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.     

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.     

26S proteasome regulatory particle mutants have increased oxidative stress tolerance

The 26S proteasome (26SP) is a multi-subunit, multi-catalytic protease that is responsible for most of the cytosolic and nuclear protein turnover. The 26SP is composed of two sub-particles, the 19S regulatory particle (RP) that binds and unfolds protein targets, and the 20S core particle (20SP) that degrades proteins into small peptides. Most 26SP targets are conjugated to a poly-ubiquitin (Ub) chain that serves as a degradation signal. However, some targets, such as oxidized proteins, do not require a poly-Ub tag for proteasomal degradation, and recent studies have shown that the main protease in this Ub-independent pathway is free 20SP. It is currently unknown how the ratio of 26SP- to 20SP-dependent proteolysis is controlled. Here we show that loss of function of the Arabidopsis RP subunits RPT2a, RPN10 and RPN12a leads to decreased 26SP accumulation, resulting in reduced rates of Ub-dependent proteolysis. In contrast, all three RP mutants have increased 20SP levels and thus enhanced Ub-independent protein degradation. As a consequence of this shift in proteolytic activity, mutant seedlings are hypersensitive to stresses that cause protein misfolding, and have increased tolerance to treatments that promote protein oxidation. Taken together, the data show that plant cells increase 20SP-dependent proteolysis when 26SP activity is impaired.     

Regulation of apoptosis by the ubiquitin and proteasome pathway

Regulated proteolysis plays important roles in cell physiology as well as in pathological conditions. In most of the cases, regulated proteolysis is carried out by the ubiquitin- and proteasome-dependent proteolytic system, which is also in charge of the bulk of cytoplasmic proteolysis. However, apoptosis or the process of programmed cell death is regulated by a different proteolytic system, i.e . by caspases, a family of specialized cysteine proteases. Nevertheless, there is plenty of evidence of a crosstalk between the apoptotic pathways and the ubiquitin and proteasome system, whose function in apoptosis appears to be very complex. Proteasome inhibitors induce apoptosis in multiple cell types, while in other they are relatively harmless or even prevent apoptosis induced by other stimuli. Proteasomes degrade specific proteins during apoptosis, but on the other hand some components of the proteasome system are degraded by caspases. The knowledge about the involvement of the ubiquitin- and proteasome-dependent system in apoptosis is already clinically exploited, since proteasome inhibitors are being tested as experimental drugs in the treatment of cancer and other pathological conditions, where manipulation of apoptosis is desirable.     

Ubiquitin-independent proteolytic functions of the proteasome

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.     

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.     

Dissecting beta-ring assembly pathway of the mammalian 20S proteasome

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). Recent studies indicated that proteasome-specific chaperones and beta-subunit appendages assist in the formation of alpha-rings and dimerization of half-proteasomes, but the process involved in the assembly of beta-rings is poorly understood. Here, we clarify the mechanism of beta-ring formation on alpha-rings by characterizing assembly intermediates accumulated in cells depleted of each beta-subunit. Starting from beta2, incorporation of beta-subunits occurs in an orderly manner dependent on the propeptides of beta2 and beta5, and the C-terminal tail of beta2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as beta2 and is required for the structural integrity of early assembly intermediates. We propose a model in which beta-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.     

Recent advances in the structural biology of the 26S proteasome

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.     

Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell‐based high‐throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin–proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell‐based reporters, detection of global ubiquitination status, and proteasome‐mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell‐based IκBα degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG‐132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition.     

Genetic analyses of the Arabidopsis 26S proteasome regulatory particle reveal its importance during light stress and a specific role for the N-Terminus of RPT2 in development.

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.     

Cell-specific inhibition of paramyxovirus maturation by proteasome inhibitors

Effects of proteasome inhibitors on the replication of a paramyxovirus in comparison with the effects on replication of an orthomyxovirus and rhabdovirus were investigated. Treatment of Sendai virus (SeV)-infected LLC-MK2 cells with 50 microM MG132 reduced virus growth to ca. 1/10,000, and treatment with different concentrations of MG132 reduced virus growth in a dose-dependent manner. Released amounts of viral proteins were reduced in correspondence with decrease in infectivity. The inhibition of virus maturation was confirmed by an SeV-like particle formation system. Lactacystin also impaired SeV growth and zLL impaired the growth to a lesser extent, suggesting involvement of proteasomes in the restriction of virus growth. In the presence of MG132, localizations of the M protein and viral F and HN glycoproteins on the cell membrane appeared to be partly dissociated, although the viral glycoproteins were normally transported to the cell surface. These results suggest that an early step of SeV assembly was disturbed by proteasome inhibitors. The relationship of the results with ubiquitin is also discussed. SeV maturation was less susceptible and resistant to MG132 in CV1 cells and A549 cells, respectively, indicating cell specificity of the drug effect. Release of vesicular stomatitis virus also showed high susceptibility to MG132 and release of influenza virus A/WSN/33 was only mildly susceptible to the drug in LLC-MK2 cells. Effects of proteasome inhibitors on virus maturation are thus highly cell-specific and partly virus-specific.     

Substrate selection by the proteasome through initiation regions

Proteins in the cell have to be eliminated once their function is no longer desired or they become damaged. Most regulated protein degradation is achieved by a large enzymatic complex called the proteasome. Many proteasome substrates are targeted for degradation by the covalent attachment of ubiquitin molecules. Ubiquitinated proteins can be bound by the proteasome, but for proteolysis to occur the proteasome needs to find a disordered tail somewhere in the target at which it initiates degradation. The initiation step contributes to the specificity of proteasomal degradation. Here, we review how the proteasome selects initiation sites within its substrates and discuss how the initiation step affects physiological processes.     

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.     

Ubiquitin‐like domains can target to the proteasome but proteolysis requires a disordered region

Ubiquitin and some of its homologues target proteins to the proteasome for degradation. Other ubiquitin‐like domains are involved in cellular processes unrelated to the proteasome, and proteins containing these domains remain stable in the cell. We find that the 10 yeast ubiquitin‐like domains tested bind to the proteasome, and that all 11 identified domains can target proteins for degradation. Their apparent proteasome affinities are not directly related to their stabilities or functions. That is, ubiquitin‐like domains in proteins not part of the ubiquitin proteasome system may bind the proteasome more tightly than domains in proteins that are bona fide components. We propose that proteins with ubiquitin‐like domains have properties other than proteasome binding that confer stability. We show that one of these properties is the absence of accessible disordered regions that allow the proteasome to initiate degradation. In support of this model, we find that Mdy2 is degraded in yeast when a disordered region in the protein becomes exposed and that the attachment of a disordered region to Ubp6 leads to its degradation.     

Functional characterization of the plant ubiquitin regulatory X (UBX) domain-containing protein AtPUX7 in Arabidopsis thaliana

p97/CDC48 is a major AAA-ATPase that acts in many cellular events such as ubiquitin-dependent degradation and membrane fusion. Its specificity depends on a set of adaptor proteins, most of them containing the ubiquitin regulatory X (UBX) domain. Using a differential hybridization system, we isolated a UBX-containing protein that is expressed during the early phase of male gametophyte development in the crop Brassica napus and isolated and characterized its closest Arabidopsis thaliana homolog, AtPUX7. The AtPUX7 gene is expressed broadly in both the sporophyte and gametophyte due to regulation inferred by its first intron. The subcellular localization of AtPUX7 was assigned mainly to the nucleus in both the sporophyte and in pollen, mirroring the AAA-ATPase AtCDC48A localization. Furthermore, AtPUX7 interacts specifically with AtCDC48A in yeast as well as in planta in the nucleus. This interaction was mediated through the AtPUX7 UBX domain, which is located at the protein C-terminus, while an N-terminal UBA domain mediated its interaction with ubiquitin. Consistent with those results, a yeast-three hybrid analysis showed that AtPUX7 can act as a bridge between AtCDC48A and ubiquitin, suggesting a role in targeted protein degradation. It is likely that AtPUX7 acts redundantly with other members of the Arabidopsis PUX family because a null Atpux7-1 mutant does not display obvious developmental defects.     

Targeted disruption of neuronal 19S proteasome subunits induces the formation of ubiquitinated inclusions in the absence of cell death

Proteasome-mediated proteolysis is a major protein degradation mechanism in cells and its dysfunction has been implicated in the pathogenesis of several neurodegenerative diseases, each with the common features of neuronal death and formation of ubiquitinated inclusions found within neurites, the cell body, or nucleus. Previous models of proteasome dysfunction have employed pharmacological inhibition of the catalytic subunits of the 20S proteasome core, or the genetic manipulation of specific subunits resulting in altered proteasome assembly. In this study, we report the use of dominant negative subunits of the 19S regulatory proteasome complex that mediate the recognition of ubiquitinated substrates as well as the removal of the poly-ubiquitin chain. Interestingly, while each mutant subunit-induced inclusion formation, like that seen with pharmacological inhibition of the 20S proteasome, none was able to induce apoptotic death, or trigger activation of macroautophagy, in either dopaminergic cell lines or primary cortical neurons. This finding highlights the dissociation between the mechanisms of neuronal inclusion formation and the induction of cell death, and represents a novel cellular model for Lewy body-like inclusion formation in neurons.     

Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast

The 20S proteasome is made up of four stacked heptameric rings, which in eucaryotes assemble from 14 different but related subunits. The rules governing subunit assembly and placement are not understood. We show that a different kind of proteasome forms in yeast when the Pre9/alpha3 subunit is deleted. Purified pre9Delta proteasomes show a two-fold enrichment for the Pre6/alpha4 subunit, consistent with the presence of an extra copy of Pre6 in each outer ring. Based on disulfide engineering and structure-guided suppressor analyses, Pre6 takes the position normally occupied by Pre9, a substitution that depends on a network of intersubunit salt bridges. When Arabidopsis PAD1/alpha4 is expressed in yeast, it complements not only pre6Delta but also pre6Delta pre9Delta mutants; therefore, the plant alpha4 subunit also can occupy multiple positions in a functional yeast proteasome. Importantly, biogenesis of proteasomes is delayed at an early stage in pre9Delta cells, suggesting an advantage for Pre9 over Pre6 incorporation at the alpha3 position that facilitates correct assembly.