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1.
The 19S regulatory complex (RC) of 26S proteasomes is a 900–1000 kDa particle composed of 18 distinct subunits (S1–S15) ranging in molecular mass from 25 to 110 kDa. This particle confers ATP-dependence and polyubiquitin (polyUb) recognition to the 26S proteasome. The symmetry and homogenous structure of the proteasome contrasts sharply with the remarkable complexity of the RC. Despite the fact that the primary sequences of all the subunits are now known, insight has been gained into the function of only eight subunits. The six ATPases within the RC constitute a subfamily (S4-like ATPases) within the AAA superfamily and we have shown that they form specific pairs in vitro[1]. We have now determined that putative coiled-coils within the variable N-terminal regions of these proteins are likely to function as recognition elements that direct the proper placement of the ATPases within the RC. We have also begun mapping putative interactions between non-ATPase subunits and S4-like ATPases. These studies have allowed us to build a model for the specific arrangement of 9 subunits within the human regulatory complex. This model agrees with recent findings by Glickman et al. [2] who have reported that two subcomplexes, termed the base and the lid, form the RC of budding yeast 26S proteasomes. 相似文献
2.
The 26S proteasome: a dynamic structure 总被引:1,自引:0,他引:1
The proteasomal system consists of a proteolytic core, the 20S proteasome, which associates in ATP-dependent and independent reactions with endogenous regulators providing specific substrate binding sites, chaperone function and regulation of activity to the protease. The best known regulators of the 20S proteasome are the 11S and the 19S complexes. Three subunits of the 20S proteasome and the two subunits of the 11S regulator are induced by -Interferon. However, there are no indications for an influence of -interferon on the subunit composition of the 19S regulator and only a few data exist about the dynamics of this complex. The analysis of 19S regulator subunits from yeast mutants reveals that the ATPases appear to be stringently organized in the 26S complex, while peripheral non-ATPases, such as S5a, might serve as subunits which shuttle substrates to the enzyme. A novel non-ATPase has been cloned, sequenced and identified in a complex besides the 19S regulator, the function of which is presently unknown. The dynamic structure of the 26S proteasome is also characterized by transient associations with components such as the modulator and isopeptidases. Certain viral proteins can also be associated with components of the proteasomal system and alter enzymatic activities. 相似文献
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4.
The Ubiquitin Proteasome System (UPS) was discovered in two steps. Initially, APF-1 (ATP-dependent proteolytic Factor 1) later identified as ubiquitin (Ub), a hitherto known protein of unknown function, was found to covalently modify proteins. This modification led to degradation of the tagged protein by – at that time – an unknown protease. This was followed later by the identification of the 26S proteasome complex which is composed of a previously identified Multi Catalytic Protease (MCP) and an additional regulatory complex, as the protease that degrades Ub-tagged proteins. While Ub conjugation and proteasomal degradation are viewed as a continued process responsible for most of the regulated proteolysis in the cell, the two processes have also independent roles. In parallel and in the years that followed, the hallmark signal that links the substrate to the proteasome was identified as an internal Lys48-based polyUb chain. However, since these initial findings were described, our understanding of both ends of the process (i.e. Ub-conjugation to proteins, and their recognition and degradation), have advanced significantly. This enabled us to start bridging the ends of this continuous process which suffered until lately from limited structural data regarding the 26S proteasomal architecture and the structure and diversity of the Ub chains. These missing pieces are of great importance because the link between ubiquitination and proteasomal processing is subject to numerous regulatory steps and are found to function improperly in several pathologies. Recently, the molecular architecture of the 26S proteasome was resolved in great detail, enabling us to address mechanistic questions regarding the various molecular events that polyubiquitinated (polyUb) substrates undergo during binding and processing by the 26S proteasome. In addition, advancement in analytical and synthetic methods enables us to better understand the structure and diversity of the degradation signal. The review summarizes these recent findings and addresses the extrapolated meanings in light of previous reports. Finally, it addresses some of the still remaining questions to be solved in order to obtain a continuous mechanistic view of the events that a substrate undergoes from its initial ubiquitination to proteasomal degradation. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf. 相似文献
5.
The 26S proteasome is an eukaryotic ATP-dependent, dumbbell-shaped protease complex with a molecular mass of approximately 2000 kDa. It consists of a central 20S proteasome, functioning as a catalytic machine, and two large V-shaped terminal modules, having possible regulatory roles, composed of multiple subunits of 25–110 kDa attached to the central portion in opposite orientations. The primary structures of all the subunits of mammalian and yeast 20S proteasomes have been determined by recombinant DNA techniques, but structural analyses of the regulatory subunits of the 26S proteasome are still in progress. The regulatory subunits are classified into two subgroups, a subgroup of at least 6 ATPases that constitute a unique multi-gene family encoding homologous polypeptides conserved during evolution and a subgroup of approximately 15 non-ATPase subunits, most of which are structurally unrelated to each other. 相似文献
6.
Sucharita Bhattacharyya Jonathan P. Renn Houqing Yu John F. Marko Andreas Matouschek 《Analytical biochemistry》2016
The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein. 相似文献
7.
26S蛋白酶体广泛分布于真核细胞中的胞质和胞核,主要是由20S核心复合物(coreparticle,CP)和19S调节复合物(regulatory particle,RP)组成,它负责细胞大多数蛋白质的降解,在几乎所有生命活动中具有关键的调控作用。26S蛋白酶体的组装是一个非常复杂且高度条理的过程,不同的分子伴侣,如PAC1-4、Ump1、p27、p28和s5b等,参与其中发挥识别及调节作用,以确保高效准确地完成蛋白酶体的组装。本文系统总结分析了20S核心复合物和19S调节复合物的组装过程及调控机制的最近研究进展。 相似文献
8.
Muneera R. Kapadia Jason W. Eng Qun Jiang Detcho A. Stoyanovsky Melina R. Kibbe 《Nitric oxide》2009,20(4):279-288
It is well established that nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation by modulating cell cycle proteins. The 26S proteasome is integral to protein degradation and tightly regulates cell cycle proteins. Therefore, we hypothesized that NO directly inhibits the activity of the 26S proteasome. The three enzymatic activities (chymotrypsin-like, trypsin-like and caspase-like) of the 26S proteasome were examined in VSMC. At baseline, caspase-like activity was approximately 3.5-fold greater than chymotrypsin- and trypsin-like activities. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited all three catalytically active sites in a time- and concentration-dependent manner (P < 0.05). Caspase-like activity was inhibited to a greater degree (77.2% P < 0.05). cGMP and cAMP analogs and inhibitors had no statistically significant effect on basal or NO-mediated inhibition of proteasome activity. Dithiothreitol, a reducing agent, prevented and reversed the NO-mediated inhibition of the 26S proteasome. Nitroso-cysteine analysis following S-nitrosoglutathione exposure revealed that the 20S catalytic core of the 26S proteasome contains 10 cysteines which were S-nitrosylated by NO. Evaluation of 26S proteasome subunit protein expression revealed differential regulation of the α and β subunits in VSMC following exposure to NO. Finally, immunohistochemical analysis of subunit expression revealed distinct intracellular localization of the 26S proteasomal subunits at baseline and confirmed upregulation of distinct subunits following NO exposure. In conclusion, NO reversibly inhibits the catalytic activity of the 26S proteasome through S-nitrosylation and differentially regulates proteasomal subunit expression. This may be one mechanism by which NO exerts its effects on the cell cycle and inhibits cellular proliferation in the vasculature. 相似文献
9.
Kwang-Hee Lee Richard S. Marshall Lucas M. Slivicke Richard D. Vierstra 《Plant signaling & behavior》2012,7(8):973-978
The 26S proteasome subunit RPT2 is a component of the hexameric ring of AAA-ATPases that forms the base of the 19S regulatory particle (RP). This subunit has specific roles in the yeast and mammalian proteasomes by helping promote assembly of the RP with the 20S core protease (CP) and gate the CP to prevent indiscriminate degradation of cytosolic and nuclear proteins. In plants, this subunit plays an important role in diverse processes that include shoot and root apical meristem maintenance, cell size regulation, trichome branching, and stress responses. Recently, we reported that mutants in RPT2 and several other RP subunits have reduced histone levels, suggesting that at least some of the pleiotropic phenotypes observed in these plants result from aberrant nucleosome assembly. Here, we expand our genetic analysis of RPT2 in Arabidopsis to shed additional light on the roles of the N- and C-terminal ends. We also present data showing that plants bearing mutations in RP subunit genes have their seedling phenotypes exacerbated by prolonged light exposure. 相似文献
10.
Ubiquitin-independent proteolytic functions of the proteasome 总被引:10,自引:0,他引:10
The discovery of the 20S proteasome (multicatalytic proteinase complex) was followed by the recognition that this multisubunit macromolecule is the proteolytic core of the 26S proteasome. Most of the research on extralysosomal proteolysis has concentrated on the role of the 26S proteasome in the ubiquitin-dependent proteolytic pathway. However, little attention has been directed toward the possible involvement of the proteasome in ubiquitin-independent proteolysis. In the past few years, many publications have provided evidence that both the 20S proteasome and the 26S proteasome can degrade some proteins in an ubiquitin-independent manner. Furthermore, it is becoming clear that demonstration of ubiquitin-protein conjugates after exposure of cells to proteasome inhibitors does not eliminate the possibility that the same protein can also be degraded by the proteasome without ubiquitination. The possible mechanisms of degradation of an unmodified protein by the 20S proteasome are discussed. These include targeting, protein unfolding, and opening of the gated channel to the catalytic sites. It is reasonable to assume that in the future the number of proteins recognized as substates of the ubiquitin-independent pathway will continue to increase, and that the metabolic significance of this pathway will be clarified. 相似文献
11.
There is growing appreciation for the fundamental role of structural dynamics in the function of macromolecules. In particular, the 26S proteasome, responsible for selective protein degradation in an ATP dependent manner, exhibits dynamic conformational changes that enable substrate processing. Recent cryo-electron microscopy (cryo-EM) work has revealed the conformational dynamics of the 26S proteasome and established the function of the different conformational states. Technological advances such as direct electron detectors and image processing algorithms allowed resolving the structure of the proteasome at atomic resolution. Here we will review those studies and discuss their contribution to our understanding of proteasome function. 相似文献
12.
The 26S proteasome, composed of the 20S core and 19S regulatory complexes, is important for the turnover of polyubiquitinated proteins. Each subunit of the complex plays a special role in proteolytic function, including substrate recruitment, deubiquitination, and structural contribution. To assess the function of some non-essential subunits in the 26S proteasome, we isolated the 26S proteasome from deletion strains of RPN13 and RPN14 using TAP affinity purification. The stability of Gcn4p and the accumulation of ubiquitinated Gcn4p were significantly increased, but the affinity in the recognition of proteasome was decreased. In addition, the subcomplexes of the isolated 26S proteasomes from deletion mutants were less stable than that of the wild type. Taken together, our findings indicate that Rpn13p and Rpn14p are involved in the efficient recognition of 26S proteasome for the proteolysis of ubiquitinated Gcn4p. 相似文献
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14.
Gang Tian Qing Lu Susanne E. Kohalmi Steven J. Rothstein Yuhai Cui 《Plant signaling & behavior》2012,7(11):1415-1419
The 26S proteasome interacts with a number of different proteins, while the TREX-2 complex is an important component of the mRNA export machinery. In animals and yeast, members of the Ubiquitin C-terminal Hydrolase 37 (UCH37) family are found to associate with the 26S proteasome, but this has not been demonstrated in plants. The Arabidopsis UCH1 and UCH2 are orthologous to UCH37. Here, we show that UCH1 and UCH2 interact with the 26S proteasome lid subunits. In addition, the two UCHs also interact with TREX-2 components. Our data suggest that Arabidopsis UCHs may serve as a link between the 26S proteasome lid complex and the TREX-2 complex. 相似文献
15.
Gaps in our knowledge exist regarding the degradation of the tropomyosin-regulated kinase A (TrkA) receptor after addition of neurotrophin, nerve growth factor (NGF). TrkA is rapidly and transiently ubiquitinated upon addition of NGF. Here, we demonstrate that the polyubiquitin tag plays a definitive role in receptor sorting. Treatment of PC12 cells with lactacystin prevented NGF-dependent deubiquitination and degradation of TrkA. However, treatment with methylamine, bafilomycin or leupeptin, did not prevent NGF-dependent deubiquitination but blocked the degradation of TrkA. Employing co-immunoprecipitation, biochemical fractionation and confocal microscopy, the kinetics of receptor trafficking post-internalization was observed to occur as a sequel from endosome/multivesicular body, proteasomes, culminating with degradation in the lysosomes. The trafficking of the polyubiquitin-deficient TrkA receptor mutant K485R was impaired and likewise failed to degrade revealing that the receptor escapes degradation. The interaction of TrkA with proteasomes was confirmed by purification and co-immunoprecipitation. We provide evidence that proteasomal deubiquitinating enzymes trim K63-ubiquitin chains from the TrkA receptor prior to its delivery to lysosomes for degradation. Taken together, our results reveal the existence of a novel proteasome-dependent step in receptor degradation. 相似文献
16.
Enenkel Cordula Lehmann Andrea Kloetzel Peter-Michael 《Molecular biology reports》1999,26(1-2):131-135
26S proteasomes are multisubunit protease complexes that play the central role in the ubiquitin-dependent protein degradation pathway. The proteolytically active core is formed by the 20S proteasome. Regulatory subunits, principally the 19S cap complex, confer the specificity towards ubiquitinated substrates and an ATP-dependence on proteolysis. Green fluorescence protein (GFP)-tagged versions of either an -subunit of the 20S core or an ATPase subunit of the 19S cap complex were functionally incorporated into the protease complex, thus allowing to monitor the subcellular distribution of 26S proteasomes in living yeast. Our localization studies suggest that proteasomal proteolysis mainly occurs at the nuclear envelope (NE)/rough ER. Implications of proteasomal functions at the NE/rough ER are discussed in the context of published work on ER degradation and with regard to possible targeting mechanisms. 相似文献
17.
26S proteasome subunits are O-linked N-acetylglucosamine-modified in Drosophila melanogaster 总被引:2,自引:0,他引:2
Sümegi M Hunyadi-Gulyás E Medzihradszky KF Udvardy A 《Biochemical and biophysical research communications》2003,312(4):1284-1289
The O-linked glycosylation of highly purified Drosophila 26S proteasome has been analyzed by immunological and lectin-binding methods. Five regulatory complex subunits and at least nine catalytic core subunits were recognized by two different monoclonal antibodies specific for O-linked N-acetylglucosamine-modified proteins, and by wheat germ agglutinin, which is specific for the N-acetylglucosamine sugar side-chain. The specificity of these reactions has been proved by competition studies with free N-acetylglucosamine. Three ATPase subunits of the regulatory complex, which are O-glycosylated, have previously been shown [FEBS Lett. 430 (1998) 269] to occur in phosphorylated form as well, indicating that several different post-translational modifications, with distinct regulatory potential, may be present on the same subunit. 相似文献
18.
ATP-dependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation 总被引:1,自引:0,他引:1
Eukaryotic cells target proteins for degradation by the 26S proteasome by attaching a ubiquitin chain. Using a rapid assay, we analyzed the initial binding of ubiquitinated proteins to purified 26S particles as an isolated process at 4°C. Subunits Rpn10 and Rpn13 contribute equally to the high-affinity binding of ubiquitin chains, but in their absence, ubiquitin conjugates bind to another site with 4-fold lower affinity. Conjugate binding is stimulated 2- to 4-fold by binding of ATP or the nonhydrolyzable analog, ATPγS (but not ADP), to the 19S ATPases. Following this initial, reversible association, ubiquitin conjugates at 37°C become more tightly bound through a step that requires ATP hydrolysis and a loosely folded domain on the protein, but appears independent of ubiquitin. Unfolded or loosely folded polypeptides can inhibit this tighter binding. This commitment step precedes substrate deubiquitination and allows for selection of ubiquitinated proteins capable of being unfolded and efficiently degraded. 相似文献
19.
Papapostolou D Coux O Reboud-Ravaux M 《Biochemical and biophysical research communications》2002,295(5):1090-1095
The implication of the released peptides in allosteric effects during protein degradation catalyzed by the proteasome is an important question not completely resolved. We present here data showing modulation of 26S proteasome activities by peptides composed of 5 or 6 natural amino acids that mimic the products generated during protein breakdown. Several of these peptides inhibit the chymotrypsin-like activity of the Xenope 26S proteasome whereas its trypsin-like activity is enhanced. The basic peptides produced competitive inhibition of the chymotrypsin-like activity and the acidic peptides, parabolic inhibition involving two different binding sites. Our results are in agreement with a model involving hypothetical non-catalytic sites interacting with effectors to modulate the peptidase activities of the proteasome. They also suggest that allosteric effects may occur in the proteasome during protein degradation. 相似文献