroder L.
      • Rubin D.M.
      • Huber R.
      • Glickman M.H.
      • Finley D.
      ) nor suppressed by potassium (
      • Köhler A.
      • Cascio P.
      • Leggett D.S.
      • Woo K.M.
      • Goldberg A.L.
      • Finley D.
      ). Thus, rates of peptide hydrolysis by 20 S proteasomes depend on whether or not these openings are in a closed or open position.
      The association of 20 S proteasomes with the 19 S regulatory complexes to form 26 S proteasomes leads to much higher rates of peptide hydrolysis (
      • Chu-Ping M., Vu, J.H.
      • Proske R.J.
      • Slaughter C.A.
      • DeMartino G.N.
      ) and confers the ability to degrade ubiquitinated proteins as well as certain non-ubiquitinated polypeptides (
      • Kisselev A.F.
      • Akopian T.N.
      • Woo K.M.
      • Goldberg A.L.
      ,
      • Verma R.
      • Deshaies R.J.
      ,
      • Tarcsa E.
      • Szymanska G.
      • Lecker S.
      • O'Connor C.M.
      • Goldberg A.L.
      ). Recent studies indicated that, in the yeast 26 S proteasome, the channel in the α-rings is primarily in an open conformation as the result of an interaction between the N terminus of the α3 subunit and the Rpt2 ATPase subunit of the adjacent 19 S particle (
      • Köhler A.
      • Cascio P.
      • Leggett D.S.
      • Woo K.M.
      • Goldberg A.L.
      • Finley D.
      ). In addition to promoting gate opening, the ATPases of the 19 S ring appear to unfold protein substrates and translocate the unfolded polypeptide into the 20 S particle (
      • Benaroudj N.
      • Goldberg A.L.
      ). A different protein complex, PA28 (also termed 11 S or REG), can attach to the α-rings and open the channel by an ATP-independent mechanism, as demonstrated by the x-ray diffraction of the complex of yeast 20 S proteasome with PA26, the PA28 homologue from Trypanosoma brucei (
      • Whitby F.G.
      • Masters E.I.
      • Kramer L.
      • Knowlton J.R.
      • Yao Y.
      • Wang C.C.
      • Hill C.P.
      ). It is noteworthy that this hexameric ring-shaped activator stimulates peptide hydrolysis but not protein breakdown by 20 S proteasomes (
      • Chu-Ping M.
      • Slaughter C.A.
      • DeMartino G.N.
      ,
      • Dubiel W.
      • Pratt G.
      • Ferrell K.
      • Rechsteiner M.
      ).
      We have reported recently (
      • Kisselev A.F.
      • Akopian T.N.
      • Castillo V.
      • Goldberg A.L.
      ) that hydrolysis of peptides by the caspase-like site of 20 S proteasomes is also stimulated by the peptide substrates of the chymotrypsin-like sites. Conversely, peptide substrates of the caspase-like sites allosterically inhibit the chymotrypsin-like activity and thereby reduce protein breakdown by the 26 S particle. These findings suggested that different proteolytic sites of proteasomes may function in an ordered, cyclical fashion in protein degradation (
      • Kisselev A.F.
      • Akopian T.N.
      • Castillo V.
      • Goldberg A.L.
      ). Because the concentration dependence of the stimulation of the caspase-like activity by hydrophobic peptides was similar to the concentration dependence of their cleavage at the chymotrypsin-like sites, we concluded that this activation of caspase-like activity is due to the binding of peptides to the chymotrypsin-like sites (
      • Kisselev A.F.
      • Akopian T.N.
      • Castillo V.
      • Goldberg A.L.
      ). However, Schmidtke et al. (
      • Schmidtke G.
      • Emch S.
      • Groettrup M.
      • Holzhutter H.G.
      ) demonstrated a similar activation even in the presence of inhibitors of the chymotrypsin-like sites and concluded that activation of the caspase-like site occurs upon peptide binding to a single unidentified “modifier” site.
      The present study was undertaken to clarify the mechanisms of allosteric stimulation of the caspase-like activity by hydrophobic peptides and to determine whether these peptides act by binding to the active site or to distinct non-catalytic sites. We report here that multiple non-catalytic sites exist in the 20 S proteasome and that the binding of hydrophobic peptides to these sites stimulates peptide hydrolysis by all three of its active sites. Furthermore, we provide evidence that this stimulation occurs by peptide-induced opening of the channel in the α-rings of the 20 S proteasome. Specifically, we show that treatments that cause channel opening eliminate the stimulatory effects by hydrophobic peptides and cause similar changes in the kinetic properties of the proteasome as do the hydrophobic peptides.

      Acknowledgments

      We thank M. Pennington, M. Bogyo, and B. Kessler for providing reagents and D. Finley for providing yeast strains. We are especially grateful to B. Kessler for help with preparation of [125I]NLVS, to A. Duff and C. Hill for providing PA26, and to M. Glickman (Technion, Haifa, Israel) for providing wild type and ΔNα3 mutant yeast proteasomes for initial experiments. We are grateful to P. Variath, T. Jagoe, and D. Finley for the critical comments on the manuscript.

      REFERENCES

        • Rock K.L.
        • Gramm C.
        • Rothstein L.
        • Clark K.
        • Stein R.
        • Dick L.
        • Hwang D.
        • Goldberg A.L.
        Cell. 1994; 78: 761-771
        • Voges D.
        • Zwickl P.
        • Baumeister W.
        Annu. Rev. Biochem. 1999; 68: 1015-1068
        • Baumeister W.
        • Walz J.
        • Zühl F.
        • Seemüller E.
        Cell. 1998; 92: 367-380
        • Chen P.
        • Hochstrasser M.
        Cell. 1996; 86: 961-972
        • Arendt C.S.
        • Hochstrasser M.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7156-7161
        • Dick T.P.
        • Nussbaum A.K.
        • Deeg M.
        • Heinemeyer W.
        • Groll M.
        • Schirle M.
        • Keilholz W.
        • Stevanovic S.
        • Wolf D.H.
        • Huber R.
        • Rammensee H.G.
        • Schild H.
        J. Biol. Chem. 1998; 273: 25637-25646
        • Heinemeyer W.
        • Fischer M.
        • Krimmer T.
        • Stachon U.
        • Wolf D.H.
        J. Biol. Chem. 1997; 272: 25200-25209
        • Kisselev A.F.
        • Akopian T.N.
        • Castillo V.
        • Goldberg A.L.
        Mol. Cell. 1999; 4: 395-402
        • Coux O.
        • Tanaka K.
        • Goldberg A.L.
        Annu. Rev. Biochem. 1996; 65: 801-847
        • Köhler A.
        • Cascio P.
        • Leggett D.S.
        • Woo K.M.
        • Goldberg A.L.
        • Finley D.
        Mol. Cell. 2001; 7: 1143-1152
        • Löwe J.
        • Stock D.
        • Jap B.
        • Zwickl P.
        • Baumeister W.
        • Huber R.
        Science. 1995; 268: 533-539
        • Groll M.
        • Ditzel L.
        • Löwe J.
        • Stock D.
        • Bochtler M.
        • Bartunik H.
        • Huber R.
        Nature. 1997; 386: 463-471
        • Wenzel T.
        • Baumeister W.
        Nat. Struct. Biol. 1995; 2: 199-204
        • Groll M.
        • Bajorek M.
        • Kohler A.
        • Moroder L.
        • Rubin D.M.
        • Huber R.
        • Glickman M.H.
        • Finley D.
        Nat. Struct. Biol. 2000; 7: 1062-1067
        • Chu-Ping M., Vu, J.H.
        • Proske R.J.
        • Slaughter C.A.
        • DeMartino G.N.
        J. Biol. Chem. 1994; 269: 3539-3547
        • Kisselev A.F.
        • Akopian T.N.
        • Woo K.M.
        • Goldberg A.L.
        J. Biol. Chem. 1999; 274: 3363-3371
        • Verma R.
        • Deshaies R.J.
        Cell. 2000; 101: 341-344
        • Tarcsa E.
        • Szymanska G.
        • Lecker S.
        • O'Connor C.M.
        • Goldberg A.L.
        J. Biol. Chem. 2000; 275: 20295-20301
        • Benaroudj N.
        • Goldberg A.L.
        Nat. Cell Biol. 2000; 2: 833-839
        • Whitby F.G.
        • Masters E.I.
        • Kramer L.
        • Knowlton J.R.
        • Yao Y.
        • Wang C.C.
        • Hill C.P.
        Nature. 2000; 408: 115-120
        • Chu-Ping M.
        • Slaughter C.A.
        • DeMartino G.N.
        J. Biol. Chem. 1992; 267: 10515-10523
        • Dubiel W.
        • Pratt G.
        • Ferrell K.
        • Rechsteiner M.
        J. Biol. Chem. 1992; 267: 22369-22377
        • Schmidtke G.
        • Emch S.
        • Groettrup M.
        • Holzhutter H.G.
        J. Biol. Chem. 2000; 275: 22056-22063
        • Bogyo M.
        • McMaster J.S.
        • Gaczynska M.
        • Tortorella D.
        • Goldberg A.L.
        • Ploegh H.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6629-6634
        • Kisselev A.F.
        • Goldberg A.L.
        Chem. Biol. 2001; 8: 739-758
        • Kessler B.M.
        • Tortorella D.
        • Altun M.
        • Kisselev A.F.
        • Fiebiger E.
        • Hekking B.G.
        • Ploegh H.L.
        • Overkleeft H.S.
        Chem. Biol. 2001; 8: 913-929
        • Bogyo M.
        • Shin S.
        • McMaster J.S.
        • Ploegh H.L.
        Chem. Biol. 1998; 5: 307-320
        • Wilk S.
        • Orlowski M.
        J. Neurochem. 1983; 40: 842-849
        • Osmulski P.A.
        • Gaczynska M.
        J. Biol. Chem. 2000; 275: 13171-13174
        • Wilk S.
        • Chen W.E.
        • Magnusson R.P.
        Arch. Biochem. Biophys. 1999; 362: 283-290
        • Li J.
        • Gao X.L.
        • Ortega J.Q.
        • Nazif T.
        • Joss L.
        • Bogyo M.
        • Steven A.C.
        • Rechsteiner M.
        EMBO J. 2001; 20: 3359-3369
        • Harris J.L.
        • Alper P.B., Li, J.
        • Rechstainer M.
        • Bakes B.J.
        Chem. Biol. 2001; 8: 1131-1141
        • Kopp F.
        • Dahlmann B.
        • Kuehn L.
        J. Mol. Biol. 2001; 313: 465-471
      1. Cascio, P., Call, M., Petre, B. M., Walz, T., and Goldberg, A. L. (2002) EMBO J., in press

        • Saric T.
        • Beninga J.
        • Graef C.I.
        • Akopian T.N.
        • Rock K.L.
        • Goldberg A.L.
        J. Biol. Chem. 2001; 276: 36474-36481
        • Myung J.
        • Kim K.B.
        • Lindsten K.
        • Dantuma N.P.
        • Crews C.M.
        Mol. Cell. 2001; 7: 411-420
        • Phillips T.A.
        • Vaughn V.
        • Bloch P.L.
        • Neidhardt F.
        Ingraham J.L. Low K.B. Magasanik B. Schaechter M. Unbarger E.H. Escherichia coli and Salmonella typhimurium. American Society for Microbiology, Washington, DC1987: 919-966