ATP hydrolysis-dependent disassembly of the 26S proteasome is part of the catalytic cycle

ATP hydrolysis is required for degradation of polyubiquitinated proteins by the 26S proteasome but is thought to play no role in proteasomal stability during the catalytic cycle. In contrast to this view, we report that ATP hydrolysis triggers rapid dissociation of the 19S regulatory particles from immunopurified 26S complexes in a manner coincident with release of the bulk of proteasome-interacting proteins. Strikingly, this mechanism leads to quantitative disassembly of the 19S into subcomplexes and free Rpn10, the polyubiquitin binding subunit. Biochemical reconstitution with purified Sic1, a prototype substrate of the Cdc34/SCF ubiquitin ligase, suggests that substrate degradation is essential for triggering the ATP hydrolysis-dependent dissociation and disassembly of the 19S and that this mechanism leads to release of degradation products. This is the first demonstration that a controlled dissociation of the 19S regulatory particles from the 26S proteasome is part of the mechanism of protein degradation.     

Differential roles of the COOH termini of AAA subunits of PA700 (19 S regulator) in asymmetric assembly and activation of the 26 S proteasome

The 26 S proteasome is an energy-dependent protease that degrades proteins modified with polyubiquitin chains. It is assembled from two multi-protein subcomplexes: a protease (20 S proteasome) and an ATPase regulatory complex (PA700 or 19 S regulatory particle) that contains six different AAA family subunits (Rpt1 to -6). Here we show that binding of PA700 to the 20 S proteasome is mediated by the COOH termini of two (Rpt2 and Rpt5) of the six Rpt subunits that constitute the interaction surface between the subcomplexes. COOH-terminal peptides of either Rpt2 or Rpt5 bind to the 20 S proteasome and activate hydrolysis of short peptide substrates. Simultaneous binding of both COOH-terminal peptides had additive effects on peptide substrate hydrolysis, suggesting that they bind to distinct sites on the proteasome. In contrast, only the Rpt5 peptide activated hydrolysis of protein substrates. Nevertheless, the COOH-terminal peptide of Rpt2 greatly enhanced this effect, suggesting that proteasome activation is a multistate process. Rpt2 and Rpt5 COOH-terminal peptides cross-linked to different but specific subunits of the 20 S proteasome. These results reveal critical roles of COOH termini of Rpt subunits of PA700 in the assembly and activation of eukaryotic 26 S proteasome. Moreover, they support a model in which Rpt subunits bind to dedicated sites on the proteasome and play specific, nonequivalent roles in the asymmetric assembly and activation of the 26 S proteasome.     

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.     

ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome

The 26S proteasome degrades polyubiquitinated proteins by an energy-dependent mechanism. Here we define multiple roles for ATP in 26S proteasome function. ATP binding is necessary and sufficient for assembly of 26S proteasome from 20S proteasome and PA700/19S subcomplexes and for proteasome activation. Proteasome assembly and activation may require distinct ATP binding events. The 26S proteasome degrades nonubiquitylated, unstructured proteins without ATP hydrolysis, indicating that substrate translocation per se does not require the energy of hydrolysis. Nonubiquitylated folded proteins and certain polyubiquitylated folded proteins were refractory to proteolysis. The latter were deubiquitylated by an ATP-independent mechanism. Other folded as well as unstructured polyubiquitylated proteins required ATP hydrolysis for proteolysis and deubiquitylation. Thus, ATP hydrolysis is not used solely for substrate unfolding. These results indicate that 26S proteasome-catalyzed degradation of polyubiquitylated proteins involves mechanistic coupling of several processes and that such coupling imposes an energy requirement not apparent for any isolated process.     

Regulatory subunit interactions of the 26S proteasome, a complex problem

The 26S proteasome is the major non-lysosomal protease in eukaryotic cells. This multimeric enzyme is the integral component of the ubiquitin-mediated substrate degradation pathway. It consists of two subcomplexes, the 20S proteasome, which forms the proteolytic core, and the 19S regulator (or PA700), which confers ATP dependency and ubiquitinated substrate specificity on the enzyme. Recent biochemical and genetic studies have revealed many of the interactions between the 17 regulatory subunits, yielding an approximation of the 19S complex topology. Inspection of interactions of regulatory subunits with non-subunit proteins reveals patterns that suggest these interactions play a role in 26S proteasome regulation and localization.     

Rpn13p and Rpn14p are involved in the recognition of ubiquitinated Gcn4p by the 26S proteasome

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.     

The 26S proteasome: ubiquitin-mediated proteolysis in the tunnel

The 26S proteasome is a self-compartmentalizing protease responsible for the degradation of intracellular proteins. This giant intracellular protease is formed by several subunits arranged into two 19S polar caps-where protein recognition and ATP-dependent unfolding occur-flanking a 20S central barrel-shaped structure with an inner proteolytic chamber. Proteins targeted to the 26S proteasome are conjugated with a polyubiquitin chain by an enzymatic cascade before delivery to the 26S proteasome for degradation into oligopeptides. As a self-compartmentalizing protease, the 26S proteasome circumvents proteins not destined for degradation and can be deployed to the cytoplasmic and nuclear compartments. The 26S proteasome is a representative of emerging group of giant proteases, including tricorn protease, multicorn protease, and TPPII (tripeptidyl peptidase II).     

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.     

Degradation of Some Polyubiquitinated Proteins Requires an Intrinsic Proteasomal Binding Element in the Substrates

Lysine 48-linked polyubiquitin chains usually target proteins for 26 S proteasomal degradation; however, this modification is not a warrant for destruction. Here, we found that efficient degradation of a physiological substrate UbcH10 requires not only an exogenous polyubiquitin chain modification but also its unstructured N-terminal region. Interestingly, the unstructured N-terminal region of UbcH10 directly binds the 19 S regulatory complex of the 26 S proteasome, and it mediates the initiation of substrate translocation. To promote ubiquitin- dependent degradation of the folded domains of UbcH10, its N-terminal region can be displaced by exogenous proteasomal binding elements. Moreover, the unstructured N-terminal region can initiate substrate translocation even when UbcH10 is artificially cyclized without a free terminus. Polyubiquitinated circular UbcH10 is completely degraded by the 26 S proteasome. Accordingly, we propose that degradation of some polyubiquitinated proteins requires two binding interactions: a polyubiquitin chain and an intrinsic proteasomal binding element in the substrates (likely an unstructured region); moreover, the intrinsic proteasomal binding element initiates substrate translocation regardless of its location in the substrates.     

Recognition of misfolding proteins by PA700, the regulatory subcomplex of the 26 S proteasome

The 26 S proteasome is a large protease complex that catalyzes the degradation of both native and misfolded proteins. These proteins are known to interact with PA700, the regulatory subcomplex of the 26 S proteasome, via a covalently attached polyubiquitin chain. Here we provide evidence for an additional ubiquitin-independent mode of substrate recognition by PA700. PA700 prevents the aggregation of three incompletely folded, nonubiquitinated substrates: the DeltaF-508 mutant form of cystic fibrosis transmembrane regulator, nucleotide binding domain 1, insulin B chain, and citrate synthase. This function does not require ATP hydrolysis. The stoichiometry required for this function, the effect of PA700 on the lag phase of aggregation, and the temporal specificity of PA700 in this process all indicate that PA700 interacts with a subpopulation of non-native conformations that is either particularly aggregation-prone or nucleates misassociation reactions. The inhibition of off-pathway self-association reactions is also reflected in the ability of PA700 to promote refolding of citrate synthase. These results provide evidence that, in addition to binding polyubiquitin chains, PA700 contains a site(s) that recognizes and interacts with misfolded or partially denatured polypeptides. This feature supplies an additional level of substrate specificity to the 26 S proteasome and a means by which substrates are maintained in a soluble state until refolding or degradation is complete.     

Complementary roles for Rpn11 and Ubp6 in deubiquitination and proteolysis by the proteasome

Substrates destined for degradation by the 26 S proteasome are labeled with polyubiquitin chains. These chains can be dismantled by deubiquitinating enzymes (DUBs). A number of reports have identified different DUBs that can hydrolyze ubiquitin from substrates bound to the proteasome. We measured deubiquitination by both isolated lid and base-core particle subcomplexes, suggesting that at least two different DUBs are intrinsic components of 26 S proteasome holoenzymes. In agreement, we find that highly purified proteasomes contain both Rpn11 and Ubp6, situated within the lid and base subcomplexes, respectively. To study their relative contributions, we purified proteasomes from a mutant in the putative metalloprotease domain of Rpn11 and from a ubp6 null. Interestingly, in both preparations we observed slower deubiquitination rates, suggesting that Rpn11 and Ubp6 serve complementary roles. In accord, the double mutant is synthetically lethal. In contrast to WT proteasomes, proteasomes lacking the lid subcomplex or those purified from the rpn11 mutant are less sensitive to metal chelators, supporting the prediction that Rpn11 may be a metalloprotein. Treatment of proteasomes with ubiquitin-aldehyde or with cysteine modifiers also inhibited deubiquitination but simultaneously promoted degradation of a monoubiquitinated substrate along with the ubiquitin tag. Degradation is unique to 26 S proteasome holoenzymes; we could not detect degradation of a ubiquitinated protein by "lidless" proteasomes, although they were competent for deubiquitination. The fascinating observation that a single ubiquitin moiety is sufficient for targeting an otherwise stable substrate to proteasomes exposes how rapid deubiquitination of poorly ubiquitinated substrates may counteract degradation.     

Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae

The 26 S proteasome, which catalyzes degradation of polyubiquitinated proteins, is composed of the 20 S proteasome and the 19 S regulatory particle (RP). The RP is composed of the lid and base subcomplexes and regulates the catalytic activity of the 20 S proteasome. In this study, we carried out affinity purification of the lid and base subcomplexes from the tagged strains of Saccharomyces cerevisiae, and we found that the lid contains a small molecular mass protein, Sem1. The Sem1 protein binds with the 26 S proteasome isolated from a mutant with deletion of SEM1 but not with the 26 S proteasome from the wild type. The lid lacking Sem1 is unstable at a high salt concentration. The 19 S RP was immunoprecipitated together with Sem1 by immunoprecipitation using hemagglutinin epitope-tagged Sem1 as bait. Degradation of polyubiquitinated proteins in vivo or in vitro is impaired in the Sem1-deficient 26 S proteasome. In addition, genetic interaction between SEM1 and RPN10 was detected. The human Sem1 homologue hDSS1 was found to be a functional homologue of Sem1 and capable of interacting with the human 26 S proteasome. The results suggest that Sem1, possibly hDSS1, is a novel subunit of the 26 S proteasome and plays a role in ubiquitin-dependent proteolysis.     

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.     

Inherent Asymmetry in the 26S Proteasome Is Defined by the Ubiquitin Receptor RPN13

The 26S double-capped proteasome is assembled in a hierarchic event that is orchestrated by dedicated set of chaperons. To date, all stoichiometric subunits are considered to be present in equal ratios, thus providing symmetry to the double-capped complex. Here, we show that although the vast majority (if not all) of the double-capped 26S proteasomes, both 19S complexes, contain the ubiquitin receptor Rpn10/S5a, only one of these 19S particles contains the additional ubiquitin receptor Rpn13, thereby defining asymmetry in the 26S proteasome. These results were validated in yeast and mammals, utilizing biochemical and unbiased AQUA-MS methodologies. Thus, the double-capped 26S proteasomes are asymmetric in their polyubiquitin binding capacity. Our data point to a potential new role for ubiquitin receptors as directionality factors that may participate in the prevention of simultaneous substrates translocation into the 20S from both 19S caps.     

Defining the geometry of the two-component proteasome degron

The eukaryotic 26S proteasome controls cellular processes by degrading specific regulatory proteins. Most proteins are targeted for degradation by a signal or degron that consists of two parts: a proteasome-binding tag, typically covalently attached polyubiquitin chains, and an unstructured region that serves as the initiation region for proteasomal proteolysis. Here we have characterized how the arrangement of the two degron parts in a protein affects degradation. We found that a substrate is degraded efficiently only when its initiation region is of a certain minimal length and is appropriately separated in space from the proteasome-binding tag. Regions that are located too close or too far from the proteasome-binding tag cannot access the proteasome and induce degradation. These spacing requirements are different for a polyubiquitin chain and a ubiquitin-like domain. Thus, the arrangement and location of the proteasome initiation region affect a protein's fate and are important in selecting proteins for proteasome-mediated degradation.     

Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry

Prion diseases are associated with the conversion of cellular prion protein (PrP(C)) to toxic β-sheet isoforms (PrP(Sc)), which are reported to inhibit the ubiquitin-proteasome system (UPS). Accordingly, UPS substrates accumulate in prion-infected mouse brains, suggesting impairment of the 26S proteasome. A direct interaction between its 20S core particle and PrP isoforms was demonstrated by immunoprecipitation. β-PrP aggregates associated with the 20S particle, but did not impede binding of the PA26 complex, suggesting that the aggregates do not bind to its ends. Aggregated β-PrP reduced the 20S proteasome's basal peptidase activity, and the enhanced activity induced by C-terminal peptides from the 19S ATPases or by the 19S regulator itself, including when stimulated by polyubiquitin conjugates. However, the 20S proteasome was not inhibited when the gate in the α-ring was open due to a truncation mutation or by association with PA26/PA28. These PrP aggregates inhibit by stabilising the closed conformation of the substrate entry channel. A similar inhibition of substrate entry into the proteasome may occur in other neurodegenerative diseases where misfolded β-sheet-rich proteins accumulate.     

Lysine 63‐linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome

Recruitment of substrates to the 26S proteasome usually requires covalent attachment of the Lys48‐linked polyubiquitin chain. In contrast, modifications with the Lys63‐linked polyubiquitin chain and/or monomeric ubiquitin are generally thought to function in proteasome‐independent cellular processes. Nevertheless, the ubiquitin chain‐type specificity for the proteasomal targeting is still poorly understood, especially in vivo. Using mass spectrometry, we found that Rsp5, a ubiquitin‐ligase in budding yeast, catalyzes the formation of Lys63‐linked ubiquitin chains in vitro. Interestingly, the 26S proteasome degraded well the Lys63‐linked ubiquitinated substrate in vitro. To examine whether Lys63‐linked ubiquitination serves in degradation in vivo, we investigated the ubiquitination of Mga2‐p120, a substrate of Rsp5. The polyubiquitinated p120 contained relatively high levels of Lys63‐linkages, and the Lys63‐linked chains were sufficient for the proteasome‐binding and subsequent p120‐processing. In addition, Lys63‐linked chains as well as Lys48‐linked chains were detected in the 26S proteasome‐bound polyubiquitinated proteins. These results raise the possibility that Lys63‐linked ubiquitin chain also serves as a targeting signal for the 26S proteaseome in vivo.     

Lead discovery and chemical biology approaches targeting the ubiquitin proteasome system

Protein degradation is critical for proteostasis, and the addition of polyubiquitin chains to a substrate is necessary for its recognition by the 26S proteasome. Therapeutic intervention in the ubiquitin proteasome system has implications ranging from cancer to neurodegeneration. Novel screening methods and chemical biology tools for targeting E1-activating, E2-conjugating and deubiquitinating enzymes will be discussed in this review. Approaches for targeting E3 ligase-substrate interactions as well as the proteasome will also be covered, with a focus on recently described approaches.     

The bipartite nuclear localization sequence of Rpn2 is required for nuclear import of proteasomal base complexes via karyopherin alphabeta and proteasome functions

26 S proteasomes fulfill final steps in the ubiquitin-dependent degradation pathway by recognizing and hydrolyzing ubiquitylated proteins. As the 26 S proteasome mainly localizes to the nucleus in yeast, we addressed the question how this 2-MDa multisubunit complex is imported into the nucleus. 26 S proteasomes consist of a 20 S proteolytically active core and 19 S regulatory particles, the latter composed of two subcomplexes, namely the base and lid complexes. We have shown that 20 S core particles are translocated into the nucleus as inactive precursor complexes via the classic karyopherin alphabeta import pathway. Here, we provide evidence that nuclear import of base and lid complexes also depends on karyopherin alphabeta. Potential classic nuclear localization sequences (NLSs) of base subunits were analyzed. Rpn2 and Rpt2, a non-ATPase subunit and an ATPase subunit of the base complex, harbor functional NLSs. The Rpt2 NLS deletion yielded wild type localization. However, the deletion of the Rpn2 NLS resulted in improper nuclear proteasome localization and impaired proteasome function. Our data support the model by which nuclear 26 S proteasomes are assembled from subcomplexes imported by karyopherin alphabeta.     

PiZ Mouse Liver Accumulates Polyubiquitin Conjugates That Associate with Catalytically Active 26S Proteasomes

Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome, possibly by inducing oxidative stress-mediated modifications that compromise substrate delivery to proteolytic core.