The complexity of recognition of ubiquitinated substrates by the 26S proteasome

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.     

The 26S proteasome: subunits and functions

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.     

The 26S proteasome: a dynamic structure

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.     

A New Inhibitor of the Chymotrypsin-Like Activity of the Multicatalytic Proteinase Complex (20S Proteasome) Induces Accumulation of Ubiquitin-Protein Conjugates in a Neuronal Cell

Abstract: Exposure of HT4 cells (a mouse neuronal cell line) to a new potent permeable peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (MPC) causes accumulation of ubiquitinylated proteins. In contrast, inhibition of calpain or treatment with a lysosomotropic agent failed to produce detectable ubiquitin-protein conjugates. The appearance of such conjugates is not a nonspecific phenomenon because incubation with the peptidyl alcohol analogue of the inhibitor does not produce accumulation of ubiquitinylated proteins. The MPC inhibitor may therefore be a useful tool for identification and study of physiological pathways involving MPC. Furthermore, the inhibitor may help develop a model for the study of neurodegeneration where accumulation of ubiquitin-protein conjugates is commonly detected in abnormal brain inclusions.     

Evidence that the Arabidopsis Ubiquitin C-terminal Hydrolases 1 and 2 associate with the 26S proteasome and the TREX-2 complex

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.     

Assembly of the regulatory complex of the 26S proteasome

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.     

泛素/26S蛋白酶体途径及其在植物生长发育中的功能

泛素/26S蛋白酶体途径是一种蛋白高效降解途径,主要负责真核细胞内蛋白的选择性降解.泛素分子主要通过泛素活化酶E1、泛素结合酶E2和泛素-蛋白连接酶E3将靶蛋白泛素化,泛素化的蛋白最后被26S蛋白酶体识别和降解.本文介绍了泛素/26S蛋白体介导的特异性蛋白质降解途经,并对其在植物激素信号、光形态建成、植物衰老、自交不亲和反应、细胞周期调控、花的发育、生物钟节律和非生物胁迫响应中的功能最新研究进展进行了综述.     

Effects of nucleotides on assembly of the 26S proteasome and degradation of ubiquitin conjugates

We have investigated three aspects of nucleotide usage by the 26S proteasome and its regulatory complex (RC). Both particles hydrolyze the four major ribonucleotides, but ATP and CTP have substantially lower K _s for hydrolysis than do GTP and UTP. The K _ for ATP hydrolysis is 15 m for the 26S proteasome and 30 m for the regulatory complex. Formation of the 26S proteasome from the RC and the 20S proteasome requires about 5 m ATP. Although measurable degradation of Ubiquitin(Ub)-lysozyme conjugates occurs in the presence of CTP, GTP, and UTP, the best nucleotide for Ub-conjugate degradation by the 26S proteasome is ATP, with an estimated K _ of 12 m. In summary, our studies show that micromolar concentrations of ATP are sufficient for several 26S proteasome activities.     

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

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.     

Changes in Proteasome Activity and Subunit Composition during Postnatal Development of Rat

The dynamics of the activities of 26S and 20S proteasomes in the rat liver and spleen have been studied during postnatal development from 1 to 90 days. The activities of proteasome forms both in spleen and in liver increased in adult animals as compared to one day rats. The activities of both proteasome forms in the liver did not differ significantly from those in the spleen at all stages of postnatal development. Using Western blot with monoclonal antibodies to Rpt6 subunit, we confirmed the presence of 26S proteasome in both organs at all stages of postnatal development. Studies with polyclonal antibodies to β1i (LMP2) subunit showed the appearance of the immune subunit in the spleen by day 9 and in the liver only by day 23 of postnatal development. This result suggests the earlier formation of the spleen as an organ with immune functions.__________Translated from Ontogenez, Vol. 36, No. 3, 2005, pp. 205–210.Original Russian Text Copyright © 2005 by Abramova, Astakhova, Sharova.     

Primary structural features of the 20S proteasome subunits of rice (Oryza sativa)

The 20S proteasome is the proteolytic complex that is involved in removing abnormal proteins, and it also has other diverse biological functions. Its structure comprises 28 subunits arranged in four rings of seven subunits, and exists as a hollow cylinder. The two outer rings and two inner rings form an 7β7β77 structure, and each subunit, and β, exists as seven different types, thus giving 14 kinds of subunits. In this study, we report the primary structures of the 14 proteasomal subunit subfamilies in rice (Oryza sativa), representing the first set for all of the subunits from monocots. Amino acid sequence homology within the rice family (-type: 28.9–42.1%; β-type: 17.2–31.9%) were lower than those between rice subunits and corresponding orthologs from Arabidopsis and yeast (-type: 49.2–94.5%; β-type: 34.8–87.7%). Structural features observed in eukaryotic proteasome subunits, i.e., - or β-type signature at the N-termini, Thr active sites in β1, β2 and β5 subunits, and nuclear localization signal-like sequences in some -type subunits, were shown to be conserved in rice.     

The ubiquitin-interacting motif of 26S proteasome subunit S5a induces A549 lung cancer cell death

The subunit S5a is a key component for the recruitment of ubiquitinated substrates to the 26S proteasome. When the full-length S5a, the N-terminal half of S5a (S5aN) containing the von Willebrand A (vWA) domain, and the C-terminal half of S5a (S5aC) containing two ubiquitin(Ub)-interacting motifs (UIMs) were ectopically expressed in HEK293 cells, Ub-conjugates accumulated most prominently in S5aC-expressing cells. In addition, S5aC induced A549 lung cancer cell death but not non-cancer BEAS-2B cell death. Similar effects were observed using only S5a-UIMs. Our data therefore suggest that S5a-UIMs can be used as upstream inhibitors of the proteasome pathway.     

Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome

Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin‐proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo‐electron microscopy and X‐ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the β‐subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven α‐subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.     

Novel internally quenched substrate of the trypsin-like subunit of 20S eukaryotic proteasome

This article describes the synthesis, using combinatorial chemistry, of internally quenched substrates of the trypsin-like subunit of human 20S proteasome. Such substrates were optimized in both the nonprime and prime regions of the peptide chain. Two were selected as the most susceptible for proteasomal proteolysis with excellent kinetic parameters: (i) ABZ-Val-Val-Ser-Arg-Ser-Leu-Gly-Tyr(3-NO₂)-NH₂ (k_cat/K_M = 934,000 M⁻¹ s⁻¹) and (ii) ABZ-Val-Val-Ser-GNF-Ala-Met-Gly-Tyr(3-NO₂)-NH₂ (k_cat/K_M = 1,980,000 M⁻¹ s⁻¹). Both compounds were efficiently hydrolyzed by the 20S proteasome at picomolar concentrations, demonstrating significant selectivity over other proteasome entities.     

Linkage between the proteasome pathway and neurodegenerative diseases and aging

During aging, the production of free radicals increases. This can result in damage to protein, the accumulation of which is characteristic of the aging process. This questions the efficacy of proteolytic systems. Among these systems, the proteasome and the adenosine triphosphate-ubiquitin-dependent pathway have been shown to play an important role in the elimination of abnormal proteins. There are two major steps in the ubiquitin-proteasome pathway: the conjugation of a polyubiquitin degradation signal to the substrate and the subsequent degradation of the tagged protein by the 26S proteasome. The 26S proteasome is build-up from the 20S proteasome, which is a cylinder-shaped multimeric complex, and two additional 19S complexes. The 20S proteasome can also bind to 11S regulator and is then implicated in antigen presentation. These regulators confer a high adaptability on proteasome. With advancing age, predisposition to neurodegenerative diseases increases. These diseases are also characterized by protein aggregation. Several findings such as the presence of ubiquinated proteins, usually broken down by proteasomes, and genetic anomalies involving the ubiquitinproteasome system (parkin, UCH-L1) suggest a link between the ubiquitin-proteasome pathway and the genesis of these diseases.     

Proteolysis is depressed during torpor in hibernators at the level of the 20S core protease

Protein synthesis is depressed during mammalian hibernation in concordance with metabolic demands. In the absence of significant protein synthesis, continued proteolysis would rapidly deplete protein pools. Since ubiquitin-dependent proteolysis is implicated in the turnover of most regulatory proteins, we examined the fate of this system during hibernation. Ubiquitin-dependent proteolysis consists of two major steps: (1) the tagging of a protein substrate by ubiquitin and (2) the protein substrates subsequent degradation by the 26S proteasome. An earlier study revealed a two to threefold elevation of ubiquitin conjugate concentrations during hibernation: an unexpected result that seemingly would suggest increased proteolytic activity. A more likely explanation for these data would be that proteolysis per se was depressed and that the increased levels of ubiquitylated proteins reflect an inability to degrade tagged proteins. We employed an assay based on the cleavage of fluorogenic substrates to address the well characterized proteolytic activities of the proteasome. All activities show little to no activity at temperatures associated with deep torpor. Coordinated depression of proteolytic activities by low temperature supports the hypothesis that the increased levels of ubiquitylated proteins during hibernation is explained by a net accumulation due to an inability to degrade the tagged proteins.