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).     

The mycobacterial Mpa–proteasome unfolds and degrades pupylated substrates by engaging Pup's N‐terminus

Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin‐like protein (Pup) that is recognized by the N‐terminal coiled‐coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substrates and establish its mechanistic features with respect to substrate recruitment, unfolding and degradation. We show that the Mpa–proteasome complex unfolds and degrades Pup‐tagged proteins and that this activity requires physical interaction of the ATPase with the proteasome. Furthermore, we establish the N‐terminal region of Pup as the structural element required for engagement of pupylated substrates into the Mpa pore. In this process, Mpa pulls on Pup to initiate unfolding of substrate proteins and to drag them toward the proteasome chamber. Unlike the eukaryotic ubiquitin, Pup is not recycled but degraded with the substrate. This assigns a dual function to Pup as both the Mpa recognition element as well as the threading determinant.     

Protein unfolding--an important process in vivo?

Protein unfolding is an important step in several cellular processes, most interestingly protein degradation by ATP-dependent proteases and protein translocation across some membranes. Unfolding can be catalyzed when the unfoldases change the unfolding pathway of substrate proteins by pulling at their polypeptide chains. The resistance of a protein to unraveling during these processes is not determined by the protein's stability against global unfolding, as measured by temperature or solvent denaturation in vitro. Instead, resistance to unfolding is determined by the local structure that the unfoldase encounters first as it follows the substrate's polypeptide chain from the targeting signal. As unfolding is a necessary step in protein degradation and translocation, the susceptibility to unfolding of substrate proteins contributes to the specificity of these important cellular processes.     

An unstructured initiation site is required for efficient proteasome-mediated degradation

The proteasome is the main ATP-dependent protease in eukaryotic cells and controls the concentration of many regulatory proteins in the cytosol and nucleus. Proteins are targeted to the proteasome by the covalent attachment of polyubiquitin chains. The ubiquitin modification serves as the proteasome recognition element but by itself is not sufficient for efficient degradation of folded proteins. We report that proteolysis of tightly folded proteins is accelerated greatly when an unstructured region is attached to the substrate. The unstructured region serves as the initiation site for degradation and is hydrolyzed first, after which the rest of the protein is digested sequentially. These results identify the initiation site as a novel component of the targeting signal, which is required to engage the proteasome unfolding machinery efficiently. The proteasome degrades a substrate by first binding to its ubiquitin modification and then initiating unfolding at an unstructured region.     

Bacterial Proteasome Activator Bpa (Rv3780) Is a Novel Ring-Shaped Interactor of the Mycobacterial Proteasome

The occurrence of the proteasome in bacteria is limited to the phylum of actinobacteria, where it is maintained in parallel to the usual bacterial compartmentalizing proteases. The role it plays in these organisms is still not fully understood, but in the human pathogen Mycobacterium tuberculosis (Mtb) the proteasome supports persistence in the host. In complex with the ring-shaped ATPase Mpa (called ARC in other actinobacteria), the proteasome can degrade proteins that have been post-translationally modified with the prokaryotic ubiquitin-like protein Pup. Unlike for the eukaryotic proteasome core particle, no other bacterial proteasome interactors have been identified to date. Here we describe and characterize a novel bacterial proteasome activator of Mycobacterium tuberculosis we termed Bpa (Rv3780), using a combination of biochemical and biophysical methods. Bpa features a canonical C-terminal proteasome interaction motif referred to as the HbYX motif, and its orthologs are only found in those actinobacteria encoding the proteasomal subunits. Bpa can inhibit degradation of Pup-tagged substrates in vitro by competing with Mpa for association with the proteasome. Using negative-stain electron microscopy, we show that Bpa forms a ring-shaped homooligomer that can bind coaxially to the face of the proteasome cylinder. Interestingly, Bpa can stimulate the proteasomal degradation of the model substrate β-casein, which suggests it could play a role in the removal of non-native or damaged proteins.     

Proteasome involvement in the degradation of the G(q) family of Galpha subunits

Metabolically unstable proteins are involved in a multitude of regulatory networks, including those that control cell signaling, the cell cycle and in many responses to physiological stress. In the present study, we have determined the stability and characterized the degradation process of some members of the G(q) class of heterotrimeric G proteins. Pulse-chase experiments in HEK293 cells indicated a rapid turnover of endogenously expressed Galpha(q) and overexpressed Galpha(q) and Galpha(16) subunits. Pretreatment with proteasome inhibitors attenuated the degradation of both G alpha subunits. In contrast, pretreatment of cells with inhibitors of lysosomal proteases and nonproteasomal cysteine proteases had very little effect on the stability of the proteins. Significantly, the turnover of these proteins is not affected by transient activation of their associated receptors. Fractionation studies showed that the rates of Galpha(q) and Galpha16 degradation are accelerated in the cytosol. In fact, we show that a mutant Galpha(q) which lacks its palmitoyl modification site, and which is localized almost entirely in the cytoplasm, has a marked increase in the rate of degradation. Taken together, these results suggest that the G(q) class proteins are degraded through the proteasome pathway and that cellular localization and/or other protein interactions determine their stability.     

PAN, the proteasome-activating nucleotidase from archaebacteria, is a protein-unfolding molecular chaperone

The proteasome-activating nucleotidase (PAN) from Methanococcus jannaschii is a complex of relative molecular mass 650,000 that is homologous to the ATPases in the eukaryotic 26S proteasome. When mixed with 20S archaeal proteasomes and ATP, PAN stimulates protein degradation. Here we show that PAN reduces aggregation of denatured proteins and enhances their refolding. These processes do not require ATP hydrolysis, although ATP binding enhances the ability of PAN to prevent aggregation. PAN also catalyses the unfolding of the green fluorescent protein with an 11-residue ssrA extension at its carboxy terminus (GFP11). This unfolding requires ATP hydrolysis, and is linked to GFP11 degradation when 20S proteasomes are also present. This unfolding activity seems to be essential for ATP-dependent proteolysis, although PAN may function by itself as a molecular chaperone.     

Slippery Substrates Impair ATP-dependent Protease Function by Slowing Unfolding

ATP-dependent proteases are responsible for most energy-dependent protein degradation across all species. Proteases initially bind an unstructured region on a substrate and then translocate along the polypeptide chain, unfolding and degrading protein domains as they are encountered. Although this process is normally processive, resulting in the complete degradation of substrate proteins to small peptides, some substrates are released prematurely. Regions of low sequence complexity within the substrate such as the glycine-rich region (GRR) from p105 or glycine-alanine repeats (GAr) from the EBNA1 (Epstein-Barr virus nuclear antigen-1) protein, can trigger partial degradation and fragment release. Loss of processivity could be due to inability to hold on to the substrate (faster release) or inability to unfold and degrade a substrate domain (slower unfolding). I previously showed that the GRR slows domain unfolding by the proteasome (Kraut, D. A., Israeli, E., Schrader, E. K., Patil, A., Nakai, K., Nanavati, D., Inobe, T., and Matouschek, A. (2012) ACS Chem. Biol. 7, 1444–1453). In contrast, a recently published study concluded that GArs increase the rate of substrate release from ClpXP, a bacterial ATP-dependent protease (Too, P. H., Erales, J., Simen, J. D., Marjanovic, A., and Coffino, P. (2013) J. Biol. Chem. 288, 13243–13257). Here, I show that these apparently contradictory results can be reconciled through a reanalysis of the ClpXP GAr data. This reanalysis shows that, as with the proteasome, low complexity sequences in substrates slow their unfolding and degradation by ClpXP, with little effect on release rates. Thus, despite their evolutionary distance and limited sequence identity, both ClpXP and the proteasome share a common mechanism by which substrate sequences regulate the processivity of degradation.     

Proteasome subunit Rpn1 binds ubiquitin-like protein domains

The yeast protein Rad23 belongs to a diverse family of proteins that contain an amino-terminal ubiquitin-like (UBL) domain. This domain mediates the binding of Rad23 to proteasomes, which in turn promotes DNA repair and modulates protein degradation, possibly by delivering ubiquitinylated cargo to proteasomes. Here we show that Rad23 binds proteasomes by directly interacting with the base subcomplex of the regulatory particle of the proteasome. A component of the base, Rpn1, specifically recognizes the UBL domain of Rad23 through its leucine-rich-repeat-like (LRR-like) domain. A second UBL protein, Dsk2, competes with Rad23 for proteasome binding, which suggests that the LRR-like domain of Rpn1 may participate in the recognition of several ligands of the proteasome. We propose that the LRR domain of Rpn1 may be positioned in the base to allow the cargo proteins carried by Rad23 to be presented to the proteasomal ATPases for unfolding. We also report that, contrary to expectation, the base subunit Rpn10 does not mediate the binding of UBL proteins to the proteasome in yeast, although it can apparently contribute to the binding of ubiquitin chains by intact proteasomes.     

Crystal structure of TET protease reveals complementary protein degradation pathways in prokaryotes

Protein degradation is an essential and strictly controlled process with proteasome and functionally related proteases representing its central part. Tricorn protease (TRI) has been shown to act downstream of the proteasome, degrading produced peptides. Recently, a novel large prokaryotic aminopeptidase oligomeric complex, named TET, has been identified. This complex degrades peptides of different length in organisms where TRI is not present. We determined the crystal structure of TET from the thermophilic archaeon Pyrococcus horikoshii at 1.6 A resolution in native form and in complex with the inhibitor amastatin. We demonstrate that, beside the novel tetrahedral oligomerisation pattern, TET possesses a unique mechanism of substrate attraction and orientation. TET sequentially degrades peptides produced by the proteasome to single amino acids. Furthermore, we reconstituted in vitro the minimal protein degradation system from initial unfolding of labelled protein substrates, up to release of free amino acids. We propose that TET and TRI act as functional analogues in different organisms, with TET being more widely distributed. Thus, TET and TRI represent two evolutionarily diverged pathways of peptide degradation in prokaryotes.     

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.     

Degradation of sarcomeric and cytoskeletal proteins in cultured skeletal muscle cells

The goal of this research was to evaluate the roles of calpains and their interactions with the proteasome and the lysosome in degradation of individual sarcomeric and cytoskeletal proteins in cultured muscle cells. Rat L8-CID muscle cells, in which we expressed a transgene calpain inhibitor (CID), were used in the study. L8-CID cells were grown as myotubes after which the relative roles of calpain, proteasome and lysosome in total protein degradation were assessed during a period of serum withdrawal. Following this, the roles of proteases in degrading cytoskeletal proteins (desmin, dystrophin and filamin) and of sarcomeric proteins (alpha-actinin and tropomyosin) were assessed. Total protein degradation was assessed by release of radioactive tyrosine from pre-labeled myotubes in the presence and absence of protease inhibitors. Effects of protease inhibitors on concentrations of individual sarcomeric and cytoskeletal proteins were assessed by Western blotting. Inhibition of calpains, proteasome and lysosome caused 20, 62 and 40% reductions in total protein degradation (P<0.05), respectively. Therefore, these three systems account for the bulk of degradation in cultured muscle cells. Two cytoskeletal proteins were highly-sensitive to inhibition of their degradation. Specifically, desmin and dystrophin concentrations increased markedly when calpain, proteasome and lysosome activities were inhibited. Conversely, sarcomeric proteins (alpha-actinin and tropomyosin) and filamin were relatively insensitive to the addition of protease inhibitors to culture media. These data demonstrate that proteolytic systems work in tandem to degrade cytoskeletal and sarcomeric protein complexes and that the cytoskeleton is more sensitive to inhibition of degradation than the sarcomere. Mechanisms, which bring about changes in the activities of the proteases, which mediate muscle protein degradation are not known and represent the next frontier of understanding needed in muscle wasting diseases and in muscle growth biology.     

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.     

Gly-Ala repeats induce position- and substrate-specific regulation of 26 S proteasome-dependent partial processing

Partial degradation or regulated ubiquitin proteasome-dependent processing by the 26 S proteasome has been demonstrated, but the underlying molecular mechanisms and the prevalence of this phenomenon remain obscure. Here we show that the Gly-Ala repeat (GAr) sequence of EBNA1 affects processing of substrates via the ubiquitin-dependent degradation pathway in a substrate- and position-specific fashion. GAr-mediated increase in stability of proteins targeted for degradation via the 26 S proteasome was associated with a fraction of the substrates being partially processed and the release of the free GAr. The GAr did not cause a problem for the proteolytic activity of the proteasome, and its fusion to the N terminus of p53 resulted in an increase in the rate of degradation of the entire chimera. Interestingly the GAr had little effect on the stability of EBNA1 protein itself, and targeting EBNA1 for 26 S proteasome-dependent degradation led to its complete degradation. Taken together, our data suggest a model in which the GAr prevents degradation or promotes endoproteolytic processing of substrates targeted for the 26 S proteasome by interfering with the initiation step of substrate unfolding. These results will help to further understand the underlying mechanisms for partial proteasome-dependent degradation.     

A Conserved Unfoldase Activity for the p97 AAA-ATPase in Proteasomal Degradation

The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. Here, we show that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended C-terminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. Our results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97^Ufd1/Npl4 complex. Our data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome.     

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.     

Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

A major fraction of intracellular protein degradation is mediated by the proteasome. Successful degradation of these substrates requires ubiquitination and delivery to the proteasome followed by protein unfolding and disassembly of the multiubiquitin chain. Enzymes, such as Rpn11, dismantle multiubiquitin chains, and mutations can affect proteasome assembly and activity. We report that different rpn11 mutations can affect proteasome interaction with ubiquitinated proteins. Moreover, proteasomes are unstable in rpn11-1 and do not form productive interactions with multiubiquitinated proteins despite high levels in cell extracts. However, increased levels of ubiquitinated proteins were found associated with shuttle factors. In contrast to rpn11-1, proteasomes expressing a catalytically inactive mutant (rpn11^AXA) were more stable and bound very high amounts of ubiquitinated substrates. Expression of the carboxyl-terminal domain of Rpn11 partially suppressed the growth and proteasome stability defects of rpn11-1. These results indicate that ubiquitinated substrates are preferentially delivered to intact proteasome.     

Dependence of proteasome processing rate on substrate unfolding

Protein degradation by eukaryotic proteasomes is a multi-step process involving substrate recognition, ATP-dependent unfolding, translocation into the proteolytic core particle, and finally proteolysis. To date, most investigations of proteasome function have focused on the first and the last steps in this process. Here we examine the relationship between the stability of a folded protein domain and its degradation rate. Test proteins were targeted to the proteasome independently of ubiquitination by directly tethering them to the protease. Degradation kinetics were compared for test protein pairs whose stability was altered by either point mutation or ligand binding, but were otherwise identical. In both intact cells and in reactions using purified proteasomes and substrates, increased substrate stability led to an increase in substrate turnover time. The steady-state time for degradation ranged from ～5 min (dihydrofolate reductase) to 40 min (I27 domain of titin). ATP turnover was 110/min./proteasome, and was not markedly changed by substrate. Proteasomes engage tightly folded substrates in multiple iterative rounds of ATP hydrolysis, a process that can be rate-limiting for degradation.     

Mitochondria unfold precursor proteins by unraveling them from their N-termini

Protein unfolding is a key step in the life cycle of many proteins, including certain proteins that are degraded by ATP-dependent proteases or translocated across membranes. The detailed mechanisms of these unfolding processes are not understood. Precursor proteins are unfolded and imported into mitochondria by a macromolecular machine that spans two membranes and contains at least nine different proteins. Here we examine import of a model precursor protein derived from the ribonuclease barnase and show that mitochondria unfold this protein by unraveling it from its N-terminus. Because barnase in free-solution unfolds by a different pathway, our results demonstrate that mitochondria catalyze unfolding in the way that enzymes catalyze reactions, namely by changing reaction pathways. The effectiveness of this mechanism depends on the structure of the N-terminal part of the precursor protein.     

Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface

Two AAA proteases, each with its catalytic site at the opposite membrane surface, mediate the ATP-dependent degradation of mitochondrial inner membrane proteins. We demonstrate here that a model substrate polypeptide containing hydrophilic domains at both sides of the membrane can be completely degraded by either of the AAA proteases, if solvent-exposed domains are in an unfolded state. A short protein tail protruding from the membrane surface is sufficient to allow the proteolytic attack of an AAA protease that facilitates domain unfolding at the opposite side. Our results provide a rationale for the membrane arrangement of AAA proteases in mitochondria and demonstrate that degradation of membrane proteins by AAA proteases involves an active extraction of transmembrane segments and transport of solvent-exposed domains across the membrane.