The Logic of the 26S Proteasome
2017; Cell Press; Volume: 169; Issue: 5 Linguagem: Inglês
10.1016/j.cell.2017.04.023
ISSN1097-4172
AutoresG. Collins, Alfred L. Goldberg,
Tópico(s)RNA modifications and cancer
ResumoThe ubiquitin proteasome pathway is responsible for most of the protein degradation in mammalian cells. Rates of degradation by this pathway have generally been assumed to be determined by rates of ubiquitylation. However, recent studies indicate that proteasome function is also tightly regulated and determines whether a ubiquitylated protein is destroyed or deubiquitylated and survives longer. This article reviews recent advances in our understanding of the proteasome's multistep ATP-dependent mechanism, its biochemical and structural features that ensure efficient proteolysis and ubiquitin recycling while preventing nonselective proteolysis, and the regulation of proteasome activity by interacting proteins and subunit modifications, especially phosphorylation. The ubiquitin proteasome pathway is responsible for most of the protein degradation in mammalian cells. Rates of degradation by this pathway have generally been assumed to be determined by rates of ubiquitylation. However, recent studies indicate that proteasome function is also tightly regulated and determines whether a ubiquitylated protein is destroyed or deubiquitylated and survives longer. This article reviews recent advances in our understanding of the proteasome's multistep ATP-dependent mechanism, its biochemical and structural features that ensure efficient proteolysis and ubiquitin recycling while preventing nonselective proteolysis, and the regulation of proteasome activity by interacting proteins and subunit modifications, especially phosphorylation. The 26S proteasome catalyzes the great majority (at least 80%) of the protein degradation in growing mammalian cells, including both the rapid degradation of misfolded and regulatory proteins and most of the slower breakdown of the bulk of cellular proteins (Zhao et al., 2015Zhao J. Zhai B. Gygi S.P. Goldberg A.L. mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy.Proc. Natl. Acad. Sci. USA. 2015; 112: 15790-15797Crossref PubMed Scopus (34) Google Scholar). Consequently, proteasome function is essential for protein homeostasis and influences the regulation of most cellular processes, and inhibitors of the proteasome have proven to be very valuable research tools and therapeutic agents that have prolonged the lives of thousands of patients with multiple myeloma (Goldberg, 2012Goldberg A.L. Development of proteasome inhibitors as research tools and cancer drugs.J. Cell Biol. 2012; 199: 583-588Crossref PubMed Scopus (88) Google Scholar). Since the discoveries of the critical role of ubiquitin (Ub) in protein turnover (Hershko et al., 1980Hershko A. Ciechanover A. Heller H. Haas A.L. Rose I.A. Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.Proc. Natl. Acad. Sci. USA. 1980; 77: 1783-1786Crossref PubMed Google Scholar) and of the 26S complex in digesting ubiquitin conjugates (Hough et al., 1987Hough R. Pratt G. Rechsteiner M. Purification of two high molecular weight proteases from rabbit reticulocyte lysate.J. Biol. Chem. 1987; 262: 8303-8313Abstract Full Text PDF PubMed Google Scholar, Waxman et al., 1987Waxman L. Fagan J.M. Goldberg A.L. Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes, one of which degrades ubiquitin conjugates.J. Biol. Chem. 1987; 262: 2451-2457Abstract Full Text PDF PubMed Google Scholar), it has been generally assumed that rates of proteolysis by this pathway are regulated solely through protein ubiquitylation. However, it is now clear that ubiquitylation and even the association of a ubiquitylated protein with the proteasome do not necessarily lead to its degradation (Finley, 2009Finley D. Recognition and processing of ubiquitin-protein conjugates by the proteasome.Annu. Rev. Biochem. 2009; 78: 477-513Crossref PubMed Scopus (837) Google Scholar, Inobe and Matouschek, 2014Inobe T. Matouschek A. Paradigms of protein degradation by the proteasome.Curr. Opin. Struct. Biol. 2014; 24: 156-164Crossref PubMed Scopus (43) Google Scholar). Thus, the proteasome is not simply a machine for efficient, automatic destruction of ubiquitin conjugates and ubiquitin recycling, but its properties also determine whether a ubiquitylated protein undergoes degradation or survives intact. In addition, the proteasome’s degradative capacity and selectivity are not fixed, but are precisely regulated by multiple post-synthetic mechanisms. In the proteasome (Figure 1), polypeptides are digested to short peptides, 90% of which range between two and ten residues in length (Kisselev et al., 1999Kisselev A.F. Akopian T.N. Woo K.M. Goldberg A.L. The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation.J. Biol. Chem. 1999; 274: 3363-3371Crossref PubMed Scopus (0) Google Scholar). Nearly all are digested in seconds to amino acids by cytosolic peptidases, but in mammals, some serve as precursors for antigenic peptides displayed on MHC-class I molecules. Because proteolysis is irreversible, the consequences for cells can be severe if proteasomes destroy proteins non-selectively or function non-processively and release partially degraded polypeptides. Therefore, proteasomes have evolved intricate mechanisms to avoid such failures and to ensure efficient, selective degradation. Genetic and biochemical studies have greatly advanced our understanding of the multiple steps in proteasomal degradation: the binding of ubiquitylated proteins, their deubiquitylation, and their ATP-driven unfolding and translocation into the 20S chamber for proteolysis (Figure 2). Although the roles of many of its 60-odd subunits and associated proteins are still unclear, dramatic progress has been made recently through cryo-EM in capturing the dynamism of the 26S complex. The goal of this article is not to summarize these various advances, as has been done in many valuable reviews (Finley et al., 2016Finley D. Chen X. Walters K.J. Gates, channels, and switches: elements of the proteasome machine.Trends Biochem. Sci. 2016; 41: 77-93Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, Inobe and Matouschek, 2014Inobe T. Matouschek A. Paradigms of protein degradation by the proteasome.Curr. Opin. Struct. Biol. 2014; 24: 156-164Crossref PubMed Scopus (43) Google Scholar, Lander et al., 2013Lander G.C. Martin A. Nogales E. The proteasome under the microscope: the regulatory particle in focus.Curr. Opin. Struct. Biol. 2013; 23: 243-251Crossref PubMed Scopus (0) Google Scholar, Liu and Jacobson, 2013Liu C.W. Jacobson A.D. Functions of the 19S complex in proteasomal degradation.Trends Biochem. Sci. 2013; 38: 103-110Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, Livneh et al., 2016Livneh I. Cohen-Kaplan V. Cohen-Rosenzweig C. Avni N. Ciechanover A. The life cycle of the 26S proteasome: from birth, through regulation and function, and onto its death.Cell Res. 2016; 26: 869-885Crossref PubMed Scopus (21) Google Scholar, Tomko and Hochstrasser, 2013Tomko Jr., R.J. Hochstrasser M. Molecular architecture and assembly of the eukaryotic proteasome.Annu. Rev. Biochem. 2013; 82: 415-445Crossref PubMed Scopus (150) Google Scholar, Wehmer and Sakata, 2016Wehmer M. Sakata E. Recent advances in the structural biology of the 26S proteasome.Int. J. Biochem. Cell Biol. 2016; 79: 437-442Crossref PubMed Scopus (1) Google Scholar). Instead, we build on many of these discoveries to try to understand both the inherent logic of proteasome function and how its catalytic and regulatory features promote efficient and selective proteolysis. This analysis also highlights important gaps in our understanding that merit further study. Although it was long believed that ubiquitylation is sufficient to mark a protein for degradation, Matouschek and colleagues provided the fundamental insight that efficient degradation of a protein by the 26S requires not only the attachment of a Ub chain or multiple single Ub molecules, but also a loosely folded region in the substrate (Lee et al., 2001Lee C. Schwartz M.P. Prakash S. Iwakura M. Matouschek A. ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal.Mol. Cell. 2001; 7: 627-637Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, van der Lee et al., 2014van der Lee R. Lang B. Kruse K. Gsponer J. Sánchez de Groot N. Huynen M.A. Matouschek A. Fuxreiter M. Babu M.M. Intrinsically disordered segments affect protein half-life in the cell and during evolution.Cell Rep. 2014; 8: 1832-1844Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, Yu et al., 2016Yu H. Singh Gautam A.K. Wilmington S.R. Wylie D. Martinez-Fonts K. Kago G. Warburton M. Chavali S. Inobe T. Finkelstein I.J. et al.Conserved sequence preferences contribute to substrate recognition by the proteasome.J. Biol. Chem. 2016; 291: 14526-14539Crossref PubMed Scopus (0) Google Scholar). Thus, protein half-lives, which generally range in mammalian cells from 10 min to several days, are determined not just by the presence of sequences in proteins recognized by Ub ligases and ligase activities but also by differences in protein folding, which influence susceptibility to the proteasome. Before the discovery of the Ub proteasome pathway (UPP), it was recognized that such wide variations in half-lives were determined by inherent structural features of the proteins, and loosely folded and misfolded proteins were degraded especially rapidly in all cells (Goldberg and Dice, 1974Goldberg A.L. Dice J.F. Intracellular protein degradation in mammalian and bacterial cells.Annu. Rev. Biochem. 1974; 43: 835-869Crossref PubMed Scopus (493) Google Scholar). Their selective destruction is thus more ancient than the UPP and evolved in prokaryotes, where degradation is catalyzed by compartmentalized proteases, which like the proteasome, rely on AAA ATPase complexes for substrate recognition. Protein ubiquitylation and a proteasome regulatory complex that recognizes Ub conjugates evolved more recently with the emergence of eukaryotes. This linkage of ubiquitylation to proteolysis enabled protein degradation to be much more selective and precisely regulated. The ability of the proteasome to recognize both a Ub chain and a loosely folded region provides the fundamental basis for how it determines which proteins to degrade and which to spare. This critical life-or-death decision depends on two stages in conjugate binding of conjugate binding: (1) an initial, reversible step in which the Ub chain undergoes high-affinity binding to receptors on the 26S particle, and (2) a subsequent tighter-binding step that depends on the ubiquitylated protein’s structure and requires ATP hydrolysis (Peth et al., 2010Peth A. Uchiki T. Goldberg A.L. ATP-dependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation.Mol. Cell. 2010; 40: 671-681Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This sequence and the “dwell-time” of the substrate on the 26S provide an opportunity for competing processes to determine the protein’s fate. On one hand, the proteasome’s multiple de-ubiquitylation enzymes (DUBs) (Figure 3) shorten the substrate’s dwell-time and promote the release of some, perhaps many, of the ubiquitylated proteins that initially bind (Lee et al., 2016Lee B.H. Lu Y. Prado M.A. Shi Y. Tian G. Sun S. Elsasser S. Gygi S.P. King R.W. Finley D. USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites.Nature. 2016; 532: 398-401Crossref PubMed Google Scholar). However, if the substrate becomes tightly bound through its loosely folded domain, the six proteasome ATPases are activated (Peth et al., 2013aPeth A. Kukushkin N. Bossé M. Goldberg A.L. Ubiquitinated proteins activate the proteasomal ATPases by binding to Usp14 or Uch37 homologs.J. Biol. Chem. 2013; 288: 7781-7790Crossref PubMed Scopus (50) Google Scholar), and the substrate becomes committed to the steps leading to its destruction—further deubiquitylation, unfolding, processive translocation, and hydrolysis to small peptides in the 20S core particle. The association of ubiquitylated proteins to the 26S complex, although of high affinity, is readily reversible and easily disrupted by competition with other Ub binding domains or high salt concentrations (Peth et al., 2010Peth A. Uchiki T. Goldberg A.L. ATP-dependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation.Mol. Cell. 2010; 40: 671-681Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This initial binding depends only on the presence of a Ub chain, is independent of ATP hydrolysis, and even occurs at 4°C. In contrast, the second tight-binding step that commits the substrate to degradation requires ATP hydrolysis and a loosely folded region in the substrate (Figure 2). Two 19S subunits, Rpn10 and Rpn13, are particularly important for initial binding of Ub chains (Finley et al., 2016Finley D. Chen X. Walters K.J. Gates, channels, and switches: elements of the proteasome machine.Trends Biochem. Sci. 2016; 41: 77-93Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Rpn10 that binds to Ub chains through its Ub-interacting motif (UIM) was the first “ubiquitin receptor” described (Deveraux et al., 1994Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. A 26 S protease subunit that binds ubiquitin conjugates.J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar). Rpn13 was more recently discovered (Qiu et al., 2006Qiu X.B. Ouyang S.Y. Li C.J. Miao S. Wang L. Goldberg A.L. hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37.EMBO J. 2006; 25: 5742-5753Crossref PubMed Scopus (131) Google Scholar, Yao et al., 2006Yao T. Song L. Xu W. DeMartino G.N. Florens L. Swanson S.K. Washburn M.P. Conaway R.C. Conaway J.W. Cohen R.E. Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1.Nat. Cell Biol. 2006; 8: 994-1002Crossref PubMed Scopus (188) Google Scholar) and binds Ub chains via its PRU domain (Husnjak et al., 2008Husnjak K. Elsasser S. Zhang N. Chen X. Randles L. Shi Y. Hofmann K. Walters K.J. Finley D. Dikic I. Proteasome subunit Rpn13 is a novel ubiquitin receptor.Nature. 2008; 453: 481-488Crossref PubMed Scopus (341) Google Scholar). Both Rpn10 and Rpn13 also bind strongly proteins bearing Ub-like (UBL) domains but have only weak affinity for free Ub. They appear to have overlapping yet distinct roles. For example, unanchored Ub chains in cells bind preferentially to Rpn13 and block degradation of certain proteins (Dayal et al., 2009Dayal S. Sparks A. Jacob J. Allende-Vega N. Lane D.P. Saville M.K. Suppression of the deubiquitinating enzyme USP5 causes the accumulation of unanchored polyubiquitin and the activation of p53.J. Biol. Chem. 2009; 284: 5030-5041Crossref PubMed Scopus (0) Google Scholar). In vitro yeast 26S lacking either Rpn13 or the UIM domain of Rpn10 show reduced binding of conjugates by ∼50% (Peth et al., 2010Peth A. Uchiki T. Goldberg A.L. ATP-dependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation.Mol. Cell. 2010; 40: 671-681Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In cells, it is necessary to knockdown Rpn13 and delete Rpn10’s UIM domain to cause an accumulation of Ub conjugates and a large decrease in the binding of UBL proteins to the 26S (Hamazaki et al., 2015Hamazaki J. Hirayama S. Murata S. Redundant roles of Rpn10 and Rpn13 in recognition of ubiquitinated proteins and cellular homeostasis.PLoS Genet. 2015; 11: e1005401Crossref PubMed Google Scholar). Unlike other 26S subunits, Rpn10 also exists in high amounts free in the cytosol and probably has roles outside the 26S (Kim et al., 2009Kim H.T. Kim K.P. Uchiki T. Gygi S.P. Goldberg A.L. S5a promotes protein degradation by blocking synthesis of nondegradable forked ubiquitin chains.EMBO J. 2009; 28: 1867-1877Crossref PubMed Scopus (0) Google Scholar, Rubin et al., 1997Rubin D.M. van Nocker S. Glickman M. Coux O. Wefes I. Sadis S. Fu H. Goldberg A. Vierstra R. Finley D. ATPase and ubiquitin-binding proteins of the yeast proteasome.Mol. Biol. Rep. 1997; 24: 17-26Crossref PubMed Google Scholar). Although often defined as a stoichiometric subunit, Rpn13 is not present on all 19S complexes and may be absent from one regulatory particle in doubly capped 26S (Berko et al., 2014Berko D. Herkon O. Braunstein I. Isakov E. David Y. Ziv T. Navon A. Stanhill A. Inherent asymmetry in the 26S proteasome is defined by the ubiquitin receptor RPN13.J. Biol. Chem. 2014; 289: 5609-5618Crossref PubMed Scopus (9) Google Scholar). Curiously, the recent cryo-EM studies of human 26S could not visualize Rpn13 (Chen et al., 2016Chen S. Wu J. Lu Y. Ma Y.B. Lee B.H. Yu Z. Ouyang Q. Finley D.J. Kirschner M.W. Mao Y. Structural basis for dynamic regulation of the human 26S proteasome.Proc. Natl. Acad. Sci. USA. 2016; 113: 12991-12996Crossref PubMed Google Scholar, Huang et al., 2016Huang X. Luan B. Wu J. Shi Y. An atomic structure of the human 26S proteasome.Nat. Struct. Mol. Biol. 2016; 23: 778-785Crossref PubMed Scopus (0) Google Scholar, Schweitzer et al., 2016Schweitzer A. Aufderheide A. Rudack T. Beck F. Pfeifer G. Plitzko J.M. Sakata E. Schulten K. Förster F. Baumeister W. Structure of the human 26S proteasome at a resolution of 3.9 Å.Proc. Natl. Acad. Sci. USA. 2016; 113: 7816-7821Crossref PubMed Google Scholar). Although Rpn10 and Rpn13 are the major sites of initial conjugate binding, other 19S subunits also bind Ub-conjugates or UBL proteins. The combined deletion of both Rpn10 and Rpn13 was not lethal in budding and fission yeast, unlike the deletion of most proteasome subunits. Therefore, another Ub binding subunit must be present on the 26S (Husnjak et al., 2008Husnjak K. Elsasser S. Zhang N. Chen X. Randles L. Shi Y. Hofmann K. Walters K.J. Finley D. Dikic I. Proteasome subunit Rpn13 is a novel ubiquitin receptor.Nature. 2008; 453: 481-488Crossref PubMed Scopus (341) Google Scholar). Accordingly, purified proteasomes lacking both Rpn13 and Rpn10’s UIM domain retain ∼20% the normal affinity of 26S for Ub conjugates (Peth et al., 2010Peth A. Uchiki T. Goldberg A.L. ATP-dependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation.Mol. Cell. 2010; 40: 671-681Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Recently, the 19S subunit Rpn1 was identified as a binding site for Ub chains (Shi et al., 2016Shi Y. Chen X. Elsasser S. Stocks B.B. Tian G. Lee B.H. Shi Y. Zhang N. de Poot S.A. Tuebing F. et al.Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome.Science. 2016; 351: aad9421Crossref PubMed Scopus (0) Google Scholar) as well as UBL domains (Elsasser et al., 2002Elsasser S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Müller B. Feng M.T. Tübing F. Dittmar G.A. Finley D. Proteasome subunit Rpn1 binds ubiquitin-like protein domains.Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (325) Google Scholar, Gomez et al., 2011Gomez T.A. Kolawa N. Gee M. Sweredoski M.J. Deshaies R.J. Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1.BMC Biol. 2011; 9: 33Crossref PubMed Scopus (0) Google Scholar). Many ubiquitylated substrates bind to the 26S indirectly via proteins that contain both a UBL-domain and a Ub-associated (UBA) domain, giving them the ability to bind simultaneously Ub conjugates and the 26S. Therefore, these proteins (in yeast: Ddi1, Dsk2, and Rad23) have been proposed to function as “shuttling factors.” Rpn1 contains two conjugate binding sites: T1, where UBL-UBA proteins and Ub chains bind, and T2, where the UBL domain of the DUB Ubp6 binds (Shi et al., 2016Shi Y. Chen X. Elsasser S. Stocks B.B. Tian G. Lee B.H. Shi Y. Zhang N. de Poot S.A. Tuebing F. et al.Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome.Science. 2016; 351: aad9421Crossref PubMed Scopus (0) Google Scholar). Another 19S subunit, Dss1, has also been implicated in Ub binding (Paraskevopoulos et al., 2014Paraskevopoulos K. Kriegenburg F. Tatham M.H. Rösner H.I. Medina B. Larsen I.B. Brandstrup R. Hardwick K.G. Hay R.T. Kragelund B.B. et al.Dss1 is a 26S proteasome ubiquitin receptor.Mol. Cell. 2014; 56: 453-461Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), but its role in conjugate degradation has not been studied, and its accessibility within the 26S structure has been questioned (Schweitzer et al., 2016Schweitzer A. Aufderheide A. Rudack T. Beck F. Pfeifer G. Plitzko J.M. Sakata E. Schulten K. Förster F. Baumeister W. Structure of the human 26S proteasome at a resolution of 3.9 Å.Proc. Natl. Acad. Sci. USA. 2016; 113: 7816-7821Crossref PubMed Google Scholar). It is bewildering why the proteasome contains so many binding sites for ubiquitylated proteins, and it is not clear that all the sites have been identified. Determining the relative affinities of Ub conjugates for these sites in intact proteasomes and whether they function cooperatively, sequentially, or are specific for certain substrates could fill important gaps in our understanding of the degradative process. Another fundamental unanswered question concerns the precise roles in conjugate degradation of the “shuttling factors.” Although genetic studies clearly support an important role in degradation of some substrates, there seems to be no a priori need to have shuttling factors, because Ub-conjugates associate with the 26S with similar affinities as the UBL-UBA proteins and bind to the same sites on the 26S as ubiquitylated proteins (Shi et al., 2016Shi Y. Chen X. Elsasser S. Stocks B.B. Tian G. Lee B.H. Shi Y. Zhang N. de Poot S.A. Tuebing F. et al.Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome.Science. 2016; 351: aad9421Crossref PubMed Scopus (0) Google Scholar). Nevertheless, Rad23 is found on many Ub conjugates binding to the proteasome after processing by the p97/VCP/Cdc48 ATPase complex (Richly et al., 2005Richly H. Rape M. Braun S. Rumpf S. Hoege C. Jentsch S. A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting.Cell. 2005; 120: 73-84Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar), and the loss of these shuttling factors, like mutations in proteasome subunits, results in increased sensitivity to conditions causing protein misfolding (Wilkinson et al., 2001Wilkinson C.R. Seeger M. Hartmann-Petersen R. Stone M. Wallace M. Semple C. Gordon C. Proteins containing the UBA domain are able to bind to multi-ubiquitin chains.Nat. Cell Biol. 2001; 3: 939-943Crossref PubMed Scopus (313) Google Scholar). Thus, these proteins must serve important functions in vivo, but efforts to reconstitute in vitro the stimulation of proteolysis by shuttling factors have failed so far. Although there is appreciable functional redundancy of these UBL-UBA proteins (Wilkinson et al., 2001Wilkinson C.R. Seeger M. Hartmann-Petersen R. Stone M. Wallace M. Semple C. Gordon C. Proteins containing the UBA domain are able to bind to multi-ubiquitin chains.Nat. Cell Biol. 2001; 3: 939-943Crossref PubMed Scopus (313) Google Scholar), mutations in certain shuttling factors produce specific phenotypes. For example, mutations in UBQLN2 (a Dsk2 homolog) cause amyotrophic lateral sclerosis (ALS) (Deng et al., 2011Deng H.X. Chen W. Hong S.T. Boycott K.M. Gorrie G.H. Siddique N. Yang Y. Fecto F. Shi Y. Zhai H. et al.Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia.Nature. 2011; 477: 211-215Crossref PubMed Scopus (501) Google Scholar), and thus it must serve some distinct role, probably in ubiquitylation and shuttling of client proteins to the 26S (Itakura et al., 2016Itakura E. Zavodszky E. Shao S. Wohlever M.L. Keenan R.J. Hegde R.S. Ubiquilins chaperone and triage mitochondrial membrane proteins for degradation.Mol. Cell. 2016; 63: 21-33Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Also, human Rad23s show a strong preference for Ub chains containing Ub molecules linked through their lysine 48 residues (K48) over chains formed by lysine 63 linkages (K63). Thus, these shuttling factors seem to help direct K48 Ub conjugates to the proteasome (Nathan et al., 2013Nathan J.A. Kim H.T. Ting L. Gygi S.P. Goldberg A.L. Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?.EMBO J. 2013; 32: 552-565Crossref PubMed Scopus (96) Google Scholar). The binding of Ub conjugates to proteasomes can be regulated by post-synthetic modifications. In response to proteasome inhibitors or conditions that impair 26S function (e.g., expression of aggregation-prone proteins), Rpn13 becomes ubiquitylated by the 26S-associated Ub ligase Ube3c (Besche et al., 2014Besche H.C. Sha Z. Kukushkin N.V. Peth A. Hock E.M. Kim W. Gygi S. Gutierrez J.A. Liao H. Dick L. Goldberg A.L. Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates.EMBO J. 2014; 33: 1159-1176Crossref PubMed Scopus (47) Google Scholar, Jacobson et al., 2014Jacobson A.D. MacFadden A. Wu Z. Peng J. Liu C.W. Autoregulation of the 26S proteasome by in situ ubiquitination.Mol. Biol. Cell. 2014; 25: 1824-1835Crossref PubMed Scopus (23) Google Scholar). This modification prevents further binding of Ub conjugates inactivating the 26S (despite the presence of other Ub-binding sites) (Besche et al., 2014Besche H.C. Sha Z. Kukushkin N.V. Peth A. Hock E.M. Kim W. Gygi S. Gutierrez J.A. Liao H. Dick L. Goldberg A.L. Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates.EMBO J. 2014; 33: 1159-1176Crossref PubMed Scopus (47) Google Scholar). This inactivation seems to occur when proteasomes encounter difficulties in degrading substrates (e.g., aggregated proteins) and presumably redirects Ub conjugates to functioning proteasomes (Besche et al., 2014Besche H.C. Sha Z. Kukushkin N.V. Peth A. Hock E.M. Kim W. Gygi S. Gutierrez J.A. Liao H. Dick L. Goldberg A.L. Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates.EMBO J. 2014; 33: 1159-1176Crossref PubMed Scopus (47) Google Scholar). It seems likely that other regulatory factors will also affect the initial binding step. In mammalian cells, Rpn13 ubiquitylation is reversed by cytosolic DUBs. However, in plants, where proteasome inhibition also causes Rpn13 ubiquitylation, this modification targets the 26S to autophagy (Marshall et al., 2015Marshall R.S. Li F. Gemperline D.C. Book A.J. Vierstra R.D. Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis.Mol. Cell. 2015; 58: 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In S. cerevisiae, the Ube3c homolog, Hul5, and Rsp5 ubiquitylate a different Ub binding protein, Rpn10 (Isasa et al., 2010Isasa M. Katz E.J. Kim W. Yugo V. González S. Kirkpatrick D.S. Thomson T.M. Finley D. Gygi S.P. Crosas B. Monoubiquitination of RPN10 regulates substrate recruitment to the proteasome.Mol. Cell. 2010; 38: 733-745Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), which may cause Rpn10 dissociation from the 26S (Zuin et al., 2015Zuin A. Bichmann A. Isasa M. Puig-Sàrries P. Díaz L.M. Crosas B. Rpn10 monoubiquitination orchestrates the association of the ubiquilin-type DSK2 receptor with the proteasome.Biochem. J. 2015; 472: 353-365Crossref PubMed Scopus (5) Google Scholar) or trigger autophagy of proteasomes (Marshall et al., 2016Marshall R.S. McLoughlin F. Vierstra R.D. Autophagic turnover of inactive 26S proteasomes in yeast is directed by the ubiquitin receptor Cue5 and the Hsp42 chaperone.Cell Rep. 2016; 16: 1717-1732Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Proteasomes face the seemingly difficult challenge of binding and disassembling many different types of Ub chains that vary both in length and linkage specificity. Despite widespread impressions to the contrary, proteasomes are not fastidious regarding the type or length of Ub chains on substrates. Isolated 26S can degrade substrates containing multiple short Ub chains (Dimova et al., 2012Dimova N.V. Hathaway N.A. Lee B.H. Kirkpatrick D.S. Berkowitz M.L. Gygi S.P. Finley D. King R.W. APC/C-mediated multiple monoubiquitylation provides an alternative degradation signal for cyclin B1.Nat. Cell Biol. 2012; 14: 168-176Crossref PubMed Scopus (66) Google Scholar) or even a single Ub (Braten et al., 2016Braten O. Livneh I. Ziv T. Admon A. Kehat I. Caspi L.H. Gonen H. Bercovich B. Godzik A. Jahandideh S. et al.Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination.Proc. Natl. Acad. Sci. USA. 2016; 113: E4639-E4647Crossref PubMed Scopus (0) Google Scholar), although these different Ub conjugates may differ widely in their affinities for the proteasome and once bound in their likelihood of being degraded. The importance of chain-length on substrate binding was first revealed by Thrower et al., 2000Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. Recognition of the polyubiquitin proteolytic signal.EMBO J. 2000; 19: 94-102Crossref PubMed Google Scholar. While often cited as indicating a requirement for only K48 tetra-Ub chains, these investigators actually found that longer chains containing five to nine ubiquitins on dihydrofolate reductase (DHFR) provided greater affinity for proteasomes but did not change its degradat
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