Proteasome-Bound UCH37/UCHL5 Debranches Ubiquitin Chains to Promote Degradation.

The linkage, length, and architecture of ubiquitin (Ub) chains are all important variables in providing tight control over many biological paradigms. There are clear roles for branched architectures in regulating proteasome-mediated degradation, but the proteins that selectively recognize and process these atypical chains are unknown. Here, using synthetic and enzyme-derived ubiquitin chains along with intact mass spectrometry, we report that UCH37/UCHL5, a proteasome-associated deubiquitinase, cleaves K48 branched chains. The activity and selectivity toward branched chains is markedly enhanced by the proteasomal Ub receptor RPN13/ADRM1. Using reconstituted proteasome complexes, we find that chain debranching promotes degradation of substrates modified with branched chains under multi-turnover conditions. These results are further supported by proteome-wide pulse-chase experiments, which show that the loss of UCH37 activity impairs global protein turnover. Our work therefore defines UCH37 as a debranching deubiquitinase important for promoting proteasomal degradation.

Small-Molecule Inhibitors Targeting Proteasome-Associated Deubiquitinases.

The 26S proteasome is the principal protease for regulated intracellular proteolysis. This multi-subunit complex is also pivotal for clearance of harmful proteins that are produced throughout the lifetime of eukaryotes. Recent structural and kinetic studies have revealed a multitude of conformational states of the proteasome in substrate-free and substrate-engaged forms. These conformational transitions demonstrate that proteasome is a highly dynamic machinery during substrate processing that can be also controlled by a number of proteasome-associated factors. Essentially, three distinct family of deubiquitinases-USP14, RPN11, and UCH37-are associated with the 19S regulatory particle of human proteasome. USP14 and UCH37 are capable of editing ubiquitin conjugates during the process of their dynamic engagement into the proteasome prior to the catalytic commitment. In contrast, RPN11-mediated deubiquitination is directly coupled to substrate degradation by sensing the proteasome's conformational switch into the commitment steps. Therefore, proteasome-bound deubiquitinases are likely to tailor the degradation events in accordance with substrate processing steps and for dynamic proteolysis outcomes. Recent chemical screening efforts have yielded highly selective small-molecule inhibitors for targeting proteasomal deubiquitinases, such as USP14 and RPN11. USP14 inhibitors, IU1 and its progeny, were found to promote the degradation of a subset of substrates probably by overriding USP14-imposed checkpoint on the proteasome. On the other hand, capzimin, a RPN11 inhibitor, stabilized the proteasome substrates and showed the anti-proliferative effects on cancer cells. It is highly conceivable that these specific inhibitors will aid to dissect the role of each deubiquitinase on the proteasome. Moreover, customized targeting of proteasome-associated deubiquitinases may also provide versatile therapeutic strategies for induced or repressed protein degradation depending on proteolytic demand and cellular context.

Deubiquitination Reactions on the Proteasome for Proteasome Versatility.

The 26S proteasome, a master player in proteolysis, is the most complex and meticulously contextured protease in eukaryotic cells. While capable of hosting thousands of discrete substrates due to the selective recognition of ubiquitin tags, this protease complex is also dynamically checked through diverse regulatory mechanisms. The proteasome's versatility ensures precise control over active proteolysis, yet prevents runaway or futile degradation of many essential cellular proteins. Among the multi-layered processes regulating the proteasome's proteolysis, deubiquitination reactions are prominent because they not only recycle ubiquitins, but also impose a critical checkpoint for substrate degradation on the proteasome. Of note, three distinct classes of deubiquitinating enzymes-USP14, RPN11, and UCH37-are associated with the 19S subunits of the human proteasome. Recent biochemical and structural studies suggest that these enzymes exert dynamic influence over proteasome output with limited redundancy, and at times act in opposition. Such distinct activities occur spatially on the proteasome, temporally through substrate processing, and differentially for ubiquitin topology. Therefore, deubiquitinating enzymes on the proteasome may fine-tune the degradation depending on various cellular contexts and for dynamic proteolysis outcomes. Given that the proteasome is among the most important drug targets, the biology of proteasome-associated deubiquitination should be further elucidated for its potential targeting in human diseases.

Proteasomal deubiquitinase UCH37 inhibits degradation of ß-catenin and promotes cell proliferation and motility.

The ubiquitin-proteasome system degrades most cellular proteins in eukaryotes. UCH37, also known as UCH-L5, is a deubiquitinase binding to Rpn13, a receptor for ubiquitinated substrates in the 26 S proteasome. But, it remains unclear how UCH37 influences the proteasomal degradation of the ubiquitinated substrates. Because deletion of UCH37 is embryonically lethal in mice, this study aims to investigate the role of UCH37 in proteasomal degradation by constructing the UCH37-deficient cell lines using CRISPR/Cas9 technology. Our results demonstrated that deletion of UCH37 decreased the levels of proteasomal Rpn13, implying that UCH37 might facilitate incorporation of Rpn13 into the proteasome. Meanwhile, deletion of UCH37 decreased the levels of ß-catenin and the early endosomal protein Rab8. ß-Catenin interacts with TCF/LEF to control transcription, and is involved in development, tissue homeostasis and tumorigenesis. We further found that deletion of UCH37 increased the levels of the ubiquitinated ß-catenin and accelerated the hydrogen peroxide-stimulated degradation of ß-catenin. Deletion of UCH37 also down-regulated the transcription of c-Myc, a downstream effector of ß-catenin, and inhibited cell proliferation and motility. These results raise the possibility that UCH37 maintains the homeostasis of proteasomal degradation reciprocally by assisting the recruitment of the ubiquitin receptor Rpn13 into the proteasome and by reversing ubiquitination of certain critical substrates of the 26 S proteasome.

The Proteasome Ubiquitin Receptor hRpn13 and Its Interacting Deubiquitinating Enzyme Uch37 Are Required for Proper Cell Cycle Progression.

Recently, we reported that bisbenzylidine piperidone RA190 adducts to Cys-88 of the proteasome ubiquitin receptor hRpn13, triggering accumulation of ubiquitinated proteins and endoplasmic reticulum stress-related apoptosis in various cancer cell lines. hRpn13 contains an N-terminal pleckstrin-like receptor for ubiquitin domain that binds ubiquitin and docks it into the proteasome as well as a C-terminal deubiquitinase adaptor (DEUBAD) domain that binds the deubiquitinating enzyme Uch37. Here we report that hRpn13 and Uch37 are required for proper cell cycle progression and that their protein knockdown leads to stalling at G0/G1 Moreover, serum-starved cells display reduced hRpn13 and Uch37 protein levels with hallmarks of G0/G1 stalling and recovery to their steady-state protein levels following release from nutrient deprivation. Interestingly, loss of hRpn13 correlates with a small but statistically significant reduction in Uch37 protein levels, suggesting that hRpn13 interaction may stabilize this deubiquitinating enzyme in human cells. We also find that RA190 treatment leads to a loss of S phase, suggesting a block of DNA replication, and G2 arrest by using fluorescence-activated cell sorting. Uch37 deprivation further indicated a reduction of DNA replication and G0/G1 stalling. Overall, this work implicates hRpn13 and Uch37 in cell cycle progression, providing a rationale for their function in cellular proliferation and for the apoptotic effect of the hRpn13-targeting molecule RA190.

Isolation and Characterization of RNA Aptamers against a Proteasome-Associated Deubiquitylating Enzyme UCH37.

Deubiquitylating (DUB) enzymes antagonize ubiquitin-dependent protein degradation both before and after the substrates are engaged with proteasomes. UCH37 is one of three proteasome-associated DUB enzymes in mammals and the only protease among them from the ubiquitin carboxyl-terminal hydrolase (UCH) family. Here, we report the identification of specific RNA aptamers for UCH37 through in vitro selection, and we describe their inhibitory effects on the DUB activity of UCH37. The RNA aptamers significantly delayed RPN13-mediated UCH37 activation and lowered total DUB activity of proteasomes, as measured by the hydrolysis of ubiquitin-rhodamine 110. In addition, the UCH37 aptamers efficiently facilitated the hydrolysis of peptide-based reporter substrates of proteasomes. Thus, the UCH37 aptamers might offer a possible strategy for removing toxic cellular proteins through enhancing proteasome activity.

Regulation of E2 promoter binding factor 1 (E2F1) transcriptional activity through a deubiquitinating enzyme, UCH37.

E2F1 is tightly controlled by multiple mechanisms, but whether ubiquitination regulates its transcriptional activity remains unknown. Here we identify UCH37 as the first, to our knowledge, deubiquitinating enzyme for E2F1. UCH37 does not deubiquitinate UbK48 chains or affect E2F1 protein stability. Instead, UCH37, but not a catalytically dead mutant, decreases the Lys-63-linked ubiquitination of E2F1 and activates its transcriptional activity. UCH37 depletion reduces the gene expression of both proliferative and pro-apoptotic E2F1 target genes. UCH37 depletion also decreases both cell proliferation and apoptosis induction in functional assays. Interestingly, UCH37 expression is induced by E2F1, and its level rises in G1/S transition and S phase, suggesting a positive feedback loop between UCH37 and E2F1. UCH37 protein and mRNA levels are also induced after DNA damage. UCH37 localizes to the promoters of E2F1 pro-apoptotic target genes such as caspase 3, caspase 7, PARP1, and Apaf-1 and activates their expression after DNA damage. Moreover, the expression of E2F1 proliferative and pro-apoptotic genes is correlated with the levels of UCH37 in many primary tumors. These results uncover a novel mechanism for E2F1 transcriptional activation through removal of its Lys-63-linked ubiquitination by UCH37.

Ubiquitination and regulation of Smad7 in the TGF-ß1/Smad signaling of aristolochic acid nephropathy.

Aristolochic acid I (AAI) affects TGF-ß1/Smad signaling, which causes AA nephropathy (AAN), but the mechanisms are not fully understood. We aimed to clarify whether Arkadia and UCH37 participate in TGF-ß1/Smad signaling via Smad7, and the regulatory mechanisms of Smad7. One side, mice and cultured mouse renal tubular epithelial cells (RTECs) were treated with various AAI doses and concentrations, respectively; on the other side, RTECs were transfected with small interfering RNA (siRNA) expression vectors against Arkadia and UCH37 and then treated with 10 µg/ml AAI. And then detect the mRNA and protein levels of Smad7, UCH37, Arkadia and any other relative factors by RT-PCR and Western blotting. In kidney tissues and RTECs, the mRNA and protein levels of Smad7 decreased with increasing AAI doses concentrations by real-time PCR and Western blotting, whereas those of Arkadia, UCH37, Smad2, Smad3 and TßRI increased. Cells transfected with the Arkadia siRNA expression vector showed reduced mRNA and protein levels of vimentin, α-SMA, Smad2, Smad3 and TßRI after AAI treatment, while those of CK18 and Smad7 increased compared with those of untransfected RTECs. Conversely, cells transfected with the UCH37 siRNA expression vector showed the opposite effect on analyzed signaling molecules after AAI treatment. Arkadia and UCH37 participate in TGF-ß1/Smad signaling-mediated renal fibrosis, and Smad7 blocks TGF-ß1 signaling by inhibiting Smad2/Smad3 phosphorylation and enhancing the degradation of TßRI.

ADRM1/RPN13 attenuates cartilage extracellular matrix degradation via enhancing UCH37-mediated ALK5 deubiquitination.

Osteoarthritis (OA) is the most common age-related joint disorder with no effective therapy, and its specific pathological mechanism remains to be fully clarified. Adhesion-regulating molecule 1 (ADRM1) has been proven to be involved in OA progression as a favorable gene. However, the exact mechanism of ADRM1 involved in OA were unknown. Here, we showed that the ADRM1 expression decreased in human OA cartilage, destabilization of the medial meniscus (DMM)-induced mouse OA cartilage, and interleukin (IL)-1ß-induced primary mouse articular chondrocytes. Global knockout (KO) ADRM1 in cartilage or ADRM1 inhibitor (RA190) could accelerate the disorders of extracellular matrix (ECM) homeostasis, thereby accelerated DMM-induced cartilage degeneration, whereas overexpression of ADRM1 protected mice from DMM-induced OA development by maintaining the homeostasis of articular cartilage. The molecular mechanism study revealed that ADRM1 could upregulate ubiquitin carboxy-terminal hydrolase 37 (UCH37) expression and bind to UCH37 to activate its deubiquitination activity. Subsequently, increased and activated UCH37 enhanced activin receptor-like kinase 5 (ALK5) deubiquitination to stabilize ALK5 expression, thereby maintaining ECM homeostasis and attenuating cartilage degeneration. These findings indicated that ADRM1 could attenuate cartilage degeneration via enhancing UCH37-mediated ALK5 deubiquitination. Overexpression of ADRM1 in OA cartilage may provide a promising OA therapeutic strategy.

A ubiquitinome analysis to study the functional roles of the proteasome associated deubiquitinating enzymes USP14 and UCH37.

The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14 (RPN11), USP14 and UCH37 (UCHL5). However, the functional roles and specificities of these proteasomal DUBs remain elusive. To reveal the specificities of proteasome associated DUBs, we used SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. We observed distinct effects on the global ubiquitinome upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggested less functional redundancy than previously anticipated. We also investigated whether the small molecule inhibitor b-AP15 has the potential to specifically target USP14 and UCH37 by comparing treatment of wild-type versus USP14/UCH37 double-knockout cells with this drug. Strikingly, broad and severe off-target effects were observed, questioning the alleged specificity of this inhibitor. In conclusion, this work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds. SIGNIFICANCE: Introduction: The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14/RPN11, USP14 and UCH37/UCHL5. However, the functional roles and specificities of these proteasomal DUBs remains elusive. MATERIALS & METHODS: We have applied a SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. Also, we have studied the function of the small molecule inhibitor b-AP15, which has the potential to specifically target USP14 and UCH37. RESULTS: We report distinct effects on the ubiquitinome and the ability of the proteasome to clear proteins upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggests less redundancy than previously anticipated. In addition, broad and severe off-target effects were observed for b-AP15, questioning the alleged specificity of this inhibitor. CONCLUSIONS: This work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds.

Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions.

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently, it was reported that UCH37 activity is stimulated by branched ubiquitin (Ub) chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and Nuclear Magnetic Resonance (NMR) structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal Ubs that emanate from a branched Ub. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear Ub chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome for the next round of substrate processing. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique Ub chain architecture is aided by a DUB.

Mechanistic Studies of the Multiple Myeloma and Melanoma Cell-Selective Toxicity of the Rpn13-Binding Peptoid KDT-11.

We previously reported a peptoid ligand for the proteasomal ubiquitin receptor Rpn13 called KDT-11 and demonstrated that this compound is toxic to multiple myeloma cells, but not non-malignant cells. Here, we show that KDT-11 decreases the viability of a variety of cancer cell lines, especially melanomas and various blood cancers. The peptoid induces selective G1 cell-cycle arrest, resulting in eventual apoptosis. While KDT-11 does not antagonize any of the known protein-protein interactions involving Rpn13, the peptoid inhibits the ability of Rpn13 to stimulate the activity of an associated deubiquitylase Uch37/UCHL5 in vitro, suggesting a high level of Uch37 activity might be important for cancer cell proliferation. However, a variety of experiments in SK-MEL-5 melanoma cells suggest that KDT-11's cytotoxic effects are mediated by interactions with proteins other than Rpn13.

Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity.

hRpn13/ADRM1 links substrate recruitment with deubiquitination at the proteasome through its proteasome- and ubiquitin-binding Pru domain and DEUBAD domain, which binds and activates deubiquitinating enzyme (DUB) UCHL5/Uch37. Here, we edit the HCT116 colorectal cancer cell line to delete part of the hRpn13 Pru, producing cells that express truncated hRpn13 (trRpn13), which is competent for UCHL5 binding but defective for proteasome interaction. trRpn13 cells demonstrate reduced levels of proteasome-bound ubiquitinated proteins, indicating that the loss of hRpn13 function at proteasomes cannot be fully compensated for by the two other dedicated substrate receptors (hRpn1 and hRpn10). Previous studies indicated that the loss of full-length hRpn13 causes a corresponding reduction of UCHL5. We find UCHL5 levels unaltered in trRpn13 cells, but hRpn11 is elevated in ΔhRpn13 and trRpn13 cells, perhaps from cell stress. Despite the ∼90 DUBs in human cells, including two others in addition to UCHL5 at the proteasome, we found deletion of UCHL5 from HCT116 cells to cause increased levels of ubiquitinated proteins in whole-cell extract and at proteasomes, suggesting that UCHL5 activity cannot be fully assumed by other DUBs. We also report anticancer molecule RA190, which binds covalently to hRpn13 and UCHL5, to require hRpn13 Pru and not UCHL5 for cytotoxicity.

Deubiquitinase UCHL5 is elevated and associated with a poor clinical outcome in lung adenocarcinoma (LUAD).

Lung cancer is one of the most common malignant tumors in the world, with a high rate of malignancy and mortality. Seeking new biomarkers and potential drug targets is urgent for effective treatment of the disease. Deubiquitinase UCHL5/UCH37, as an important component of the 26S proteasome, plays critical roles in ubiquitinated substrate degradation. Although previous studies have shown that UCHL5 promotes tumorigenesis, its role in lung cancer remains largely unknown. In this study, we evaluated the expression and clinical significance of UCHL5 in non-small cell lung cancer (NSCLC). The results demonstrated that the UCHL5 expression level was significantly upregulated in NSCLC tissues compared with the adjacent noncancerous tissues. The level of UCHL5 was associated with tumor size, lymph node invasion, TNM stage and malignant tumor history in patients with lung adenocarcinoma (LUAD). Importantly, high UCHL5 expression predicted a poor overall survival (OS) and a poor disease-free survival (DFS) in patients with LUAD. Univariate regression analysis showed that tumor size, lymph node invasion, TNM stage and UCHL5 expression were associated with OS and DFS in patients with LUAD. The multivariate analysis indicated that the UCHL5 expression level was an independent prognostic factor for OS (HR=1.171, 95% CI=1.052-1.303) and DFS (HR=1.143, 95% CI=1.031-1.267) in these patients. UCHL5 knockdown in LUAD cells significantly inhibited cell proliferation and reduced the expression of key cell cycle proteins. These findings indicate that UCHL5 may serve as a potential prognostic marker and a new therapeutic target for patients with LUAD.

Peroxiredoxin 1, restraining cell migration and invasion, is involved in hepatocellular carcinoma recurrence.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a high-burden disease. Peroxiredoxin 1 (PRDX1) is a member of the peroxiredoxin family of antioxidant enzymes. The aim of this study was to assess the value of PRDX1 for predicting HCC recurrence after curative resection and to explore the role of PRDX1 in HCC cell migration and invasion. METHODS: Data of patients with HCC who had undergone complete resection between 2002 and 2006 were collected. Immunohistochemical detection of PRDX1 in HCC tissue and adjacent non-cancerous tissue was conducted. Kaplan-Meier survival estimate and log-rank test were used to assess the relationship between PRDX1 expression and prognostic significance. HCC cell migration and invasion together with the interaction between PRDX1 and ubiquitin C-terminal hydrolase 37 (UCH37) were studied in vitro. RESULTS: PRDX1 was expressed at lower levels in HCC tissues than in adjacent non-cancerous tissues, and PRDX1 was found to be an independent risk factor for disease-free survival and overall survival. PRDX1 restrained cell migration and invasion in vitro. PRDX1 was found to interact with UCH37 to affect HCC cell migration and invasion. CONCLUSION: PRDX1 restrains cell migration and invasion in HCC cell lines and that may be involved in a UCH37-relevant pathway, suggesting that PRDX1 may be a new marker for HCC recurrence after surgery.

Meddling with Fate: The Proteasomal Deubiquitinating Enzymes.

Three deubiquitinating enzymes-Rpn11, Usp14, and Uch37-are associated with the proteasome regulatory particle. These enzymes allow proteasomes to remove ubiquitin from substrates before they are translocated into the core particle to be degraded. Although the translocation channel is too narrow for folded proteins, the force of translocation unfolds them mechanically. As translocation proceeds, ubiquitin chains bound to substrate are drawn to the channel's entry port, where they can impede further translocation. Rpn11, situated over the port, can remove these chains without compromising degradation because substrates must be irreversibly committed to degradation before Rpn11 acts. This coupling between deubiquitination and substrate degradation is ensured by the Ins-1 loop of Rpn11, which controls ubiquitin access to its catalytic site. In contrast to Rpn11, Usp14 and Uch37 can rescue substrates from degradation by promoting substrate dissociation from the proteasome prior to the commitment step. Uch37 is unique in being a component of both the proteasome and a second multisubunit assembly, the INO80 complex. However, only recruitment into the proteasome activates Uch37. Recruitment to the proteasome likewise activates Usp14. However, the influence of Usp14 on the proteasome depends on the substrate, due to its marked preference for proteins that carry multiple ubiquitin chains. Usp14 exerts complex control over the proteasome, suppressing proteasome activity even when inactive in deubiquitination. A major challenge for the field will be to elucidate the specificities of Rpn11, Usp14, and Uch37 in greater depth, employing not only model in vitro substrates but also their endogenous targets.

Polypharmacology of small molecules targeting the ubiquitin-proteasome and ubiquitin-like systems.

Targeting the ubiquitin-proteasome system (UPS) and ubiquitin-like signalling systems (UBL) has been considered a promising therapeutic strategy to treat cancer, neurodegenerative and immunological disorders. There have been multiple efforts recently to identify novel compounds that efficiently modulate the activities of different disease-specific components of the UPS-UBL. However, it is evident that polypharmacology (the ability to affect multiple independent protein targets) is a basic property of small molecules and even highly potent molecules would have a number of "off target" effects. Here we have explored publicly available high-throughput screening data covering a wide spectrum of currently accepted drug targets in order to understand polypharmacology of small molecules targeting different components of the UPS-UBL. We have demonstrated that molecules targeting a given UPS-UBL protein also have high odds to target a given off target spectrum. Moreover, the off target spectrum differs significantly between different components of UPS-UBL. This information can be utilized further in drug discovery efforts, to improve drug efficiency and to reduce the risk of potential side effects of the prospective drugs designed to target specific UPS-UBL components.