Modeling the in vitro 20S proteasome activity: the effect of PA28-alphabeta and of the sequence and length of polypeptides on the degradation kinetics

Proteasomes are fundamental for the degradation of intracellular proteins, having a key role in several important metabolic and signaling pathways, in the cell cycle and in antigen presentation. In vitro proteasomal digestion assays are widely used in molecular biology and immunology. We developed a model, ProteaMAlg (proteasome modeling algorithm) that describes the kinetics of specific protein fragments generated by 20S proteasomes in different conditions, once the substrate cleavage strengths are provided. ProteaMAlg was tested on a variety of data available in the literature as well as on new degradation experiments performed with polypeptides of different sequences and lengths. The comparison between in vitro and in silico experiments was used to quantify the effect on degradation of the sequence and the length of target polypeptides, of the presence of regulatory molecules such as PA28-αβ, and of the type of 20S proteasome (constitutive- or immunoproteasome). The model showed that the effect of the PA28 regulatory subunit results in a modification of the gating functions of the proteasome core particle. Immunoproteasome digestion experiments suggested that this form of proteasome, which is involved in generating MHC-class I epitopes, presents modified cleavage and gating activities. Our analysis improves the current understanding of the kinetics of proteasome functioning, and provides a tool to quantify and predict the effect of key parameters during in vitro digestion. ProteaMAlg is publicly available on the web (http://www.proteamalg.com).     

Deubiquitination by proteasome is coordinated with substrate translocation for proteolysis in vivo

The 26S proteasome mediates degradation of protein substrates labeled with polyUb chains. After recognition by the 19S proteasome regulatory complex, polyUb chains are disassembled and substrates are processed in the 20S core of proteasome. However, the exact relationship of degradation-associated deubiquitination to substrate processing remains unclear. Here, using Ub-based tagging strategies, we provided evidence that removable polyUb chains serve as the signal for proteolytic processing of ubiquitinated substrates. We showed that inhibition of the proteasome by proteasome inhibitor MG132 results in trapping of the substrate in the proteasome. Such a trapping allows proteasomal cleavage of attached non-removable Ub mutant (UbV75,76), which is otherwise a "difficult" deubiquitination substrate. Characterization of deubiquitination and degradation intermediates, generated due to incomplete proteolytic inhibition, revealed changes in proteolytic cleavage sites, within the Gal4-VP16 model substrate, suggesting that the copy number of attached UbV75,76 affects substrate processing. Conversion of lysine48 to arginine48 in UbV75,76 did not have significant effect on in vivo polyubiquitination of multiple Ub-fused substrates, but considerably reduced proteolytic intermediates. Taken together, the results support a model in which deubiquitination process is a crucial event for proteolysis of ubiquitinated substrates and such an event is coordinated with substrate translocation.     

Feeding the machine: mechanisms of proteasome-catalyzed degradation of ubiquitinated proteins

The proteasome plays a role in a myriad of intracellular processes from cell-cycle control to antigen presentation. Central to these processes is the targeting of selected proteins for proteasomal degradation via their conjugation to ubiquitin. The mechanisms by which the ubiquitin-dependent proteasomal proteolysis occurs can be divided into four steps: first, substrate protein recognition by its cognate E3 ubiquitin ligase; second, polyubiquitinated protein substrate recruitment to the proteasome; third, protein substrate deubiquitination; and four, proteolytic chamber pore opening/substrate translocation followed by proteolysis. Recent advances include the identification of novel E3 ubiquitin ligase recognition determinants, a new isopeptidase activity, and a better understanding of how the proteasome's axial channels are gated.     

Degradation of hypochlorite-damaged glucose-6-phosphate dehydrogenase by the 20S proteasome.

Glucose-6-phosphate dehydrogenase (G6PD) was treated with various concentrations of hypochlorite, which is produced by myeloperoxidase and is one of the most important oxidants during inflammatory processes. Inhibition of enzymatic activity, protein fragmentation, and proteolytic susceptibility toward the isolated 20S proteasome of G6PD were investigated. With rising hypochlorite concentrations, an increased proteasomal degradation of G6PD was measured. This occurred at higher hypochlorite concentrations than G6PD inactivation and at lower levels than G6PD fragmentation. The proteolytic activities of the 20S proteasome itself was determined by degradation of oxidized model proteins and cleavage of the synthetic proteasome substrate suc-LLVY-MCA. Proteasome activities remained intact at hypochlorite concentrations in which G6PD is maximally susceptible to proteasomal degradation. Only higher hypochlorite concentrations could decrease the proteolytic activities of the proteasome, which was accompanied by disintegration and fragmentation of the proteasome and proteasome subunits. Therefore, we conclude that the 20S proteasome can degrade proteins moderately damaged by hypochlorite and could contribute to an increased protein turnover in cells exposed to inflammatory stress.     

Sequence- and species-dependence of proteasomal processivity

The proteasome is the degradation machine at the center of the ubiquitin-proteasome system and controls the concentrations of many proteins in eukaryotes. It is highly processive so that substrates are degraded completely into small peptides, avoiding the formation of potentially toxic fragments. Nonetheless, some proteins are incompletely degraded, indicating the existence of factors that influence proteasomal processivity. We have quantified proteasomal processivity and determined the underlying rates of substrate degradation and release. We find that processivity increases with species complexity over a 5-fold range between yeast and mammalian proteasome, and the effect is due to slower but more persistent degradation by proteasomes from more complex organisms. A sequence stretch that has been implicated in causing incomplete degradation, the glycine-rich region of the NFκB subunit p105, reduces the proteasome's ability to unfold its substrate, and polyglutamine repeats such as found in Huntington's disease reduce the processivity of the proteasome in a length-dependent manner.     

The Proteasome-associated Protein Ecm29 Inhibits Proteasomal ATPase Activity and in Vivo Protein Degradation by the Proteasome

Several proteasome-associated proteins regulate degradation by the 26 S proteasome using the ubiquitin chains that mark most substrates for degradation. The proteasome-associated protein Ecm29, however, has no ubiquitin-binding or modifying activity, and its direct effect on substrate degradation is unclear. Here, we show that Ecm29 acts as a proteasome inhibitor. Besides inhibiting the proteolytic cleavage of peptide substrates in vitro, it inhibits the degradation of ubiquitin-dependent and -independent substrates in vivo. Binding of Ecm29 to the proteasome induces a closed conformation of the substrate entry channel of the core particle. Furthermore, Ecm29 inhibits proteasomal ATPase activity, suggesting that the mechanism of inhibition and gate regulation by Ecm29 is through regulation of the proteasomal ATPases. Consistent with this, we identified through chemical cross-linking that Ecm29 binds to, or in close proximity to, the proteasomal ATPase subunit Rpt5. Additionally, we show that Ecm29 preferentially associates with both mutant and nucleotide depleted proteasomes. We propose that the inhibitory ability of Ecm29 is important for its function as a proteasome quality control factor by ensuring that aberrant proteasomes recognized by Ecm29 are inactive.     

Ubiquitin-mediated degradation of the proapoptotic active form of bid. A functional consequence on apoptosis induction

Under basal conditions, the proapoptotic protein Bid is a long-lived protein. Pro-apoptotic stimuli such as tumor necrosis factor-alpha (TNFalpha) or Fas induce its caspase-8-mediated cleavage into two fragments. The COOH-terminal cleavage fragment of Bid (tBid) becomes localized to mitochondrial membranes and triggers the release of cytochrome c. Here we show that tBid is ubiquitinated and subsequently degraded by the 26 S proteasome. Degradation of tBid is significantly inhibited by the proteasome inhibitors MG-132 and lactacystin. In contrast, caspase-specific or lysosomal inhibitors do not affect tBid stability. Furthermore, mutation of the putative ubiquitin acceptor sites within tBid results in a stabilized protein as assessed by pulse-chase analysis. To address whether tBid degradation might be regulated by interaction with other Bcl-2-like proteins, cotransfection studies were performed. However, neither the presence of proapoptotic Bax nor antiapoptotic Bcl-2 or Bcl-XL affected tBid degradation. Finally, we determined the functional role of tBid degradation. Overexpression of stabilized tBid proteins significantly enhanced cytochrome c release and subsequent apoptosis induction approximately 2-fold compared with wild type tBid. Similarly, tBid-induced apoptosis was considerably amplified by inhibition of tBid degradation using the proteasome-specific inhibitor MG-132. Thus, proteasomal degradation of tBid limits the extent of apoptosis in living cells.     

A kinetic model of vertebrate 20S proteasome accounting for the generation of major proteolytic fragments from oligomeric peptide substrates

There is now convincing evidence that the proteasome contributes to the generation of most of the peptides presented by major histocompatibility complex class I molecules. Here we present a model-based kinetic analysis of fragment patterns generated by the 20S proteasome from 20 to 40 residues long oligomeric substrates. The model consists of ordinary first-order differential equations describing the time evolution of the average probabilities with which fragments can be generated from a given initial substrate. First-order rate laws are used to describe the cleavage of peptide bonds and the release of peptides from the interior of the proteasome to the external space. Numerical estimates for the 27 unknown model parameters are determined across a set of five different proteins with known cleavage patterns. Testing the validity of the model by a jack knife procedure, about 80% of the observed fragments can be correctly identified, whereas the abundance of false-positive classifications is below 10%. From our theoretical approach, it is inferred that double-cleavage fragments of length 7-13 are predominantly cut out in "C-N-order" in that the C-terminus is generated first. This is due to striking differences in the further processing of the two fragments generated by the first cleavage. The upstream fragment exhibits a pronounced tendency to escape from second cleavage as indicated by a large release rate and a monotone exponential decline of peptide bond accessibility with increasing distance from the first scissile bond. In contrast, the release rate of the downstream fragment is about four orders of magnitude lower and the accessibility of peptide bonds shows a sharp peak in a distance of about nine residues from the first scissile bond. This finding strongly supports the idea that generation of fragments with well-defined lengths is favored in that temporary immobilization of the downstream fragment after the first cleavage renders it susceptible for a second cleavage.     

Expanded Polyglutamine-containing N-terminal Huntingtin Fragments Are Entirely Degraded by Mammalian Proteasomes

Huntington disease is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat within the protein huntingtin (Htt). N-terminal fragments of the mutant Htt (mHtt) proteins containing the polyQ repeat are aggregation-prone and form intracellular inclusion bodies. Improving the clearance of mHtt fragments by intracellular degradation pathways is relevant to obviate toxic mHtt species and subsequent neurodegeneration. Because the proteasomal degradation pathway has been the subject of controversy regarding the processing of expanded polyQ repeats, we examined whether the proteasome can efficiently degrade Htt-exon1 with an expanded polyQ stretch both in neuronal cells and in vitro. Upon targeting mHtt-exon1 to the proteasome, rapid and complete clearance of mHtt-exon1 was observed. Proteasomal degradation of mHtt-exon1 was devoid of polyQ peptides as partial cleavage products by incomplete proteolysis, indicating that mammalian proteasomes are capable of efficiently degrading expanded polyQ sequences without an inhibitory effect on the proteasomal activity.     

alpha-Synuclein protofibrils inhibit 26 S proteasome-mediated protein degradation: understanding the cytotoxicity of protein protofibrils in neurodegenerative disease pathogenesis

The impaired ubiquitin-proteasome activity is believed to be one of the leading factors that contribute to Parkinson disease pathogenesis partially by causing alpha-synuclein aggregation. However, the relationship between alpha-synuclein aggregation and the impaired proteasome activity is yet unclear. In this study, we examined the effects of three soluble alpha-synuclein species (monomer, dimer, and protofibrils) on the degradation activity of the 26 S proteasome by reconstitution of proteasomal degradation using highly purified 26 S proteasomes and model substrates. We found that none of the three soluble alpha-synuclein species impaired the three distinct peptidase activities of the 26 S proteasome when using fluorogenic peptides as substrates. In striking contrast, alpha-synuclein protofibrils, but not monomer and dimer, markedly inhibited the ubiquitin-independent proteasomal degradation of unstructured proteins and ubiquitin-dependent degradation of folded proteins when present at 5-fold molar excess to the 26 S proteasome. Together these results indicate that alpha-synuclein protofibrils have a pronounced inhibitory effect on 26 S proteasome-mediated protein degradation. Because alpha-synuclein is a substrate of the proteasome, impaired proteasomal activity could further cause alpha-synuclein accumulation/aggregation, thus creating a vicious cycle and leading to Parkinson disease pathogenesis. Furthermore we found that alpha-synuclein protofibrils bound both the 26 S proteasome and substrates of the 26 S proteasome. Accordingly we propose that the inhibitory effect of alpha-synuclein protofibrils on 26 S proteasomal degradation might result from impairing substrate translocation by binding the proteasome or sequestrating proteasomal substrates by binding the substrates.     

Limited degradation of oxidized calmodulin by proteasome: formation of peptides

Oxidized proteins are recognized and degraded preferentially by the proteasome. This is true for numerous proteins including calmodulin (CaM). The degradation of CaM was investigated in a human fibroblast cell line under conditions of oxidative stress. Low molecular CaM fragments or peptides were found under such conditions. In in vitro experiments it was investigated whether this CaM breakdown product formation is induced by protein oxidation or is due to a limited proteolysis-derived degradation by the 20S proteasome. Native unoxidized CaM was not degraded by 20S proteasome, oxidized CaM was degraded in a time- and H₂O₂ concentration-dependent manner. Peptides of similar molecular weight were detected in isolated calmodulin as in oxidatively stressed fibroblasts. The peptides were identified using isolated calmodulin. Therefore, in oxidatively stressed fibroblasts and in vitro CaM is forming oxidation-driven fragments and proteasomal cleavage peptides of approximately 30 amino acids which undergo a slow or no degradation.     

Rapid degradation of solid‐phase bound peptides by the 20S proteasome

Proteasomes are cellular proteases involved in the degradation of numerous cellular proteins. The 20S proteasome is a cylindrical 28‐mer protein complex composed of two outer heptameric α‐rings forming the entrance for the protein substrate and two inner heptameric β‐rings carrying the catalytic sites. Numerous in vitro studies have provided evidence that the 20S proteasome may degrade peptides of various lengths and even unfolded full‐length polypeptide chains. However, a direct demonstration that the 20S proteasome may also cleave surface‐attached immobilized peptides is lacking so far. To this end, we used a model system by coupling peptides from different source proteins covalently to the surface of glass beads and applied nanoLC/MS analysis to monitor the generation of proteolytic fragments in the presence of the 20S proteasome. Detectable amounts of cleavage products occurred within a few minutes indicating a much higher cleavage rate than observed with the same substrates in solution. Our finding lends support to the idea that proteasomes may directly degrade segments of membrane‐bound proteins protruding into the aqueous phase. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The human 26 S and 20 S proteasomes generate overlapping but different sets of peptide fragments from a model protein substrate

Intracellular protein degradation is a major source of short antigenic peptides that can be presented on the cell surface in the context of major histocompatibility class I molecules for recognition by cytotoxic T lymphocytes. The capacity of the most important cytosolic protease, the 20 S proteasome, to generate peptide fragments with an average length of 7-8 amino acid residues has been thoroughly investigated. It has been shown that the cleavage products are not randomly generated, but originate from the commitment of the catalytically active subunits to complex recognition motifs in the primary amino acid sequence. The role of the even larger 26 S proteasome is less well defined, however. It has been demonstrated that the 26 S proteasome can bind and degrade ubiquitin-tagged proteins and minigene translation products in vivo and in vitro, but the nature of the degradation products remains elusive. In this study, we present the first analysis of cleavage products from in vitro digestion of the unmodified model substrate beta-casein with both the 26 S and 20 S proteasome. The data we obtained show that 26 S and 20 S proteasomes generate overlapping, but at the same time substantially different, sets of fragments by following very similar instructions.     

Direct ubiquitin independent recognition and degradation of a folded protein by the eukaryotic proteasomes-origin of intrinsic degradation signals

Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases. Apomyoglobin emerges as a new model substrate to further explore the role of ATPases and protein structure in proteasomal degradation.     

Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination

The Cdk inhibitor p21Cip1 is an unstable protein. Pharmacologic inhibition of the proteasome increases the half-life of p21 from less than 30 min to more than 2 hr and results in the accumulation of p21-ubiquitin conjugates. To determine whether ubiquitination was required for proteasomal degradation of p21, we constructed mutant versions of p21 that were not ubiquitinated in vivo. Remarkably, these mutants remained unstable and increased in abundance upon proteasome inhibition, indicating that direct ubiquitination of p21 is not necessary for its turnover by the proteasome. The frequently observed correlation between protein ubiquitination and proteasomal degradation is insufficient to conclude that ubiquitination is a prerequisite for degradation.     

Extracellular proteasome in the human alveolar space: a new housekeeping enzyme?

We hypothesized that 20S proteasome is present and functional in the extracellular alveolar space in humans. Proteasomal activity was measured in bronchoalveolar lavage (BAL) supernatant from eight humans using specific proteasomal fluorogenic substrates and I(125)-albumin with and without specific proteasome inhibitors. Furthermore, gelfiltration, Western blot technique, and mass spectrometry were applied for proteasome characterization. All proteasomal fluorogenic substrates were hydrolyzed by BAL supernatant, with hydrolysis inhibited by epoxomicin (P = 0.024) and other proteasome inhibitors as well. E64, a lysosomal inhibitor, did not inhibit enzyme activity. The majority of proteolytic activity was detected in BAL supernatant rather than in the cell pellet. No correlation was found between proteasomal hydrolysis in BAL supernatant and lactate dehydrogenase activity, the total cell count in the cell pellet, and the fraction of avital cells in the cell pellet, ruling out cell lysis as a major source of proteasomal activity. Gelfiltration revealed hydrolyzing activity in the supernatant at 660 kDa and proteasome core proteins after analysis by ESI-QqTOF mass spectrometry. Furthermore, Western blots using a polyclonal antibody against proteasomal alpha-/beta-subunits detected proteasomal proteins in the typical 20- to 30-kDa range in BAL supernatant. Incubation of BAL supernatant with I(125)-albumin showed a high mean cleavage rate (101.8 microg/ml x h lavage +/- 46 SD) that was inhibited by epoxomicin (P = 0.013) and was ATP and ubiquitin independent. We identified for the first time extracellular, biologically active, ATP- and ubiquitin-independent 20S proteasome in the human alveolar space, with a high albumin cleavage rate. Possibly, the proteasome assists in maintenance of a low intra-alveolar oncotic pressure and/or alveolar protein degradation.     

Stabilization signals: a novel regulatory mechanism in the ubiquitin/proteasome system

The turnover of cellular proteins is a highly organized process that involves spatially and temporally regulated degradation by the ubiquitin/proteasome system. It is generally acknowledged that the specificity of the process is determined by constitutive or conditional protein domains, the degradation signals, that target the substrate for proteasomal degradation. In this review, we discuss a new type of regulatory domain: the stabilization signal. A model is proposed according to which protein half-lives are determined by the interplay of counteracting degradation and stabilization signals.     

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.     

Conformational remodeling of proteasomal substrates by PA700, the 19 S regulatory complex of the 26 S proteasome

PA700, the 19 S regulatory complex of the 26 S proteasome, plays a central role in the recognition and efficient degradation of misfolded proteins. PA700 promotes degradation by recruiting proteasomal substrates utilizing polyubiquitin chains and chaperone-like binding activities and by opening the access to the core of the 20 S proteasome to promote degradation. Here we provide evidence that PA700 in addition to binding misfolded protein substrates also acts to remodel their conformation prior to proteolysis. Scrambled RNase A (scRNase A), a misfolded protein, only slowly refolds spontaneously into an active form because of the rate-limiting unfolding of misfolded disulfide isomers. Notably, PA700 accelerates the rate of reactivation of scRNase A, consistent with its ability to increase the exposure of these disulfide bonds to the solvent. In this regard, PA700 also exposes otherwise buried sites to digestion by exogenous chymotrypsin in a polyubiquitinated enzymatically active substrate, pentaubiquitinated dihydrofolate reductase, Ub(5)DHFR. The dihydrofolate reductase ligand methotrexate counters the ability of PA700 to promote digestion by chymotrypsin. Together, these results indicate that in addition to increasing substrate affinity and opening the access channel to the catalytic sites, PA700 activates proteasomal degradation by remodeling the conformation of protein substrates.