Multiubiquitin Chain Research Articles

RNF149 negatively regulates LPS/TLR4 signal transduction by ubiquitination-mediated CD63 degradation

This study aims to investigate the role of RNF149 and tetraspanin CD63 in lipopolysaccharide/Toll-like receptor 4 (LPS/TLR4) signal transduction. TNF-α was assessed using enzyme-linked immunosorbent assay. The distribution of TLR4 was examined through flow cytometry after CD63 knockdown. Real-time polymerase chain reaction was used to analyze the expression of the target genes RNF149 and CD63 under different conditions. Western blotting was employed to detect gene expression, while immunoprecipitation and confocal microscopy were used to evaluate protein interactions. Transcriptome array data from stimulated monocytes (GSE7547) was obtained from GEO and subjected to bioinformatic analysis. It is suggested that CD63 may serve as a substrate of RNF149, with RNF149 capable of directly interacting with CD63. RNF149 degrades CD63 through covalent modification of CD63 at lysine 29 of the ubiquitin monomer, leading to the formation of a multiubiquitin chain. Both RNF149 and CD63 interact with TLR4, with CD63 promoting LPS/TLR4 signaling and RNF149 inhibits it. CD63 does not impact the distribution of TLR4 on the cell surface and does not directly interact with TIRAP, IRAK4, or TRAF6, but does interact with Myd88.RNF149 plays a negative regulatory role in LPS/TLR4 signal transduction by mediating ubiquitination-induced CD63 degradation.

Insight into the physiological and pathological roles of USP44, a potential tumor target (Review).

Ubiquitin-specific peptidase 44 (USP44) is a member of the ubiquitin-specific proteases (USPs) family and its functions in various biological processes have been gradually elucidated in recent years. USP44 targets multiple downstream factors and regulates multiple mechanisms through its deubiquitination activity. Ubiquitination is, in essence, a process in which a single ubiquitin molecule or a multiubiquitin chain binds to a substrate protein to form an isopeptide bond. Deubiquitination is the catalyzing of the isopeptide bonds between ubiquitin and substrate proteins through deubiquitylating enzymes. These two processes serve an important role in the regulation of the expression, conformation, localization and function of substrate proteins by regulating their binding to ubiquitin. Based on existing research, this paper summarized the current state of knowledge about USP44. The physiological roles of USP44 in various cellular events and its pathophysiological roles in different cancer types are evaluated and the therapeutic potential of USP44 for cancer treatment is evaluated.

A New Cofilin-Dependent Mechanism for the Regulation of Brain Mitochondria Biogenesis and Degradation.

The aim Was to study the role of post-translational modifications of cofilin in the regulation of respiration and autophagy in murine brain mitochondria.Materials and MethodsThe experiments were performed with C57BL/6 mice. To obtain cytoplasmic and mitochondrial fractions of the brain tissue, differential centrifugation was used. Expressions of cofilin, phospho-cofilin, K48- and K63-associated chains of ubiquitin, and the autophagy marker LC3B were determined using electrophoresis, immunoprecipitation and Western blot methods. To study the processes of ubiquitination, we used PR619 — the inhibitor of deubiquitinating enzymes. Respiratory activity of brain mitochondria was evaluated using high-resolution fluorespirometry.ResultsModification of cofilin by non-canonical K63 multiubiquitin chains in the cytoplasm and mitochondria from murine brain was demonstrated. Different levels of phospho-cofilin, cofilin, and its ubiquitinated proteoforms were found. PR619, the inhibitor of deubiquitinating enzymes, affects the expression of phosphorylated and ubiquitinated forms of cofilin in the mitochondria and cytoplasm, at the same time it changes the activity of tissue respiration and mitophagy.ConclusionThe sensitivity of cofilin to the inhibitor of deubiquitinating enzymes indicates the existence of a new non-catabolic mechanism of cofilin modification, which may be involved in the regulation of mitochondrial functions, specifically, the mitochondrial respiration and autophagy. The data help understand the molecular mechanisms of mitochondrial function in normal and pathological conditions, which may be useful in developing novel methods for the treatment of diseases of the nervous system.

Rad23 Interaction with the Proteasome Is Regulated by Phosphorylation of Its Ubiquitin-Like (UbL) Domain

Rad23 was identified as a DNA repair protein, although a role in protein degradation has been described. The protein degradation function of Rad23 contributes to cell cycle progression, stress response, endoplasmic reticulum proteolysis, and DNA repair. Rad23 binds the proteasome through a UbL (ubiquitin-like) domain and contains UBA (ubiquitin-associated) motifs that bind multiubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. This property is shared by structurally similar proteins (Dsk2 and Ddi1) and is conserved among the human and mouse counterparts of Rad23. Despite much effort, the regulation of Rad23 interactions with ubiquitinated substrates and the proteasome is unknown. We report here that Rad23 is extensively phosphorylated in vivo and in vitro. Serine residues in UbL are phosphorylated and influence Rad23 interaction with proteasomes. Replacement of these serine residues with acidic residues, to mimic phosphorylation, reduced proteasome binding. We reported that when UbL is overexpressed, it can compete with Rad23 for proteasome interaction and can inhibit substrate turnover. This effect is not observed with UbL containing acidic substitutions, consistent with results that phosphorylation inhibits interaction with the proteasome. Loss of both Rad23 and Rpn10 caused pleiotropic defects that were suppressed by overexpressing either Rad23 or Rpn10. Rad23 bearing a UbL domain with acidic substitutions failed to suppress rad23Δ rpn10Δ, confirming the importance of regulated Rad23/proteasome binding. Strikingly, threonine 75 in human HR23B also regulates interaction with the proteasome, suggesting that phosphorylation is a conserved mechanism for controlling Rad23/proteasome interaction.

Deubiquitinating enzymes in oocyte maturation, fertilization and preimplantation embryo development.

Post-translational modifications of cellular proteins by ubiquitin and ubiquitin-like protein modifiers are important regulatory events involved in diverse aspects of gamete and embryo physiology including oocyte maturation, fertilization and development of embryos to term. Deubiquitinating enzymes (DUBs) regulate proteolysis by reversing ubiquitination, which targets proteins to the 26S proteasome. The ubiquitin C-terminal hydrolases (UCHs) comprise are DUBs that play a role in the removal of multi-ubiquitin chains. We review here the roles of UCHs in oocytes maturation, fertilization and development in mouse, bovine, porcine and rhesus monkeys. Oocyte UCHs contributes to fertilization and embryogenesis by regulating the physiology of the oocyte and blastomere cortex as well as oocyte spindle. Lack of UCHs in embryos reduces fertilization, while mutant embryos fail to undergo compaction and blastocyst formation. In addition to advancing our understanding of reproductive process, research on the role of deubiquitinating enzymes will allow us to better understand and treat human infertility, and to optimize reproductive performance in agriculturally important livestock species.

The N-Glycosylation Modification of LHBs (Large Surface Proteins of HBV) Effects on Endoplasmic Reticulum Stress, Cell Proliferation and its Secretion

BackgroundThe mutations of LHBs in pre-S, especially in pre-S2, are definitive in hepatocellular carcinoma (HCC) associated with HBV. However, the mechanisms of the N-glycosylation modification in LHBs are unclear. The N-glycosylation modification of LHBs affects Endoplasmic Reticulum stress, cell proliferation and its secretion which was further studied.ObjectivesThe objectives of our studies was to indentified that modification of LHBs by N glycosylation modulate their secretion, affect ER stress or expression of cycling, cell cycle and proliferation.Materials and MethodsThe LHBs was mutated; then expression of proteins related to endoplasmic reticulum stress and EAED path of L02 cells affected by LHBs and its mutations was evaluated. LHBs proteins bound to multiubiquitin chains and its glycosylation motif were studied. The subcellular localization and secretion of LHBs and its mutations were identified. The effect on cell cycle and proliferation by LHBs and its mutations were detected.ResultsThese data demonstrated that the N-glycosylation motifs of LHBs were associated with ER stress. The N15S, N123S, and N177S mutated LHBs proteins could induce overexpression of EDEM in L02 cells. LHBs and its mutated proteins contained p62-derived UBA domain, which could affect expression of cyclins. The subcellular localization of LHBs in endoplasmic reticulum was similar to its mutations. The secretion of LHBs was blocked by N320K mutation, which could induce an increase in G1 phase and inhibition of S phase, and inhibited mitotic entry.ConclusionsIn conclusion, our studies powerfully demonstrated that modification of LHBs by N glycosylation could modulate their secretion, affect ER stress or expression of cycling, cell cycle and proliferation. The N320K may be the key sites N-linked glycosylation modification of LHBs. It may be a mechanism of HBV-induced HCC.

Phosphorylations of Sds23/Psp1/Moc1 by stress‐activated kinase and cAMP‐dependent kinase are essential for regulating cell viability in prolonged stationary phase

Under nutritional deprivation caused by prolonged culture, actively growing cells prepare to enter stationary phase. We showed here that Sds23/Psp1/Moc1 was phosphorylated by both cAMP-dependent kinase and stress-activated MAP kinase Sty1 upon entry into stationary phase. Overexpression of the phosphorylation-defective mutant Sds23/Psp1/Moc1 induced cell death in prolonged culture and blocked re-entry into the cell division cycle. These phosphorylations are likely to be required for cell survival during stationary phase and for binding of Ufd2, a Schizosaccharomyces pombe homologue of multi-ubiquitin chain assembly factor E4. Deletion of the Ufd2 gene and overexpression of Sds23/Psp1/Moc1 increased cell viability in prolonged stationary phase. These results suggested that Ufd2 induces cell death in prolonged nutrient deprivation, that Sds23/Psp1/Moc1 may be a target protein of the ubiquitin-fusion degradation pathway for regulation of cell viability under this stress condition, and that Sty1 and PKA activity in stationary phase is essential for interaction between Sds23/Psp1/Moc1 and Ufd2.

Regulation of the proteasome by ubiquitin chain editing

Error‐correction mechanisms in protein synthesis are well‐known. In an interesting analogy, the proteasome contains activities that can remodel, or edit, multiubiquitin chains on substrates. These activities regulate degradation rates in yeast and mammals. The effects are powerful but vary in strength from substrate to substrate. The main editing factors are the deubiquitinating enzyme Ubp6 and the ubiquitin ligase Hul5 (in yeast). Ubp6 suppresses degradation while Hul5 promotes it. Regulation of degradation may reflect that more heavily ubiquitinated substrates bind the proteasome more avidly. These factors can transduce signals to modulate proteasome activity positively or negatively. For example, UBP6 is induced when ubiquitin levels fall, while HUL5 is not. In addition to modifying ubiquitin chains, both factors regulate degradation noncatalytically. Hul5's noncatalytic effect is apparently to suppress Ubp6 deubiquitinating activity. Also, of the 910 residues in Hul5, only a short 13‐residue segment at the N‐terminus is required for the noncatalytic effect. The deubiquitinating activity of Usp14 (the mammalian ortholog of Ubp6) can be suppressed by small‐molecule inhibitors identified by high‐throughput screening. These compounds allow one to enhance the degradation rates of many proteins, including toxic proteins such as mutant forms of tau that have been implicated in neurodegeneration.

E6AP/UBE3A Ubiquitin Ligase Harbors Two E2∼ubiquitin Binding Sites

By exploiting (125)I-polyubiquitin chain formation as a functional readout of enzyme activity, we have quantitatively examined the mechanism of human E6AP/UBE3A for the first time. Initial rate studies identify UbcH7 as the cognate E2 carrier protein for E6AP, although related Ubc5 isoforms and the ISG15-specific UbcH8 paralog also support E6AP with reduced efficacy due to impaired binding and catalytic competence. Initial rates of polyubiquitin chain formation displayed hyperbolic kinetics with respect to UbcH7 concentration (K(m) = 57.6 ± 5.7 nM and kcat = 0.032 ± 0.001 s(-1)) and substrate inhibition above 2 μM. Competitive inhibition by an isosteric UbcH7C86S-ubiquitin oxyester substrate analog (K(i) = 64 ± 18 nM) demonstrates that Km reflects intrinsic substrate affinity. In contrast, noncompetitive inhibition by a UbcH7C86A product analog (K(i) = 7 ± 0.7 μM) and substrate inhibition at high concentrations require two functionally distinct E2∼ubiquitin substrate binding sites. The kinetics of polyubiquitin chain formation reflect binding at a cryptic Site 1 not previously recognized that catalyzes E6AP∼ubiquitin thioester formation. Subsequent binding of E2∼ubiquitin at the canonical Site 2 present in the extant crystal structure is responsible for polyubiquitin chain elongation. Other rate studies show that the conserved -4 Phe(849) residue is required for polyubiquitin chain formation rather than target protein conjugation as originally suggested. The present studies unambiguously preclude earlier models for the mechanism of Hect domain-catalyzed conjugation through the canonical binding site suggested by the crystal structure and define a novel two-step mechanism for formation of the polyubiquitin degradation signal.

Essential role of ubiquitin C‐terminal hydrolases UCHL1 and UCHL3 in mammalian oocyte maturation

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of deubiquitinating enzymes that play a role in the removal of multi-ubiquitin chains from proteins that are posttranslationally modified by ubiquitination to be targeted for proteolysis by the 26S proteasome. The UCH-enzymes also generate free monomeric ubiquitin from precursor multi-ubiquitin chains and, in some instances, may rescue ubiquitinated proteins from degradation. This study examined the roles of two oocyte-expressed UCHs, UCHL1, and UCHL3 in murine and rhesus monkey oocyte maturation. The Uchl1 and Uchl3 mRNAs were highly expressed in GV and MII oocytes, and were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Microinjection of the UCH-family enzyme inhibitor, ubiquitin-aldehyde (UBAL) to GV oocytes prevented oocyte meiotic progression beyond metaphase I in a majority of treated oocytes and caused spindle and first polar body anomalies. Injection of antibodies against UCHL3 disrupted oocyte maturation and caused meiotic anomalies, including abnormally long meiotic spindles. A selective, cell permeant inhibitor of UCHL3, 4, 5, 6, 7-tetrachloroidan-1, 3-dione also caused meiotic defects and chromosome misalignment. Cortical granule localization in the oocyte cortex was disrupted by UBAL injected after oocyte maturation. We conclude that the activity of oocyte UCHs contributes to oocyte maturation by regulating the oocyte cortex and meiotic spindle.

A novel conserved isoform of the ubiquitin ligase UFD2a/UBE4B is expressed exclusively in mature striated muscle cells.

Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish), UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3′ introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis.

Preparation of Distinct Ubiquitin Chain Reagents of High Purity and Yield

The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.

A Proteasome Assembly Defect in rpn3 Mutants Is Associated with Rpn11 Instability and Increased Sensitivity to Stress

Rpn11 is a proteasome-associated deubiquitinating enzyme that is essential for viability. Recent genetic studies showed that Rpn11 is functionally linked to Rpn10, a major multiubiquitin chain binding receptor in the proteasome. Mutations in Rpn11 and Rpn10 can reduce the level and/or stability of proteasomes, indicating that both proteins influence its structural integrity. To characterize the properties of Rpn11, we examined its interactions with other subunits in the 19S regulatory particle and detected strong binding to Rpn3. Two previously described rpn3 mutants are sensitive to protein translation inhibitors and an amino acid analog. These mutants also display a mitochondrial defect. The abundance of intact proteasomes was significantly reduced in rpn3 mutants, as revealed by strongly reduced binding between 20S catalytic with 19S regulatory particles. Proteasome interaction with the shuttle factor Rad23 was similarly reduced. Consequently, higher levels of multiUb proteins were associated with Rad23, and proteolytic substrates were stabilized. The availability of Rpn11 is important for maintaining adequate levels of intact proteasomes, as its depletion caused growth and proteolytic defects in rpn3. These studies suggest that Rpn11 is stabilized following its incorporation into proteasomes. The instability of Rpn11 and the defects of rpn3 mutants are apparently caused by a failure to recruit Rpn11 into mature proteasomes.

Proteome Dynamics and Proteome Function of Cardiac 19S Proteasomes

Myocardial proteasomes are comprised of 20S core particles and 19S regulatory particles, which together carry out targeted degradation of cardiac proteins. The 19S complex is unique among the regulators of proteasomes in that it affects both the capacity and specificity of protein degradation. However, a comprehensive molecular characterization of cardiac 19S complexes is lacking. In this investigation, we tailored a multidimensional chromatography-based purification strategy to isolate structurally intact and functionally viable 19S complexes from murine hearts. Two distinct subpopulations of 19S complexes were isolated based upon (1) potency of activating 20S proteolytic activity, and (2) molecular composition using a combination of immuno-detection, two-dimensional-differential gel electrophoresis, and MS-based approaches. Heat shock protein 90 (Hsp90) was identified to be characteristic to 19S subpopulation I. The physical interaction of Hsp90 with 19S complexes was demonstrated via multiple approaches. Inhibition of Hsp90 activity using geldanamycin or BIIB021 potentiated the ability of subpopulation I to activate 20S proteasomes in the murine heart, thus demonstrating functional specificity of Hsp90 in subpopulation I. This investigation has advanced our understanding of the molecular heterogeneity of cardiac proteasomes by identifying molecularly and functionally distinct cardiac 19S complexes. The preferential association of Hsp90 with 19S subpopulation I unveils novel targets for designing proteasome-based therapeutic interventions for combating cardiac disease.

The Steady-State Repertoire of Human SCF Ubiquitin Ligase Complexes Does Not Require Ongoing Nedd8 Conjugation

The human genome encodes 69 different F-box proteins (FBPs), each of which can potentially assemble with Skp1-Cul1-RING to serve as the substrate specificity subunit of an SCF ubiquitin ligase complex. SCF activity is switched on by conjugation of the ubiquitin-like protein Nedd8 to Cul1. Cycles of Nedd8 conjugation and deconjugation acting in conjunction with the Cul1-sequestering factor Cand1 are thought to control dynamic cycles of SCF assembly and disassembly, which would enable a dynamic equilibrium between the Cul1-RING catalytic core of SCF and the cellular repertoire of FBPs. To test this hypothesis, we determined the cellular composition of SCF complexes and evaluated the impact of Nedd8 conjugation on this steady-state. At least 42 FBPs assembled with Cul1 in HEK 293 cells, and the levels of Cul1-bound FBPs varied by over two orders of magnitude. Unexpectedly, quantitative mass spectrometry revealed that blockade of Nedd8 conjugation led to a modest increase, rather than a decrease, in the overall level of most SCF complexes. We suggest that multiple mechanisms including FBP dissociation and turnover cooperate to maintain the cellular pool of SCF ubiquitin ligases.

Synthetic lethality of rpn11-1 rpn10Δ is linked to altered proteasome assembly and activity

An rpn11-1 temperature-sensitive mutant shows defect in proteolysis, mitochondrial function and proteasome assembly. The Rpn11 protein is a proteasome subunit that deubiquitinates proteolytic substrates. Multiubiquitinated proteins interact with proteasome receptors, such as Rpn10, which intriguingly is also required for promoting proteasome stability. We report here that Rpn10 binds Rpn11, and genetic studies revealed synthetic lethality of an rpn11-1 rpn10Δ double mutant. The carboxy-terminus of Rpn11 is critical for function, as deletion of 7 C-terminal residues prevented suppression of rpn11-1 rpn10Δ. Native gel electrophoresis showed increased levels of the proteasome 20S catalytic particle in rpn11-1 rpn10Δ, and altered assembly. The inviability of rpn11-1 rpn10Δ was suppressed by rpn10(uim), a mutant that can bind the proteasome, but not multiubiquitin chains. rpn10(uim) reduced the levels of free 20S, and increased formation of intact proteasomes. In contrast, rpn10(vwa), which binds multiubiquitin chains but not the proteasome, failed to suppress rpn11-1 rpn10Δ. Moreover, high levels of multiubiquitinated proteins were bound to rpn10(vwa), but were not delivered to the proteasome. Based on these findings, we propose that the lethality of rpn11-1 rpn10Δ results primarily from altered proteasome integrity. It is conceivable that Rpn10/Rpn11 interaction couples proteasome assembly to substrate binding.

Proteasomal Degradation Is Transcriptionally Controlled by TCF11 via an ERAD-Dependent Feedback Loop

Coordinated regulation of the ubiquitin-proteasome system (UPS) is crucial for the cell to adjust its protein degradation capacity to changing proteolytic requirements. We have shown previously that mammalian cells upregulate proteasome gene expression in response to proteasome inhibition. Here, we report the identification of the transcription factor TCF11 (long isoform of Nrf1) as a key regulator for 26S proteasome formation in human cells to compensate for reduced proteolytic activity. Under noninducing conditions, TCF11 resides in the endoplasmic reticulum (ER) membrane. There, TCF11 is targeted to ER-associated protein degradation requiring the E3 ubiquitin ligase HRD1 and the AAA ATPase p97. Proteasome inhibitors trigger the accumulation of oxidant-damaged proteins and promote the nuclear translocation of TCF11 from the ER, permitting activation of proteasome gene expression by binding to antioxidant response elements in their promoter regions. Thus, we uncovered the transcriptional control loop regulating human proteasome-dependent protein degradation to counteract proteotoxic stress caused by proteasome inhibition.

Trimming of Ubiquitin Chains by Proteasome-associated Deubiquitinating Enzymes

The proteasome generally recognizes substrate via its multiubiquitin chain followed by ATP-dependent unfolding and translocation of the substrate from the regulatory particle into the proteolytic core particle to be degraded. Substrate-bound ubiquitin groups are for the most part not delivered to the core particle and broken down together with substrate but instead recovered as intact free ubiquitin and ubiquitin chains. Substrate deubiquitination on the proteasome is mediated by three distinct deubiquitinating enzymes associated with the regulatory particle: RPN11, UCH37, and USP14. RPN11 cleaves at the base of the ubiquitin chain where it is linked to the substrate, whereas UCH37 and apparently USP14 mediate a stepwise removal of ubiquitin from the substrate by disassembling the chain from its distal tip. In contrast to UCH37 and USP14, RPN11 shows degradation-coupled activity; RPN11-mediated deubiquitination is apparently delayed until the proteasome is committed to degrade the substrate. Accordingly, RPN11-mediated deubiquitination promotes substrate degradation. In contrast, removal of ubiquitin prior to commitment could antagonize substrate degradation by promoting substrate dissociation from the proteasome. Emerging evidence suggests that USP14 and UCH37 can both suppress substrate degradation in this way. One line of study has shown that small molecule USP14 inhibitors can enhance proteasome function in cells, which is consistent with this model. Enhancing protein degradation could potentially have therapeutic applications for diseases involving toxic proteins that are proteasome substrates. However, the responsiveness of substrates to inhibition of proteasomal deubiquitinating enzymes may vary substantially. This substrate specificity and its mechanistic basis should be addressed in future studies.

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.

In Vitro Analysis of Hrd1p-mediated Retrotranslocation of Its Multispanning Membrane Substrate 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase

Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for the ubiquitin-mediated destruction of both misfolded and normal ER-resident proteins. ERAD substrates must be moved from the ER to the cytoplasm for ubiquitination and proteasomal destruction by a process called retrotranslocation. Many aspects of retrotranslocation are poorly understood, including its generality, the cellular components required, the energetics, and the mechanism of transfer through the ER membrane. To address these questions, we have developed an in vitro assay, using the 8-transmembrane span ER-resident Hmg2p isozyme of HMG-CoA reductase fused to GFP, which undergoes regulated ERAD mediated by the Hrd1p ubiquitin ligase. We have now directly demonstrated in vitro retrotranslocation of full-length, ubiquitinated Hmg2p-GFP to the aqueous phase. Hrd1p was rate-limiting for Hmg2p-GFP retrotranslocation, which required ATP, the AAA-ATPase Cdc48p, and its receptor Ubx2p. In addition, the adaptors Dsk2p and Rad23p, normally implicated in later parts of the pathway, were required. Hmg2p-GFP retrotranslocation did not depend on any of the proposed ER channel candidates. To examine the role of the Hrd1p transmembrane domain as a retrotranslocon, we devised a self-ubiquitinating polytopic substrate (Hmg1-Hrd1p) that undergoes ERAD in the absence of Hrd1p. In vitro retrotranslocation of full-length Hmg1-Hrd1p occurred in the absence of the Hrd1p transmembrane domain, indicating that it did not serve a required channel function. These studies directly demonstrate polytopic membrane protein retrotranslocation during ERAD and delineate avenues for mechanistic understanding of this general process.