The emerging role of deubiquitylating enzymes as therapeutic targets in cancer metabolism.

Cancer cells must rewire cellular metabolism to satisfy the unbridled proliferation, and metabolic reprogramming provides not only the advantage for cancer cell proliferation but also new targets for cancer treatment. However, the plasticity of the metabolic pathways makes them very difficult to target. Deubiquitylating enzymes (DUBs) are proteases that cleave ubiquitin from the substrate proteins and process ubiquitin precursors. While the molecular mechanisms are not fully understood, many DUBs have been shown to be involved in tumorigenesis and progression via controlling the dysregulated cancer metabolism, and consequently recognized as potential drug targets for cancer treatment. In this article, we summarized the significant progress in understanding the key roles of DUBs in cancer cell metabolic rewiring and the opportunities for the application of DUBs inhibitors in cancer treatment, intending to provide potential implications for both research purpose and clinical applications.

ABPP-HT*-Deep Meets Fast for Activity-Based Profiling of Deubiquitylating Enzymes Using Advanced DIA Mass Spectrometry Methods.

Activity-based protein profiling (ABPP) uses a combination of activity-based chemical probes with mass spectrometry (MS) to selectively characterise a particular enzyme or enzyme class. ABPP has proven invaluable for profiling enzymatic inhibitors in drug discovery. When applied to cell extracts and cells, challenging the ABP-enzyme complex formation with a small molecule can simultaneously inform on potency, selectivity, reversibility/binding affinity, permeability, and stability. ABPP can also be applied to pharmacodynamic studies to inform on cellular target engagement within specific organs when applied to in vivo models. Recently, we established separate high depth and high throughput ABPP (ABPP-HT) protocols for the profiling of deubiquitylating enzymes (DUBs). However, the combination of the two, deep and fast, in one method has been elusive. To further increase the sensitivity of the current ABPP-HT workflow, we implemented state-of-the-art data-independent acquisition (DIA) and data-dependent acquisition (DDA) MS analysis tools. Hereby, we describe an improved methodology, ABPP-HT* (enhanced high-throughput-compatible activity-based protein profiling) that in combination with DIA MS methods, allowed for the consistent profiling of 35-40 DUBs and provided a reduced number of missing values, whilst maintaining a throughput of 100 samples per day.

PR-619, a General Inhibitor of Deubiquitylating Enzymes, Diminishes Cisplatin Resistance in Urothelial Carcinoma Cells through the Suppression of c-Myc: An In Vitro and In Vivo Study.

Cisplatin-based chemotherapy is the standard treatment for bladder urothelial carcinoma (UC). Most patients experience chemoresistance, the primary cause of treatment failure, which leads to disease relapse. The underlying mechanism of chemoresistance involves reduced apoptosis. In this study, we investigated the antitumor effect of the deubiquitylating enzyme inhibitor PR-619 in cisplatin-resistant bladder UC. Deubiquitinase (ubiquitin-specific protease 14 (USP14) and USP21) immunohistochemical staining demonstrated that deubiquitination is related to chemoresistance in patients with metastatic UC and may be a target for overcoming chemoresistance. Cytotoxicity and apoptosis were assessed using fluorescence-activated flow cytometry and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay, and PR-619 was found to enhance the cytotoxic and apoptotic effects of cisplatin in cisplatin-resistant T24/R cells. Mitigated cisplatin chemoresistance was associated with the concurrent suppression of c-Myc expression in T24/R cells. Moreover, the expression of c-Myc was upregulated in human bladder UC specimens from patients with chemoresistance. Experiments in a xenograft nude mouse model confirmed that PR-619 enhanced the antitumor effects of cisplatin. These results are promising for the development of therapeutic strategies to prevent UC chemoresistance through the combined use of chemotherapeutic agents/deubiquitination inhibitors (PR-619) by targeting the c-Myc pathway.

Regulation of Ubiquitin Enzymes in the TGF-ß Pathway.

The transforming growth factor-ß (TGF-ß) pathway has a tumor suppressor role in normal and premalignant cells but promotes oncogenesis in advanced cancer cells. Components of the pathway are tightly controlled by ubiquitin modifying enzymes and aberrations in these enzymes are frequently observed to dysregulate the pathway causing diseases such as bone disorders, cancer and metastasis. These enzymes and their counterparts are increasingly being tested as druggable targets, and thus a deeper understanding of the enzymes is required. This review summarizes the roles of specific ubiquitin modifying enzymes in the TGF-ß pathway and how they are regulated.

Specificity profiling of deubiquitylases against endogenously generated ubiquitin-protein conjugates.

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high-impact DUBs (USP7, USP9X, USP36, USP15, and USP24) that each reduced ubiquitylation of over 10% of the isolated proteins. Candidate substrates of high-impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

UCHL5 is a putative prognostic marker in renal cell carcinoma: a study of UCHL family.

A macroscopic perspective is indispensable for understanding the intricate relationship between deubiquitinases and tumorigenesis. Proteomics has been proposed as a viable approach for elucidating the complex role of deubiquitylation in cellular progression. Instead of studying the function of a single ubiquitinase, research on a deubiquitinase family with similar catalytic core(s) may provide a new perspective for the pathological understanding of cancer. The Ubiquitin C-terminal hydrolase L (UCHL) family consists of four members: UCHL1, UCHL3, UCHL5, and BRAC1 associated protein-1 (BAP1), and they have been implicated in tumorigenesis and metastasis. Some members are considered hallmarks of intracranial lesions, colon cancer, chromatin remodeling, and histone stability. The present study uncovered an unknown correlation between the UCHL family and renal cancer. We discovered that UCHLs exhibit diverse regulatory effects in renal cancer, establishing connections between the renal cancer and truncated gene mutations, mitochondrial energetic metastasis, immune cell infiltration, and chromosomal stability of UCHLs family. Notably, we found that the increase of UCHL5 expression in renal cancer cells decreases the antigen processing and presentation of RCC tumor-infiltrating B cells. Further research identified that the expression of UCHL5 in RCC tumors is correlated with transport proteins, which led us to find that the abundance of UCHL5 in the blood of late-stage renal cell cancer patients is upregulated from 18 ng/L to 500 ng/L. Therefore, we propose that the abundance of UCHL5 in patients' blood can be a possible indicator of poor prognosis for renal cell cancer.

USP7 inhibitors suppress tumour neoangiogenesis and promote synergy with immune checkpoint inhibitors by downregulating fibroblast VEGF.

BACKGROUND: Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts. METHODS: To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co-cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs). RESULTS: Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8-positive T-lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival. CONCLUSIONS: USP7-mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well-characterized VEGF transcription factor, HIF-1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform "immune desert" tumors into "immune responsive" tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs).

Deubiquitylating Enzymes in Cancer and Immunity.

Deubiquitylating enzymes (DUBs) maintain relative homeostasis of the cellular ubiquitome by removing the post-translational modification ubiquitin moiety from substrates. Numerous DUBs have been demonstrated specificity for cleaving a certain type of ubiquitin linkage or positions within ubiquitin chains. Moreover, several DUBs perform functions through specific protein-protein interactions in a catalytically independent manner, which further expands the versatility and complexity of DUBs' functions. Dysregulation of DUBs disrupts the dynamic equilibrium of ubiquitome and causes various diseases, especially cancer and immune disorders. This review summarizes the Janus-faced roles of DUBs in cancer including proteasomal degradation, DNA repair, apoptosis, and tumor metastasis, as well as in immunity involving innate immune receptor signaling and inflammatory and autoimmune disorders. The prospects and challenges for the clinical development of DUB inhibitors are further discussed. The review provides a comprehensive understanding of the multi-faced roles of DUBs in cancer and immunity.

Structural characterization of human Uch37.

Uch37 is a de-ubiquitylating enzyme that is functionally linked with the 26S proteasome via Rpn13, and is essential for metazoan development. Here, we report the X-ray crystal structure of full-length human Uch37 at 2.95 Å resolution. Uch37's catalytic domain is similar to those of all UCH enzymes characterized to date. The C-terminal extension is elongated, predominantly helical and contains coiled coil interactions. Additionally, we provide an initial characterization of Uch37's oligomeric state and identify a systematic error in previous analyses of Uch37 activity. Taken together, these data provide a strong foundation for further analysis of Uch37's several functions.

UCH-L3 structure and function: Insights about a promising drug target.

In the past few years, researchers have shed light on the immense importance of ubiquitin in numerous regulatory pathways. The post-translational addition of mono or poly-ubiquitin molecules namely "ubiquitinoylation" is therefore pivotal to maintain the cell's vitality, maturation, differentiation, and division. Part of conserving homeostasis stems from maintaining the ubiquitin pool in the vicinity of the cell's intracellular environment; this crucial role is played by deubiquitylating enzymes (DUBs) that cleave ubiquitin molecules from target molecules. To date, they are categorized into 7 families with ubiquitin carboxyl c-terminal de-hydrolase family (UCH) as the most common and well-studied. Ubiquitin C-terminal hydrolase L (UCH-L3) is a significant protein in this family as it has been implicated in many molecular and cellular processes with its mRNA identified in a range of body tissues including the brain. It goes without saying that it manifests in maintaining health and when abnormally regulated in disease. As it is an attractive small molecule drug target, scientists have used high throughput screening (HTS) and other drug discovery methods to discover inhibitors for this enzyme for the treatment of cancer and neurodegenerative diseases. In this review we present an overview of UCH-L3 catalytic mechanism, structure, its role in DNA repair and cancer along with the inhibitors discovered so far to halt its activity.

Cellular Deubiquitylating Enzyme: A Regulatory Factor of Antiviral Innate Immunity.

Deubiquitylating enzymes (DUBs) are proteases that crack the ubiquitin code from ubiquitylated substrates to reverse the fate of substrate proteins. Recently, DUBs have been found to mediate various cellular biological functions, including antiviral innate immune response mediated by pattern-recognition receptors (PRRs) and NLR Family pyrin domain containing 3 (NLRP3) inflammasomes. So far, many DUBs have been identified to exert a distinct function in fine-tuning antiviral innate immunity and are utilized by viruses for immune evasion. Here, the recent advances in the regulation of antiviral responses by DUBs are reviewed. We also discussed the DUBs-mediated interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and antiviral innate immunity. The understanding of the mechanisms on antiviral innate immunity regulated by DUBs may provide therapeutic opportunities for viral infection.

Deubiquitinating Enzymes Orchestrate the Cancer Stem Cell-Immunosuppressive Niche Dialogue: New Perspectives and Therapeutic Potential.

Cancer stem cells (CSCs) are sparks for igniting tumor recurrence and the instigators of low response to immunotherapy and drug resistance. As one of the important components of tumor microenvironment, the tumor associated immune microenvironment (TAIM) is driving force for the heterogeneity, plasticity and evolution of CSCs. CSCs create the inhibitory TAIM (ITAIM) mainly through four stemness-related signals (SRSs), including Notch-nuclear factor-κB axis, Hedgehog, Wnt and signal transducer and activator of transcription. Ubiquitination and deubiquitination in proteins related to the specific stemness of the CSCs have a profound impact on the regulation of ITAIM. In regulating the balance between ubiquitination and deubiquitination, it is crucial for deubiquitinating enzymes (DUBs) to cleave ubiquitin chains from substrates. Ubiquitin-specific peptidases (USPs) comprise the largest family of DUBs. Growing evidence suggests that they play novel functions in contribution of ITAIM, including regulating tumor immunogenicity, activating stem cell factors, upregulating the SRSs, stabilizing anti-inflammatory receptors, and regulating anti-inflammatory cytokines. These overactive or abnormal signaling may dampen antitumor immune responses. The inhibition of USPs could play a regulatory role in SRSs and reversing ITAIM, and also have great potential in improving immune killing ability against tumor cells, including CSCs. In this review, we focus on the USPs involved in CSCs signaling pathways and regulating ITAIM, which are promising therapeutic targets in antitumor therapy.

Ubiquitin-specific peptidase 37: an important cog in the oncogenic machinery of cancerous cells.

Protein ubiquitination is one of the most crucial posttranslational modifications responsible for regulating the stability and activity of proteins involved in homeostatic cellular function. Inconsistencies in the ubiquitination process may lead to tumorigenesis. Ubiquitin-specific peptidases are attractive therapeutic targets in different cancers and are being evaluated for clinical development. Ubiquitin-specific peptidase 37 (USP37) is one of the least studied members of the USP family. USP37 controls numerous aspects of oncogenesis, including stabilizing many different oncoproteins. Recent work highlights the role of USP37 in stimulating the epithelial-mesenchymal transition and metastasis in lung and breast cancer by stabilizing SNAI1 and stimulating the sonic hedgehog pathway, respectively. Several aspects of USP37 biology in cancer cells are yet unclear and are an active area of research. This review emphasizes the importance of USP37 in cancer and how identifying its molecular targets and signalling networks in various cancer types can help advance cancer therapeutics.

ABPP-HT - High-Throughput Activity-Based Profiling of Deubiquitylating Enzyme Inhibitors in a Cellular Context.

The potency and selectivity of a small molecule inhibitor are key parameters to assess during the early stages of drug discovery. In particular, it is very informative for characterizing compounds in a relevant cellular context in order to reveal potential off-target effects and drug efficacy. Activity-based probes are valuable tools for that purpose, however, obtaining cellular target engagement data in a high-throughput format has been particularly challenging. Here, we describe a new methodology named ABPP-HT (high-throughput-compatible activity-based protein profiling), implementing a semi-automated proteomic sample preparation workflow that increases the throughput capabilities of the classical ABPP workflow approximately ten times while preserving its enzyme profiling characteristics. Using a panel of deubiquitylating enzyme (DUB) inhibitors, we demonstrate the feasibility of ABPP-HT to provide compound selectivity profiles of endogenous DUBs in a cellular context at a fraction of time as compared to previous methodologies.

Proteomics of broad deubiquitylase inhibition unmasks redundant enzyme function to reveal substrates and assess enzyme specificity.

Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control stability or activity of substrates. Identification of DUB substrates is challenging because multiple DUBs can act on the same substrate, thwarting genetic approaches. Here, we circumvent redundancy by chemically inhibiting multiple DUBs simultaneously in Xenopus egg extract. We used quantitative mass spectrometry to identify proteins whose ubiquitylation or stability is altered by broad DUB inhibition, and confirmed their DUB-dependent regulation with human orthologs, demonstrating evolutionary conservation. We next extended this method to profile DUB specificity. By adding recombinant DUBs to extract where DUB activity was broadly inhibited, but ubiquitylation and degradation were active at physiological rates, we profiled the ability of DUBs to rescue degradation of these substrates. We found that USP7 has a unique ability to broadly antagonize degradation. Together, we present an approach to identify DUB substrates and characterize DUB specificity that overcomes challenges posed by DUB redundancy.

USP25 Regulates EGFR Fate by Modulating EGF-Induced Ubiquitylation Dynamics.

Deregulated epidermal growth factor receptor (EGFR) signaling is a key feature in different stages of oncogenesis. One important mechanism whereby cancer cells achieve increased and uncontrolled EGFR signaling is escaping down-modulation of the receptor. Ubiquitylation of the EGFR plays a decisive role in this process, as it regulates receptor internalization, trafficking and degradation. Deubiquitinating enzymes (DUBs) may oppose the ubiquitylation process, antagonizing or even promoting receptor degradation. Here, we use qualitative and quantitative assays to measure EGFR internalization and degradation after Ubiquitin Specific Peptidase 25 (USP25) depletion. We show that, by acting at the early steps of EGFR internalization, USP25 restrains the degradation of the EGFR by assisting in the association of the E3 ubiquitin ligase c-Cbl with EGFR, thereby modulating the amplitude of ubiquitylation on the receptor. This study establishes USP25 as a negative regulator of the EGFR down-modulation process and suggests that it is a promising target for pharmacological intervention to hamper oncogenic growth signals in tumors that depend on the EGFR.

Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized.

Activation of the integrated stress response (ISR) or the ribosome-associated quality control (RQC) pathway stimulates regulatory ribosomal ubiquitylation (RRub) on distinct 40S ribosomal proteins, yet the cellular role and fate of ubiquitylated proteins remain unclear. We demonstrate that uS10 and uS5 ubiquitylation are dependent upon eS10 or uS3 ubiquitylation, respectively, suggesting that a hierarchical relationship exists among RRub events establishing a ubiquitin code on ribosomes. We show that stress dependent RRub events diminish after initial stimuli and that demodification by deubiquitylating enzymes contributes to reduced RRub levels during stress recovery. Utilizing an optical RQC reporter we identify OTUD3 and USP21 as deubiquitylating enzymes that antagonize ZNF598-mediated 40S ubiquitylation and can limit RQC activation. Critically, cells lacking USP21 or OTUD3 have altered RQC activity and delayed eS10 deubiquitylation indicating a functional role for deubiquitylating enzymes within the RQC pathway.

Ribosomes are cellular machines that build proteins by latching on and then reading template molecules known as mRNAs. Several ribosomes may be moving along the same piece of mRNA at the same time, each making their own copy of the same protein. Damage to an mRNA or other problems may cause a ribosome to stall, leading to subsequent collisions. A quality control pathway exists to identify stalled ribosomes and fix the 'traffic jams'. It relies on enzymes that tag halted ribosomes with molecules known as ubiquitin. The cell then removes these ribosomes from the mRNA and destroys the proteins they were making. Afterwards, additional enzymes take off the ubiquitin tags so the cell can recycle the ribosomes. These enzymes are key to signaling the end of the quality control event, yet their identity was still unclear. Garshott et al. used genetic approaches to study traffic jams of ribosomes in mammalian cells. The experiments showed that cells added sets of ubiquitin tags to stalled ribosomes in a specific order. Two enzymes, known as USP21 and OTUD3, could stop this process; this allowed ribosomes to carry on reading mRNA. Further work revealed that the ribosomes in cells that produce higher levels of USP21 and OTUD3 were less likely to stall on mRNA. On the other hand, ribosomes in cells lacking USP1 and OTUD3 retained their ubiquitin tags for longer and were more likely to stall. The findings of Garshott et al. reveal that USP21 and OTUD3 are involved in the quality control pathway which fixes ribosome traffic jams. In mice, problems in this pathway have been linked with neurons dying or being damaged because toxic protein products start to accumulate in cells; this is similar to what happens in human conditions such as Alzheimer's and Parkinson's diseases. Using ubiquitin to target and potentially fix the pathway could therefore open the door to new therapies.

Comprehensive Landscape of Active Deubiquitinating Enzymes Profiled by Advanced Chemoproteomics.

Enzymes that bind and process ubiquitin, a small 76-amino-acid protein, have been recognized as pharmacological targets in oncology, immunological disorders, and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition to the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation, and more potent/selective ubiquitin active-site probes with propargylic-based electrophiles to profile 74 DUBs including distinguishable isoforms for 5 DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents and pan-DUB inhibitors combined with probe labeling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39, and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small-molecule DUB selectivity profiling.