Proteasome Activator 200: The HEAT is on…
2011; Elsevier BV; Volume: 10; Issue: 5 Linguagem: Inglês
10.1074/mcp.r110.006890
ISSN1535-9484
AutoresAnca F. Savulescu, Michael H. Glickman,
Tópico(s)Protein Degradation and Inhibitors
ResumoProteasomes play a key regulatory role in all eukaryotic cells by removing proteins in a timely manner. There are two predominant forms: The 20S core particle (CP) can hydrolyze peptides and certain unstructured proteins, and the 26S holoenzyme is able to proteolyse most proteins conjugated to ubiquitin. The 26S complex consists of a CP barrel with a 19S regulatory particle (RP; a.k.a PA700) attached to its outer surface. Several studies purified another proteasome activator with a MW of 200 kDa (PA200) that attaches to the same outer ring of the CP. A role for PA200 has been demonstrated in spermatogenesis, in response to DNA repair and in maintenance of mitochondrial inheritance. Enhanced levels of PA200-CP complexes are observed under conditions in which either activated or disrupted CP prevail, suggesting it participates in regulating overall proteolytic activity. PA200, or its yeast ortholog Blm10, may also incorporate into 26S proteasomes yielding PA200-CP-RP hybrids. A three-dimensional molecular structure determined by x-ray crystallography of Blm10-CP provides a model for activation. The carboxy terminus of Blm10 inserts into a dedicated pocket in the outer ring of the CP surface, whereas multiple HEAT-like repeats fold into an asymmetric solenoid wrapping around the central pore to stabilize a partially open conformation. The resulting hollow domelike structure caps the entire CP surface. This asymmetric structure may provide insight as to how the 19S RP, with two HEAT repeatlike subunits (Rpn1, Rpn2) alongside six ATPases (Rpt1–6), attaches to the same surface of the CP ring, and likewise, induces pore opening. Proteasomes play a key regulatory role in all eukaryotic cells by removing proteins in a timely manner. There are two predominant forms: The 20S core particle (CP) can hydrolyze peptides and certain unstructured proteins, and the 26S holoenzyme is able to proteolyse most proteins conjugated to ubiquitin. The 26S complex consists of a CP barrel with a 19S regulatory particle (RP; a.k.a PA700) attached to its outer surface. Several studies purified another proteasome activator with a MW of 200 kDa (PA200) that attaches to the same outer ring of the CP. A role for PA200 has been demonstrated in spermatogenesis, in response to DNA repair and in maintenance of mitochondrial inheritance. Enhanced levels of PA200-CP complexes are observed under conditions in which either activated or disrupted CP prevail, suggesting it participates in regulating overall proteolytic activity. PA200, or its yeast ortholog Blm10, may also incorporate into 26S proteasomes yielding PA200-CP-RP hybrids. A three-dimensional molecular structure determined by x-ray crystallography of Blm10-CP provides a model for activation. The carboxy terminus of Blm10 inserts into a dedicated pocket in the outer ring of the CP surface, whereas multiple HEAT-like repeats fold into an asymmetric solenoid wrapping around the central pore to stabilize a partially open conformation. The resulting hollow domelike structure caps the entire CP surface. This asymmetric structure may provide insight as to how the 19S RP, with two HEAT repeatlike subunits (Rpn1, Rpn2) alongside six ATPases (Rpt1–6), attaches to the same surface of the CP ring, and likewise, induces pore opening. The proteasome is the regulatory protease for removal of cellular proteins selected by the ubiquitin (Ub) 1The abbreviations used are:UbubiquitinCP20S core particleHEATHuntingtin–elongation factor 3–PR65/A subunit of PP2A–lipid kinase TORPAproteasome activatorRP19S regulatory particleRpnregulatory particle non-ATPaseRptregulatory particle triple A protein. system (1Finley D. Recognition and processing of ubiquitin-protein conjugates by the proteasome.Annu. Rev. Biochem. 2009; 78: 477-513Crossref PubMed Scopus (1275) Google Scholar). At the heart of the proteasome structure lies the barrel-shaped 20S core particle (CP) into which substrates must enter if they are to be proteolysed (2Pickart C.M. Cohen R.E. Proteasomes and their kin: proteases in the machine age.Nat. Rev. Mol. Cell Biol. 2004; 5: 177-187Crossref PubMed Scopus (605) Google Scholar). Substrates access the central catalytic chamber through narrow pores defined by the outer α-rings of the 20S CP (3Bajorek M. Glickman M.H. The proteasome portal and regulation of proteolysis.in: Protein Degradation. Vol II. Wiley-VCH Press, 2005: 111-127Crossref Scopus (1) Google Scholar). In the absence of activators, these channels are closed by the N termini of the seven different α subunits that forge these rings, and proteolysis rates are repressed (4Bajorek M. Glickman M.H. Proteasome regulatory particles: Keepers of the gates.Cell Mol. Life Sci. 2004; 61: 1579-1588Crossref PubMed Google Scholar, 5Rechsteiner M. Hill C.P. Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors.Trends in Cell Biology. 2005; 15: 27-33Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Disordering of the blocking residues or opening the channel accelerates entry of peptides, however in order to degrade globular or ubiquitinated substrates, attachment of proteasome activators (PAs) to the outer surface of the 20S CP is required (6Groll M. Bajorek M. Köhler A. Moroder L. Rubin D.M. Huber R. Glickman M.H. Finley D. A gated channel into the proteasome core particle.Nat. Struct. Biol. 2000; 7: 1062-1067Crossref PubMed Scopus (651) Google Scholar, 7Förster A. Whitby F.G. Hill C.P. The pore of activated 20S proteasomes has an ordered 7-fold symmetric conformation.EMBO J. 2003; 22: 4356-4364Crossref PubMed Scopus (95) Google Scholar, 8Schmidt M. Hanna J. Elsasser S. Finley D. Proteasome-associated proteins: regulation of a proteolytic machine.Biol. Chem. 2005; 386: 725-737Crossref PubMed Scopus (145) Google Scholar, 9Huang L. Chen C.H. Proteasome regulators: activators and inhibitors.Curr. Med. Chem. 2009; 16: 931-939Crossref PubMed Scopus (76) Google Scholar). The Ub-binding, ATPase-containing, 19S regulatory particle (RP) is the best studied such activator (a.k.a "proteasome activator 700"; PA700) and is required for regulated proteolysis (Fig. 1). Attachment of the 19S RP to the outer surface of the α-ring induces an open pore, thereby enhancing the basal peptidase activity of the proteasome (2Pickart C.M. Cohen R.E. Proteasomes and their kin: proteases in the machine age.Nat. Rev. Mol. Cell Biol. 2004; 5: 177-187Crossref PubMed Scopus (605) Google Scholar, 5Rechsteiner M. Hill C.P. Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors.Trends in Cell Biology. 2005; 15: 27-33Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 10Glickman M.H. Raveh D. Proteasome plasticity.FEBS Letters. 2005; 579: 3214-3223Crossref PubMed Scopus (93) Google Scholar). Ubiquitin-binding subunits in the 19S RP anchor the tagged substrate whereas six ATPases (Rpt1–6) unfold and translocate it through the channel into the 20S CP for proteolysis. This RP-CP holoenzyme, termed the 26S proteasome, is thought to be the most abundant species in rapidly growing cells, although it is found in equilibrium with free 20S CP (11Bajorek M. Finley D. Glickman M.H. Proteasome Disassembly and Downregulation Is Correlated with Viability during Stationary Phase.Current Biology. 2003; 13: 1140-1144Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). ubiquitin 20S core particle Huntingtin–elongation factor 3–PR65/A subunit of PP2A–lipid kinase TOR proteasome activator 19S regulatory particle regulatory particle non-ATPase regulatory particle triple A protein. In vitro, many unfolded or loosely structured proteins are substrates for proteolysis by the isolated 20S CP, however it is unclear whether in cells, free 20S CP is biologically active as a regulatory protease on its own or whether it serves primarily as a reserve of core particles for rapid assembly with activators as needed. Nevertheless, there are increasing reports of 20S CP capable of proteolysis without requirement for ATP (12Hoyt M.A. Coffino P. Ubiquitin-free routes into the proteasome.Cell Mol. Life Sci. 2004; 61: 1596-1600Crossref PubMed Google Scholar, 13Asher G. 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Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors.Trends in Cell Biology. 2005; 15: 27-33Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 7Förster A. Whitby F.G. Hill C.P. The pore of activated 20S proteasomes has an ordered 7-fold symmetric conformation.EMBO J. 2003; 22: 4356-4364Crossref PubMed Scopus (95) Google Scholar, 17Cascio P. Call M. Petre B.M. Walz T. Goldberg A.L. Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes.EMBO J. 2002; 21: 2636-2645Crossref PubMed Scopus (183) Google Scholar). 11S-Regs form a hollow cone-shaped ring of seven 28-kDa subunits. The family includes the heteromeric interferon-γ induced PA28α/β involved in immune response and antigen presentation, the nuclear PA28γ, and a distantly related complex unique to trypanosomes termed PA26 (18Hill C.P. Masters E.I. Whitby F.G. The 11S regulators of 20S proteasome activity.Curr. Top. Microbiol. Immunol. 2002; 268: 73-89PubMed Google Scholar, 19Masters E.I. Pratt G. Förster A. Hill C.P. Purification and analysis of recombinant 11S activators of the 20S proteasome: Trypanosoma brucei PA26 and human PA28 alpha, PA28 beta, and PA28 gamma.Methods Enzymol. 2005; 398: 306-321Crossref PubMed Scopus (7) Google Scholar, 20Förster A. Masters E.I. Whitby F.G. Robinson H. Hill C.P. The 1.9 A structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions.Mol. Cell. 2005; 18: 589-599Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Another ATP-independent proteasome activator is the 250-kDa monomeric PA200 (Blm10 in yeast) that competes for the same binding sites on the 20S α-ring surface (21Ustrell V. Hoffman L. Pratt G. Rechsteiner M. PA200, a nuclear proteasome activator involved in DNA repair.EMBO J. 2002; 21: 3516-3525Crossref PubMed Scopus (270) Google Scholar, 22Schmidt M. Haas W. Crosas B. Santamaria P.G. Gygi S.P. Walz T. Finley D. The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle.Nat. Struct. Mol. Biol. 2005; 12: 294-303Crossref PubMed Scopus (149) Google Scholar, 23Ortega J. Bernard Heymann J. Kajava A.V. Ustrell V. Rechsteiner M. Steven A.C. The Axial Channel of the 20 S Proteasome Opens Upon Binding of the PA200 Activator.J. of Mol. Biol. 2005; 3461221 Crossref PubMed Scopus (89) Google Scholar, 24Sadre-Bazzaz K. Whitby F.G. Robinson H. Formosa T. Hill C.P. Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening.Mol. Cell. 2010; 37: 728-735Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Non-ATPase activators probably do not promote general proteolysis of intact globular proteins (9Huang L. Chen C.H. Proteasome regulators: activators and inhibitors.Curr. Med. Chem. 2009; 16: 931-939Crossref PubMed Scopus (76) Google Scholar, 25Stadtmueller B.M. Hill C.P. Proteasome activators.Mol. Cell. 2011; 41: 8-19Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). However, degradation of specific, loosely folded substrates aided by PA28/11S-Reg has been documented and may reflect a more general feature of non-ATPase activators (26Schwartz K. Eggers M. Soza A. Koszinowski U.H. Kloetzel P.M. Groettrup M. The proteasome regulator PA28a/b can enhance antigen presentation without affecting 20S proteasome subunit composition.Eur. J. Immunol. 2000; 12: 3672-3679Crossref Scopus (55) Google Scholar, 27Stohwasser R. Salzmann U. Ruppert T. Kloetzel P.M. Holzhüetter H.G. Kinetic evidences for facilitation of peptide channeling by the proteasomal activator PA28.Eur. J. Biochem. 2000; 267: 6221-6230Crossref PubMed Scopus (64) Google Scholar, 28Zhang Z. Zhang R. Proteasome activator PA28 gamma regulates p53 by enhancing its MDM2-mediated degradation.EMBO J. 2008; 27: 852-864Crossref PubMed Scopus (131) Google Scholar). Attachment of a single copy of PA200 (or Blm10) to 20S CP to generate the asymmetric PA200-CP (Fig. 1) is correlated with enhanced hydrolysis of small peptides, hence its name "proteasome activator 200" (PA200) (21Ustrell V. Hoffman L. Pratt G. Rechsteiner M. PA200, a nuclear proteasome activator involved in DNA repair.EMBO J. 2002; 21: 3516-3525Crossref PubMed Scopus (270) Google Scholar, 22Schmidt M. Haas W. Crosas B. Santamaria P.G. Gygi S.P. Walz T. Finley D. The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle.Nat. Struct. Mol. Biol. 2005; 12: 294-303Crossref PubMed Scopus (149) Google Scholar, 23Ortega J. Bernard Heymann J. Kajava A.V. Ustrell V. Rechsteiner M. Steven A.C. The Axial Channel of the 20 S Proteasome Opens Upon Binding of the PA200 Activator.J. of Mol. Biol. 2005; 3461221 Crossref PubMed Scopus (89) Google Scholar). Homologs of PA200 are found in many eukaryotes, though in some (fruit flies and fission yeast for example) it may have diverged beyond recognition or replaced by another non-ATPase activator. A large portion of our knowledge comes from the budding yeast model, where the gene encoding for the PA200 ortholog is termed BLM10 (YFL007w; previously known as BLM3). In this review we will use the term PA200 in reference to general properties of this protein regardless of origin, and revert to Blm10 to denote observations unique to yeast. PA200 is poorly conserved; yeast and mammalian orthologs maintain significantly less than 20% identity, and multiple sequence alignments are limited to key residues and structural motifs (23Ortega J. Bernard Heymann J. Kajava A.V. Ustrell V. Rechsteiner M. Steven A.C. The Axial Channel of the 20 S Proteasome Opens Upon Binding of the PA200 Activator.J. of Mol. Biol. 2005; 3461221 Crossref PubMed Scopus (89) Google Scholar, 24Sadre-Bazzaz K. Whitby F.G. Robinson H. Formosa T. Hill C.P. Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening.Mol. Cell. 2010; 37: 728-735Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 29Kajava A.V. Gorbea C. Ortega J. Rechsteiner M. Steven A.C. New HEAT-like repeat motifs in proteins regulating proteasome structure and function.J. Struct. Biol. 2004; 146: 425-430Crossref PubMed Scopus (67) Google Scholar). In budding yeast, Blm10 was initially identified as a nuclear protein associated with nascent and de novo synthesized 20S CP and is considered to be the functional ortholog of PA200 (30Fehlker M. Wendler P. Lehmann A. Enenkel C. Blm3 is part of nascent proteasomes and is involved in a late stage of nuclear proteasome assembly.EMBO Rep. 2003; 4: 959-963Crossref PubMed Scopus (98) Google Scholar). A later study in yeast suggested a role for Blm10 in the maintenance of mitochondrial functions (24Sadre-Bazzaz K. Whitby F.G. Robinson H. Formosa T. Hill C.P. Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening.Mol. Cell. 2010; 37: 728-735Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). PA200 was detected in most mammalian organs by immuno-blotting, and found particularly abundant in testis (21Ustrell V. Hoffman L. Pratt G. Rechsteiner M. PA200, a nuclear proteasome activator involved in DNA repair.EMBO J. 2002; 21: 3516-3525Crossref PubMed Scopus (270) Google Scholar). Knockdown cells (PA200ΔΔ) exhibited a decrease in the level of mature spermatozoa and multiple defects in spermatogenesis. However, PA200 deficiency did not lead to significant defects in either embryonic development or viability of adult mice, as shown with PA200 null mice (31Khor B. Bredemeyer A.L. Huang C.Y. Turnbull I.R. Evans R. Maggi Jr., L.B. White J.M. Walker L.M. Carnes K. Hess R.A. Sleckman B.P. Proteasome activator PA200 is required for normal spermatogenesis.Mol. Cell Biol. 2006; 26: 2999-3007Crossref PubMed Scopus (115) Google Scholar). Even in Arabidopsis, PA200 null mutants are phenotypically indistinguishable from wild type under a variety of growth conditions, which include darkness, and short and long photoperiods (32Book A.J. Gladman N.P. Lee S.S. Scalf M. Smith L.M. Vierstra R.D. Affinity purification of the Arabidopsis 26 S proteasome reveals a diverse array of plant proteolytic complexes.J. Biol. Chem. 2010; 285: 25554-25569Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). These observations suggest that PA200 is not strictly essential in most model organisms. One condition that seems to require PA200 is response to radiation damage and in DNA repair. Following γ-irradiation, the predominantly nuclear PA200 relocalizes into subnuclear foci and associates with chromatin, suggesting a role in response to DNA repair (21Ustrell V. Hoffman L. Pratt G. Rechsteiner M. PA200, a nuclear proteasome activator involved in DNA repair.EMBO J. 2002; 21: 3516-3525Crossref PubMed Scopus (270) Google Scholar). Relocalization is mediated by DNA-PK, which is a PI3-related kinase that responds to infra-red (IR)-induced damage, but not on ATM or p53 (33Blickwedehl J. McEvoy S. Wong I. Kousis P. Clements J. Elliott R. Cresswell P. Liang P. Bangia N. Proteasomes and proteasome activator 200 kDa (PA200) accumulate on chromatin in response to ionizing radiation.Radiat. Res. 2007; 167: 663-674Crossref PubMed Scopus (46) Google Scholar, 34Blickwedehl J. Agarwal M. Seong C. Pandita R.K. Melendy T. Sung P. Pandita T.K. Bangia N. Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 16165-16170Crossref PubMed Scopus (82) Google Scholar). Accumulation of PA200 on chromatin correlates with formation of hybrid PA200-CP-RP proteasomes and an increase in general and postacidic proteolytic activity. However, in mammalian cells PA200 is not essential for the repair of DNA double-strand breaks generated upon treatment with ionizing radiation or bleomycin (31Khor B. Bredemeyer A.L. Huang C.Y. Turnbull I.R. Evans R. Maggi Jr., L.B. White J.M. Walker L.M. Carnes K. Hess R.A. Sleckman B.P. Proteasome activator PA200 is required for normal spermatogenesis.Mol. Cell Biol. 2006; 26: 2999-3007Crossref PubMed Scopus (115) Google Scholar). Likewise, Δblm10 yeast mutants and null PA200 mutants in Arabidopsis survive stress conditions that accumulate damaged proteins or DNA damage such as induced by bleomycin or IR exposure (35Iwanczyk J. Sadre-Bazzaz K. Ferrell K. Kondrashkina E. Formosa T. Hill C.P. Ortega J. Structure of the Blm10–20 S Proteasome Complex by Cryo-electron Microscopy. Insights into the Mechanism of Activation of Mature Yeast Proteasomes.J. Mol. Biol. 2006; 363: 648-659Crossref PubMed Scopus (46) Google Scholar). In contrast, deletion of a C-terminal domain of Blm10/PA200 prevents its nuclear import and compromises survival of cells exposed to bleomycin, a property attributed by the authors to dominant-negative cytoplasmic sequestration (22Schmidt M. Haas W. Crosas B. Santamaria P.G. Gygi S.P. Walz T. Finley D. The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle.Nat. Struct. Mol. Biol. 2005; 12: 294-303Crossref PubMed Scopus (149) Google Scholar), though defects in nuclear assembly of proteasomes cannot be ruled out (30Fehlker M. Wendler P. Lehmann A. Enenkel C. Blm3 is part of nascent proteasomes and is involved in a late stage of nuclear proteasome assembly.EMBO Rep. 2003; 4: 959-963Crossref PubMed Scopus (98) Google Scholar). Taken together, the studies above suggest that although not absolutely essential for survival, PA200 may play a supporting role in DNA repair in parallel with other factors (34Blickwedehl J. Agarwal M. Seong C. Pandita R.K. Melendy T. Sung P. Pandita T.K. Bangia N. Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 16165-16170Crossref PubMed Scopus (82) Google Scholar). PA200 is composed almost entirely of α-turn-α modules identified as HEAT (Huntingtin - elongation factor 3 -PR65/A subunit of PP2A - lipid kinase TOR) repeats (29Kajava A.V. Gorbea C. Ortega J. Rechsteiner M. Steven A.C. New HEAT-like repeat motifs in proteins regulating proteasome structure and function.J. Struct. Biol. 2004; 146: 425-430Crossref PubMed Scopus (67) Google Scholar, 36Andrade M.A. Petosa C. O'Donoghue S.I. Müller C.W. Bork P. Comparison of ARM and HEAT protein repeats.J. Mol. Biol. 2001; 309: 1-18Crossref PubMed Scopus (396) Google Scholar, 37Kajava A.V. What Curves alpha -Solenoids? Evidence For An Alpha -Helical Toroid Structure of Rpn1 and Rpn2 Proteins of The 26S Proteasome.J. Biol. Chem. 2002; 277: 49791-49798Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Because of their concave nature, 32 such HEAT repeats in PA200 wrap around into a two-layered snail-like dome situated over the 20S surface, covering the translocation channel and well positioned to regulate traffic into the 20S (24Sadre-Bazzaz K. Whitby F.G. Robinson H. Formosa T. Hill C.P. Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening.Mol. Cell. 2010; 37: 728-735Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 29Kajava A.V. Gorbea C. Ortega J. Rechsteiner M. Steven A.C. New HEAT-like repeat motifs in proteins regulating proteasome structure and function.J. Struct. Biol. 2004; 146: 425-430Crossref PubMed Scopus (67) Google Scholar) (see Fig. 2). It is still unclear whether PA200 functions solely as a proteasome regulator or might harbor independent roles as well. A significant increase in cellular PA200 levels, followed by accumulation of PA200-CP, was obtained upon exposure of Arabidopsis seedlings to the proteasome chemical inhibitor MG132 or in a mutant that dampens RP activity, suggesting response of PA200 to cellular proteolytic demand (32Book A.J. Gladman N.P. Lee S.S. Scalf M. Smith L.M. Vierstra R.D. Affinity purification of the Arabidopsis 26 S proteasome reveals a diverse array of plant proteolytic complexes.J. Biol. Chem. 2010; 285: 25554-25569Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). However, a Δblm10 mutant showed no synthetic sensitivity to accumulation of damaged or miss folded proteins, indicating that Blm10 might not be involved in general protein degradation, but rather have a more specific role. One example whereby PA200 may participate in degradation of a select subset of substrates is during the maintenance of mitochondrial functions (24Sadre-Bazzaz K. Whitby F.G. Robinson H. Formosa T. Hill C.P. Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening.Mol. Cell. 2010; 37: 728-735Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). A double mutant deleted for BLM10 and the nonessential stress-induced proteasome transcriptional activator RPN4 showed synthetic sensitivity to accumulation of damaged or miss folded proteins that must be removed by the proteasome, indicating that Blm10 might have a positive role as a proteasome regulator when regulation of the proteasome is defective (22Schmidt M. Haas W. Crosas B. Santamaria P.G. Gygi S.P. Walz T. Finley D. The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle.Nat. Struct. Mol. Biol. 2005; 12: 294-303Crossref PubMed Scopus (149) Google Scholar). In addition (or instead) of being an activator of entry rates into the 20S CP chamber, PA200 may be a flusher, aiding exit of peptide products through a widened orifice as has been suggested for 11S-Regs (5Rechsteiner M. Hill C.P. Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors.Trends in Cell Biology. 2005; 15: 27-33Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 7Förster A. Whitby F.G. Hill C.P. The pore of activated 20S proteasomes has an ordered 7-fold symmetric conformation.EMBO J. 2003; 22: 4356-4364Crossref PubMed Scopus (95) Google Scholar). Such a role would be a particularly appealing option for PA200–20S-19S hybrids (because the 19S RP would activate uptake of protein substrates and PA200 would accelerate excretion of peptide products). But PA200 may also be a bona fide activator of proteolysis, facilitating entry of substrates into PA200–20S complexes. The question is what for? Proteolysis of what kind of substrates would be enhanced by a non-ATPase activator? It has been shown that opening of the channel alone can permit 20S CP to proteolyse unstructured proteins, thus gating and substrate translocation through the open channel are not mandated to be ATP-dependent (11Bajorek M. Finley D. Glickman M.H. Proteasome Disassembly and Downregulation Is Correlated with Viability during Stationary Phase.Current Biology. 2003; 13: 1140-1144Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Through its ability to open the channel, could PA200 be involved in proteolysis of stress-induced damaged or miss folded proteins, or possibly of defective unfolded polypeptides leaching off ribosomes (defective ribosomal intermediate products) (38Yewdell J.W. Immunoproteasomes: regulating the regulator.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 9089-9090Crossref PubMed Scopus (45) Google Scholar)? If so, one could imagine that PA200 may be involved in Ub-independent proteolysis. So far, there is no evidence for this. Alternatively (and this is utter speculation), PA200 may yet be involved in more complex (and Ub-dependent) reactions by serving as a docking site for enzymes that feed into the Ub system, possibly forming an ersatz RP with faux lidlike complexes. Finally, Blm10/PA200 seems to be involved in proteasome assembly, although its specific role still remains an open question (39Li X. Kusmierczyk A.R. Wong P. Emili A. Hochstrasser M. beta-Subunit appendages promote 20S proteasome assembly by overcoming an Ump1-dependent checkpoint.EMBO J. 2007; 26: 2339-2349Crossref PubMed Scopus (115) Google Scholar, 40Marques A.J. Glanemann C. Ramos P.C. Dohmen R.J. The C-terminal extension of the beta7 subunit and activator complexes stabilize nascent 20 S proteasomes and promote their maturation.J. Biol. Chem. 2007; 282: 34869-34876Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 41Lehmann A. Jechow K. Enenkel C. Blm10 binds to pre-activated proteasome core particles with open gate conformation.EMBO Rep. 2008; 9: 1237-1243Crossref PubMed Scopus (48) Google Scholar, 42Rosenzweig R. Glickman M.H. Forging a proteasome alpha-ring with dedicated proteasome chaperones.Nat. Struct. Mol. Biol. 2008; 15: 218-220Crossref PubMed Scopus (8) Google Scholar, 43Bedford L. Paine S. Sheppard P.W. Mayer R.J. Roelofs J. Assembly, structure, and function of the 26S proteasome.Trends Cell Biol. 2010; 20: 391-401Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The role of Blm10/PA200 in proteasome maturation is a matter of debate. In an early study, the transition from the nascent to matured 20S CP was actually accelerated in yeast upon deletion of BLM10 (YFL007w; the gene coding for Blm10) pointing to a complicated relationship between Blm10 and proteasomes (30Fehlker M. Wendler P. Lehmann A. Enenkel C. Blm3 is part of nascent proteasomes and is involved in a late stage of nuclear proteasome assembly.EMBO Rep. 2003; 4: 959-963Crossref PubMed Scopus (98) Google Scholar). In other studies, Blm10 was found to promote proteasome maturation. For example, Blm10 was detected in the inactive 15S-proteasome precursor (Fig. 1), which contained the following subunits: all α subunits, precursor forms of β2, β3, β4, and the assembly factors Ump1 and Pba1 and Pba2 (39Li X. Kusmierczyk A.R. Wong P. Emili A. Hochstrasser M. beta-Subunit appendages promote 20S proteasome assembly by overcoming an Ump1-dependent checkpoint.EMBO J. 2007; 26: 2339-2349Crossref PubMed Scopus (115) Google Scholar). Blm10 was also found in the next assembly intermediate containing all 20S subunits except for the β7/Pre4 subunit, which is believed to be the last subunit to incorporate prior to dimerization of the so-called half proteasome (two α7β7 rings coming together) (40Marques A.J. Glanemann C. Ramos P.C. Dohmen R.J. The C-terminal extension of the beta7 subunit and activator complexes stabilize nascent 20 S proteasomes and promote their maturation.J. Biol. Chem. 2007; 282: 34869-34876Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, deletion of the BLM10 gene alone had little effect on 20S CP formation from these half proteasomes, although a combined deletion of BLM10 with a C-terminal truncation in β7/Pre4 led to accumulation of half proteasomes, suggesting that both the C-terminal extension of β7/Pre4 and Blm10 have additive roles in stabilizing 20S CPs. Similarly, a
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