Mode of targeting to the proteasome determines GFP fate

The ubiquitin–proteasome system is the canonical pathway for protein degradation in eukaryotic cells. GFP is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome''s ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub₄ degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, whereas the Ub₄ degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Difficult-to-unfold substrates are released and re-engaged multiple times, with removal of the degradation initiation region providing an alternative clipping pathway that precludes unfolding and degradation; the UBL degron favors degradation of even difficult-to-unfold substrates, whereas the Ub₄ degron favors clipping. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates. Our results indicate that the choice of targeting method and reporter protein are critical to the design of protein degradation experiments.     

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.     

VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome

The 26S proteasome recognizes a vast number of ubiquitin-dependent degradation signals linked to various substrates. This recognition is mediated mainly by the stoichiometric proteasomal resident ubiquitin receptors S5a and Rpn13, which harbor ubiquitin-binding domains. Regulatory steps in substrate binding, processing, and subsequent downstream proteolytic events by these receptors are poorly understood. Here we demonstrate that mammalian S5a is present in proteasome-bound and free states. S5a is required for efficient proteasomal degradation of polyubiquitinated substrates and the recruitment of ubiquitin-like (Ubl) harboring proteins; however, S5a-mediated ubiquitin and Ubl binding occurs only on the proteasome itself. We identify the VWA domain of S5a as a domain that limits ubiquitin and Ubl binding to occur only upon proteasomal association. Multiubiquitination events within the VWA domain can further regulate S5a association. Our results provide a molecular explanation to how ubiquitin and Ubl binding to S5a is restricted to the 26S proteasome.     

Human T-cell leukemia virus type 1 HBZ protein bypasses the targeting function of ubiquitination

Human T-cell leukemia virus type 1 (HTLV-1) encodes an antisense viral gene product termed HTLV-1 basic leucine-zipper factor (HBZ). HBZ forms heterodimers with c-Jun, a member of the AP-1 family, and promotes its proteasomal degradation. Although most proteasomal substrates are targeted for degradation via conjugation of polyubiquitin chains, we show that ubiquitination is not required for HBZ-mediated proteasomal degradation of c-Jun. We demonstrate that HBZ directly interacts with both the 26 S proteasome and c-Jun and facilitates the delivery of c-Jun to the proteasome without ubiquitination. HBZ acts as a tethering factor between the 26 S proteasome and its substrate, thereby bypassing the targeting function of ubiquitination. These findings disclose a novel viral strategy to utilize the cellular proteolytic system for viral propagation.     

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.     

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.     

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.     

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.     

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.     

Ubiquitin-independent degradation of proteins by the proteasome

The proteasome is the main proteolytic machinery of the cell and constitutes a recognized drugable target, in particular for treating cancer. It is involved in the elimination of misfolded, altered or aged proteins as well as in the generation of antigenic peptides presented by MHC class I molecules. It is also responsible for the proteolytic maturation of diverse polypeptide precursors and for the spatial and temporal regulation of the degradation of many key cell regulators whose destruction is necessary for progression through essential processes, such as cell division, differentiation and, more generally, adaptation to environmental signals. It is generally believed that proteins must undergo prior modification by polyubiquitin chains to be addressed to, and recognized by, the proteasome. In reality, however, there is accumulating evidence that ubiquitin-independent proteasomal degradation may have been largely underestimated. In particular, a number of proto-oncoproteins and oncosuppressive proteins are privileged ubiquitin-independent proteasomal substrates, the altered degradation of which may have tumorigenic consequences. The identification of ubiquitin-independent mechanisms for proteasomal degradation also poses the paramount question of the multiplicity of catabolic pathways targeting each protein substrate. As this may help design novel therapeutic strategies, the underlying mechanisms are critically reviewed here.     

A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly

Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves a ubiquitin-activating enzyme, E1, a ubiquitin-conjugating enzyme, E2, and often a substrate-specific ubiquitin-protein ligase, E3. Here we show that efficient multiubiquitination needed for proteasomal targeting of a model substrate requires an additional conjugation factor, named E4. This protein, previously known as UFD2 in yeast, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjunction with E1, E2, and E3. Intriguingly, E4 defines a novel protein family that includes two human members and the regulatory protein NOSA from Dictyostelium required for fruiting body development. In yeast, E4 activity is linked to cell survival under stress conditions, indicating that eukaryotes utilize E4-dependent proteolysis pathways for multiple cellular functions.     

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.     

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.     

Proteasomal degradation of the nuclear targeting growth factor midkine

It is widely held that growth factor signaling is terminated by lysosomal degradation of its activated receptor and the endocytosed growth factor is transported to lysosomes. Nuclear targeting is another important pathway through which signals of growth factors are mediated. However, mechanisms underlying desensitization of nuclear targeting growth factors are poorly understood. Here we report that the nuclear targeting pathway is down-regulated by the proteasome system. Degradation of endocytosed midkine, a heparin-binding growth factor, was suppressed by both proteasome and lysosome inhibitors to similar extents. By contrast, a proteasome inhibitor, but not lysosome ones, accelerated the nuclear accumulation of midkine. An expression vector of signal sequence-less midkine, which is produced in the cytosol, was constructed because endocytosed midkine may be translocated to the cytosol from cellular compartments before entering the nucleus. The cytosol-produced midkine underwent proteasomal degradation and accumulated in the nucleus as did the endocytosed midkine. It was polyubiquitinated, and its nuclear accumulation was enhanced by a proteasome inhibitor. We further dissected the midkine molecule to investigate roles in degradation and trafficking. The N-terminal half-domain of midkine was significantly more susceptible to proteasomal degradation, whereas the C-terminal half-domain was sufficient for nuclear localization. Together, these data highlight the desensitization of nuclear targeting by growth factors and indicate a critical role of the proteasome system in it.     

Substrate receptors of proteasomes

Proteasomes are responsible for the turnover of most cellular proteins, and thus are critical to almost all cellular activities. A substrate entering the proteasome must first bind to a substrate receptor. Substrate receptors can be classified as ubiquitin receptors and non‐ubiquitin receptors. The intrinsic ubiquitin receptors, including proteasome regulatory particle base subunits 1, 10 and 13 (Rpn1, Rpn10, and Rpn13), determine the capability of the proteasome to recognize a ubiquitin chain, and thus provide selectivity for the 26S proteasome. However, the non‐ubiquitin receptors, including proteasome activator 200 (PA200) and PA28γ, have received great attention due to their remarkable compensatory roles relative to canonical ubiquitin‐mediated proteasomal degradation. Herein we review recent advances in understanding the contributions of these substrate receptors to proteasomal degradation, and introduce their substrates and interacting factors. We also provide insights into their biological functions related to spermatogenesis, immune responses, cellular homeostasis, and tumour development. Finally, we summarize advances in developing small‐molecule inhibitors of these substrate receptors and discuss their potential as drug targets.     

Evidence for an interaction between ubiquitin-conjugating enzymes and the 26S proteasome

The targeting of proteolytic substrates is accomplished by a family of ubiquitin-conjugating (E2) enzymes and a diverse set of substrate recognition (E3) factors. The ligation of a multiubiquitin chain to a substrate can promote its degradation by the proteasome. However, the mechanism that facilitates the translocation of a substrate to the proteasome in vivo is poorly understood. We have discovered that E2 proteins, including Ubc1, Ubc2, Ubc4, and Ubc5, can interact with the 26S proteasome. Significantly, the interaction between Ubc4 and the proteasome is strongly induced by heat stress, consistent with the requirement for this E2 for efficient stress tolerance. A catalytically inactive derivative of Ubc4 (Ubc4(C86A)), which causes toxicity in yeast cells, can also bind the proteasome. Purified proteasomes can ligate ubiquitin to a test substrate without the addition of exogenous E2 protein, suggesting that the ubiquitylation of some proteolytic substrates might be directly coupled to degradation by the proteasome.     

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 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.