doi: 10.1161/01.RES.0000239412.40685.61
© 2006 American Heart Association, Inc.
Editorials |
The Cardiac 26S Proteasome
Regulating the Regulator
From The Feinstein Institute for Medical Research, Long Island Jewish Medical Center Campus, New Hyde Park; and Department of Medicine, The Albert Einstein College of Medicine, Bronx, NY.
Correspondence to Saul R. Powell, PhD, FAHA, Department of Medicine, Long Island Jewish Medical Center, 270-05 76th Ave, Rm B-387, New Hyde Park, NY 11042. E-mail spowell{at}lij.edu
See related articles, pages 362–371 and 372–380
Key Words: cardiac • ubiquitin–proteasome system • regulation • immunoproteasome • phosphorylation
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Significance of Ubiquitin–Proteasome–Mediated Degradation of Proteins |
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 Top Significance of Ubiquitin... New Regulatory InsightsPerspectivesConcluding StatementReferences |
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The ubiquitin–proteasome system (UPS) is the main pathway for the nonlysosomal degradation of intracellular proteins, representing upwards of 80% of all intracellular proteins. A key component of the UPS is the 26S proteasome, a macromolecular multisubunit complex that has the responsibility of recognizing, unfolding, and the ultimately destroying proteins that have been tagged by polyubiquitin chains generally at the
-NH2 group of an internal substrate lysine residue. This is a multistep process that has been reviewed in detail elsewhere.1,2 What has become clear is that the rapid destruction of these proteins, some with high biological activity, actually represents a form of regulation of cellular processes. The UPS is now recognized as a regulator of the cell cycle and cell division,3 immune response and antigen presentation,4 apoptosis,5 and cell signaling.6,7 Moreover, the UPS plays critical roles in protein quality by removal of damaged, oxidized, and/or misfolded proteins.8,9 Ciechanover et al10 have aptly referred to this process as "biological regulation via destruction." Over the past few years, it has become increasingly clear that this critical regulatory system becomes dysfunctional in certain disease states, such as Alzheimer’s disease, Huntingdon’s disease, and amyotrophic lateral sclerosis.11 With respect to the cardiovascular system, recent studies have suggested that myocardial ischemia,12,13 certain mutant protein–associated cardiomyopathies,14 atherosclerosis,15 and even diabetes16 may be examples of proteasomal dysfunction disorders. Despite all of the evidence supporting the critical role that the UPS plays in these processes, little is know about how the 26S proteasome itself is regulated. The majority of studies have focused on substrate specificity and selectivity, function of the ubiquitin protein ligases or E3s, as a means of regulating activity of the UPS. Specificity can be accounted for by the sheer number of E3s, probably numbering in the thousands, with each recognizing specific motifs on individual substrates. Regulation of the UPS in this manner has been reviewed extensively elsewhere.1,17,18 Very few studies have examined regulation of proteasome activity once a substrate is ubiquitinated.
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New Regulatory Insights |
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TopSignificance of Ubiquitin...New Regulatory InsightsPerspectivesConcluding StatementReferences |
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In this issue of Circulation Research, Ping and colleagues19,20 present companion articles that provide new insights into some of the factors that may regulate substrate selectivity and activity of the murine cardiac 26S proteasome. Both of these studies have used an exhaustive, exceedingly well-executed proteomics approach to characterize the murine cardiac 26S proteasome and describe associating proteins that play a role in regulating activity. The magnitude of this analysis and exhaustive attention to detail prompted one reviewer to quite appropriately refer to these studies as a "tour de force."
In the study by Gomes et al,19 multidimensional chromatography was used to obtain highly purified, functional 20S proteasome complexes and 19S regulatory particles, the 2 main components of the 26S proteasome (reviews on proteasome structure have been published previously1,2). These were subjected to 2D gel electrophoresis and tandem mass spectrometry analyses, which revealed rather surprising heterogeneity in expression of the constitutive ß1, ß2, and ß5 subunits and their so-called inducible immunoforms (ß1i, ß2i, and ß5i). These subunits represent the catalytic sites within the 20S proteasome and are responsible for the caspase-like, tryptic, and chymotryptic activities of the proteasome, respectively, and can be replaced by immunoforms in response to the cytokine, 
-interferon, to form the immunoproteasome.4 This is an extraordinary observation as it suggests a level of complexity heretofore unknown for the cardiac proteasome and also suggested that ongoing processing of cell-surface antigens might be occurring in the heart and that, perhaps, this organ is more immunologically active than previously thought. These investigators then demonstrate the existence of a novel alternate splice isoform of the 19S regulatory particle subunit, Rpn10, the primary ubiquitin recognition site of the 26S proteasome. Whether this splice is specific to murine heart is unclear at this time. Lastly, 3 distinct posttranslational modifications of various 20S proteasome and 19S regulatory particle subunits were characterized, including: N-terminal acetylation, which is thought to play a role in protein stabilization21,22; N-terminal myristoylation, which may target proteins to membranes23; and phosphorylation, which is further examined in the companion study.
The companion study by Zong et al20 is a further examination of the regulatory role of posttranslational modifications of proteasome, in this case, phosphorylation. Using 2D gel electrophoresis, immunoblotting, and tandem mass spectrometry, the first phosphorylation profile for murine cardiac 20S proteasome subunits was developed. Phosphorylation was detected on multiple subunits (
1, 6: serine, threonine, and tyrosine; 2, 3, 6, 7, ß2: serine). Separation and analysis of purified, functional 20S proteasome revealed the presence of the , ß, and c subunits of protein phosphatase 2A (PP2A), and the catalytic subunit of protein kinase A (PKA) residing in close association with the native 20S proteasome complex. Inhibition of PP2A with okadaic acid marginally (20%) enhanced all 3 proteolytic activities of the proteasome to similar degrees, suggesting a nonspecific general effect of phosphatase inhibition. In contrast, incubation of purified 20S proteasome with different amounts of recombinant PKA increased caspase-like (ß1) and chymotryptic (ß5) activities by a maximum of 2-fold, whereas tryptic (ß2) activity was only marginally increased. The specific targeting of 20S proteasome subunits by PKA and PP2A was documented by examination of the phosphorylation profile in the presence of these 2 proteins. PP2A was observed to diminish serine phosphorylation on multiple subunits, and threonine phosphorylation on only the 1 subunit. In contrast, PKA markedly changed the profile and revealed additional subunits subject to phosphorylation. PKA enhanced serine phosphorylation of the 1, 2, 3, and ß2, ß3, and ß7 subunits, whereas threonine phosphorylation was enhanced on the 3, ß3, and ß7 subunits. PKA has previously been shown to copurify with and phosphorylate at least 2 subunits of 26S proteasome isolated from bovine pituitary.24 The identities of these subunits were unclear, but the nomenclature used suggests these to have been part of the 19S regulatory particle. Thus the study by Gomes et al19 is the first to unequivocally show that PKA has specific molecular targets on the murine cardiac 20S proteasome, several of which (1, ß2, ß3, and ß7) are unique to this organ and that phosphorylation is regulatory.
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Perspectives |
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TopSignificance of Ubiquitin...New Regulatory InsightsPerspectivesConcluding StatementReferences |
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The studies by Gomes et al19 and Zong et al20 suggest intriguing possibilities for altering activity and function of the 26S proteasome, including: (1) altering proteasome subunit configuration through directed assembly to change substrate specificity; or (2) changing activity via posttranslational modification, eg, phosphorylation, of proteasome subunits.
Assembly
The catalytic core 20S proteasome is composed of 4 stacked heptameric rings; 2 outer homologous rings, and 2 inner ß rings previously thought to be homologous. Assembly of this structure involves the chaperone-mediated mating of 2 half-proteasomes, each containing one ring and one ß ring.25 Formation of the "immunoproteasome" involves the substitution of the immunoform for one or more of the constitutive catalytic ß subunits, which all evidence suggests to occur during the formation of the half-proteasome.4 To obtain the degree of intraproteasome ß-subunit heterogeneity suggested by Gomes et al19 would require the mating of 2 half-proteasomes with distinctly different configurations (see Figure 1 for example). This heterogeneity could conceivably alter specificity and selectivity of the 26S proteasome for various substrates, as has been suggested for immunoproteasome to account for cell surface antigen diversity.26 Dahlmann et al27 have previously reported the presence of multiple subtypes of 20S proteasome in skeletal muscle that vary with respect to their enzymatic characteristics and substrate cleavage sites. Furthermore, a shift from constitutive to immunoproteasome has been reported in aging muscle,28,29 possibly to compensate for altered protein metabolism. In light of these reports, it is tempting to speculate that one way proteasome activity and function are regulated is via changes in catalytic subunit configuration, or, to put it another way, not all proteasomes are the same. What is not clear is the conditions that would signal for a half-proteasome with one particular configuration to join with another half-proteasome with a totally different configuration; or, for that matter, to incorporate the observed alternate splice isoform of the Rpn10 subunit into the 26S proteasome, which could alter recognition of ubiquitinated substrates. This is a critical question, as the observations of Gomes et al19 suggest the rather intriguing possibility of altering proteasome configuration through either genetic or mutagenic manipulations to direct degradation of specific classes of proteins, which could conceivably have profound effects on cardiac pathophysiologies. Clearly, additional studies are necessary to better define the signals and/or conditions that regulate assembly of the various subtypes of 26S proteasome and whether this process can be altered to achieve some beneficial effect.
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Posttranslational Modification of Proteasome Subunits
Proteasome is known to be phosphorylated on numerous subunits exhibiting diverse domain, species, and tissue variability. Before the study of Zong et al,20 the known phosphorylated subunits on mammalian 20S proteasome included 2, 3, 4, 5, 6, and ß6.30 The studies of Zong et al20 have identified several additional phosphorylated subunits on the 20S proteasome, and it is likely that with continued research more subunits will be identified, particularly on the 19S regulatory particle. With few exceptions, the functional significance of phosphorylation is unknown. Mason et al31 identified the 3 and 7 subunits of 20S proteasome isolated from Rat-1 fibroblasts as being phosphorylated by the associating protein kinase, casein kinase II. Dephosphorylation of these subunits resulted in decreased peptidase activities, suggesting a regulatory role for phosphorylation. Bose et al32 have shown that phosphorylation of the 7 subunit may also play a role in stabilizing the 26S proteasome. There have been even fewer studies of functional significance of phosphorylation of the 19S regulatory particle, even though this component contains a multitude of phosphorylation sites. In this regard, phosphorylation of the ATPase subunit Rpt6 and subsequent interaction with the 2 subunit plays a critical role in attachment of the 19S regulatory particle to the 20S proteasome.33 The critical role of subunit phosphorylation in regulating assembly, function, and activity of 26S proteasome is an area meriting intense scrutiny. However, just as critical is identification of the protein kinases that phosphorylate proteasome, their molecular targets, and effects on function of this critical regulator.
The studies of Zong et al20 unequivocally show that PKA regulates activity of murine cardiac 20S proteasome through direct phosphorylation of multiple subunits some unique to the murine heart. PP2A had a more subtle, generalized effect diminishing phosphorylation of several subunits independent of the action of PKA, suggesting the presence of additional associating protein kinases whose identities are unknown at this time. Perhaps the most intriguing implication of this study is the suggestion that cellular signal transduction pathways regulate function and activity of the 26S proteasome. The UPS is already known to regulate numerous cellular signaling pathways, including NF-
B,34 JNK and JAK-STAT,7 and PKC pathways,35 to name a few. Although it is not clear that the same is true for the PKA pathway, many of these pathways interact through extensive crosstalk networks, suggesting the exciting possibility that signaling pathways that are regulated by proteasome also feedback and regulate proteasome. If this is true, it would indicate a previously unknown complex level of regulation of signal transduction pathways in which the interaction of signaling molecules with proteasome would regulate degradation of these same molecules, which could act to either amplify or dampen a biological signal. In theory, this could represent a means by which a gene product could feedback to regulate its own availability (Figure 2).
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Obviously, many questions remain and much research needs to done. One of the more pressing questions is how does subunit phosphorylation affect proteasome peptidase activity? Does this result in alteration of -subunit configuration such that entrance into the catalytic chamber is more or less efficient or is it an allosteric effect that changes the kinetics at the catalytic site? Could another mechanism be alteration in recognition or unfolding of ubiquitinated proteins by phosphorylation of 19S regulatory particle subunits? Lastly, a critical step would be to develop profiles of the known proteasome subunit protein kinase consensus sites with determination of the effect phosphorylation has on 26S proteasome activity.
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Concluding Statement |
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TopSignificance of Ubiquitin...New Regulatory InsightsPerspectivesConcluding StatementReferences |
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The studies by Gomes et al19 and Zong et al20 have provided new insights into different levels of regulation of the murine cardiac 26S proteasome, suggesting potentially intriguing possibilities for altering function and substrate selectivity. The 26S proteasome is intimately involved in regulation of many cell functions that it accomplishes via a degradative process. Any research increasing current knowledge of processes regulating this critical regulator could provide insights into disease processes. The current cardiac disease research and treatment paradigms focus on activation and upregulation of signaling pathways. The concept that degradation plays just as important a role in regulating these pathways is often overlooked even though at any given point in time, the levels of a signaling molecule is dependent on its formation and degradation. Recognition of this reality could shift this paradigm and have vast implications for treatment of cardiac diseases.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grant R01 HL 68936 and American Heart Association Heritage Affiliate Grant 0455856T.
Disclosures
S.R.P. is the principal investigator of NIH grant R01 HL 68936 and American Heart Association Heritage Affiliate Grant 0455856T.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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1. Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev. 2002; 82: 373–428.
2. Powell SR. The ubiquitin proteasome system in cardiac physiology and pathology. Am J Physiol Heart Circ Physiol. 2006; 291: 1–19.
3. Hershko A. Roles of ubiquitin-mediated proteolysis in cell cycle control. Curr Opin Cell Biol. 1997; 9: 788–799.[CrossRef][Medline] [Order article via Infotrieve]
4. Goldberg AL, Cascio P, Saric T, Rock KL. The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides. Mol Immunol. 2002; 39: 147–164.[CrossRef][Medline] [Order article via Infotrieve]
5. Jesenberger V, Jentsch S. Deadly encounter: ubiquitin meets apoptosis. Nat Rev Mol Cell Biol. 2002; 3: 112–121.[CrossRef][Medline] [Order article via Infotrieve]
6. Desterro JM, Rodriguez MS, Hay RT. Regulation of transcription factors by protein degradation. Cell Mol Life Sci. 2000; 57: 1207–1219.[CrossRef][Medline] [Order article via Infotrieve]
7. Fuchs SY, Fried VA, Ronai Z. Stress-activated kinases regulate protein stability. Oncogene. 1998; 17: 1483–1490.[CrossRef][Medline] [Order article via Infotrieve]
8. Divald A, Powell SR. Proteasome mediates removal of proteins oxidized during myocardial ischemia. Free Radic Biol Med. 2006; 40: 156–164.[CrossRef][Medline] [Order article via Infotrieve]
9. Goldberg AL. Protein degradation and protection against misfolded or damaged proteins. Nature. 2003; 426: 895–899.[CrossRef][Medline] [Order article via Infotrieve]
10. Ciechanover A, Orian A, Schwartz AL. Ubiquitin-mediated proteolysis: biological regulation via destruction. Bioessays. 2000; 22: 442–451.[CrossRef][Medline] [Order article via Infotrieve]
11. Xu C, Bailly-Maitre B, Reed JC. Endoplasmic reticulum stress: cell life and death decisions. J Clin Invest. 2005; 115: 2656–2664.[CrossRef][Medline] [Order article via Infotrieve]
12. Bulteau AL, Lundberg KC, Humphries KM, Sadek HA, Szweda PA, Friguet B, Szweda LI. Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion. J Biol Chem. 2001; 276: 30057–30063.
13. Powell SR, Wang P, Katzeff HL, Shringarpure R, Teoh C, Khaliulin I, Das DK, Schwalb H. Oxidized and ubiquitinated proteins may predict recovery of postischemic cardiac function. Essential role of the proteasome. Antioxid Redox Signal. 2005; 7: 538–535.[CrossRef][Medline] [Order article via Infotrieve]
14. Liu J, Chen Q, Huang W, Horak KM, Zheng H, Mestril R, Wang X. Impairment of the ubiquitin-proteasome system in desminopathy mouse hearts. FASEB J. 2006; 20: 362–364.
15. Herrmann J, Ciechanover A, Lerman LO, Lerman A. The ubiquitin-proteasome system in cardiovascular diseases-a hypothesis extended. Cardiovasc Res. 2004; 61: 11–21.
16. Hayashi T, Faustman DL. Implications of altered apoptosis in diabetes mellitus and autoimmune disease. Apoptosis. 2001; 6: 31–45.[CrossRef][Medline] [Order article via Infotrieve]
17. Cao PR, Kim HJ, Lecker SH. Ubiquitin-protein ligases in muscle wasting. Int J Biochem Cell Biol. 2005; 37: 2088–2097.[CrossRef][Medline] [Order article via Infotrieve]
18. Cardozo T, Pagano M. The SCF ubiquitin ligase: insights into a molecular machine. Nat Rev Mol Cell Biol. 2004; 5: 739–751.[CrossRef][Medline] [Order article via Infotrieve]
19. Gomes AV, Zong C, Edmondson RD, Li X, Stefani E, Zhang J, Jones RC, Thyparambil S, Wang G-W, Qiao X, Bardag-Gorce F, Ping P. Mapping the murine cardiac 26S Proteasome complexes. Circ Res. 2006; 99: 362–371.
20. Zong C, Gomes AV, Drews O, Li X, Young GW, Berhane B, Qiao X, French SW, Bardag-Gorce F, Ping P. Regulation of murine cardiac 20S proteasomes. Role of associating partners. Circ Res. 2006; 99: 372–380.
21. Chen X, Chi Y, Bloecher A, Aebersold R, Clurman BE, Roberts JM. N-acetylation and ubiquitin-independent proteasomal degradation of p21(Cip1). Mol Cell. 2004; 16: 839–847.[CrossRef][Medline] [Order article via Infotrieve]
22. Caron C, Boyault C, Khochbin S. Regulatory cross-talk between lysine acetylation and ubiquitination: role in the control of protein stability. Bioessays. 2005; 27: 408–415.[CrossRef][Medline] [Order article via Infotrieve]
23. Resh MD. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta. 1999; 1451: 1–16.[Medline] [Order article via Infotrieve]
24. Pereira ME, Wilk S. Phosphorylation of the multicatalytic proteinase complex from bovine pituitaries by a copurifying cAMP-dependent protein kinase. Arch Biochem Biophys. 1990; 283: 68–74.[CrossRef][Medline] [Order article via Infotrieve]
25. Hirano Y, Hendil KB, Yashiroda H, Iemura S, Nagane R, Hioki Y, Natsume T, Tanaka K, Murata S. A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes. Nature. 2005; 437: 1381–1385.[CrossRef][Medline] [Order article via Infotrieve]
26. Kingsbury DJ, Griffin TA, Colbert RA. Novel propeptide function in 20 S proteasome assembly influences beta subunit composition. J Biol Chem. 2000; 275: 24156–24162.
27. Dahlmann B, Ruppert T, Kuehn L, Merforth S, Kloetzel PM. Different proteasome subtypes in a single tissue exhibit different enzymatic properties. J Mol Biol. 2000; 303: 643–653.[CrossRef][Medline] [Order article via Infotrieve]
28. Ferrington DA, Husom AD, Thompson LV. Altered proteasome structure, function, and oxidation in aged muscle. FASEB J. 2005; 19: 644–646.
29. Husom AD, Peters EA, Kolling EA, Fugere NA, Thompson LV, Ferrington DA. Altered proteasome function and subunit composition in aged muscle. Arch Biochem Biophys. 2004; 421: 67–76.[CrossRef][Medline] [Order article via Infotrieve]
30. Bose S, Mason GG, Rivett AJ. Phosphorylation of proteasomes in mammalian cells. Mol Biol Rep. 1999; 26: 11–14.[CrossRef][Medline] [Order article via Infotrieve]
31. Mason GG, Hendil KB, Rivett AJ. Phosphorylation of proteasomes in mammalian cells. Identification of two phosphorylated subunits and the effect of phosphorylation on activity. Eur J Biochem. 1996; 238: 453–462.[Medline] [Order article via Infotrieve]
32. Bose S, Stratford FL, Broadfoot KI, Mason GG, Rivett AJ. Phosphorylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by gamma-interferon. Biochem J. 2004; 378: 177–184.[CrossRef][Medline] [Order article via Infotrieve]
33. Satoh K, Sasajima H, Nyoumura KI, Yokosawa H, Sawada H. Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit. Biochemistry. 2001; 40: 314–319.[CrossRef][Medline] [Order article via Infotrieve]
34. Krappmann D, Scheidereit C. A pervasive role of ubiquitin conjugation in activation and termination of IkappaB kinase pathways. EMBO Rep. 2005; 6: 321–326.[CrossRef][Medline] [Order article via Infotrieve]
35. Lu Z, Liu D, Hornia A, Devonish W, Pagano M, Foster DA. Activation of protein kinase C triggers its ubiquitination and degradation. Mol Cell Biol. 1998; 18: 839–845.
36. Gomes AV, Zong C, Edmondson RD, Berhane BT, Wang GW, Le S, Young G, Zhang J, Vondriska TM, Whitelegge JP, Jones RC, Joshua IG, Thyparambil S, Pantaleon D, Qiao J, Loo J, Ping P. The murine cardiac 26S proteasome: an organelle awaiting exploration. Ann N Y Acad Sci. 2005; 1047: 197–207.[CrossRef][Medline] [Order article via Infotrieve]
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