Specific Deubiquitinases Research Articles

A review of deubiquitinases and thier roles in tumorigenesis and development.

Ubiquitin is a small protein that can be added onto target protein for inducing target degradation, thereby modulating the activity and stability of protein. Relatively, deubiquitinases (DUBs), a class catalase that can remove ubiquitin from substrate protein, provide a positive regulation of the protein amount at transcription level, post-translational modification, protein interaction, etc. The reversible and dynamic ubiquitination-deubiquitination process plays an essential role in maintaining protein homeostasis, which is critical to almost all the biological processes. Therefore, the metabolic dysregulation of deubiquitinases often lead to serious consequences, including the growth and metastasis of tumors. Accordingly, deubiquitinases can be served as key drug targets for the treatment of tumors. The small molecule inhibitors targeting deubiquitinases has become one of the hot spots of anti-tumor drug research areas. This review concentrated on the function and mechanism of deubiquitinase system in the proliferation, apoptosis, metastasis and autophagy of tumor cells. The research status of small molecule inhibitors of specific deubiquitinases in tumor treatment is introduced, aiming to provide reference for the development of clinical targeted drugs.

Identification of Deubiquitinases (DUBs) that modulate PAR1‐mediated p38 MAPK inflammatory signaling in endothelial cells

Protease‐activated receptor‐1 (PAR1), a GPCR for thrombin, induces endothelial dysfunction via multiple signal transduction pathways including p38 mitogen‐activated protein kinase (MAPK). We showed that thrombin‐stimulates K63‐linked ubiquitination of PAR1 that drives recruitment of transforming growth factor‐β‐activated kinase‐1 binding protein‐2 (TAB2), an adaptor protein that binds TAB1, which triggers p38 activation on endosomes and promotes endothelial barrier disruption. However, the regulatory processes that control thrombin‐stimulated PAR1 ubiquitin‐dependent p38 inflammatory signaling are not known. To address this gap in knowledge, we sought to identify specific deubiquitinases (DUBs) that counter thrombin‐induced ubiquitin‐mediated p38 signaling by conducting an unbiased genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells. We hypothesize that a PAR1‐specific DUB is essential for controlling proper dynamics of thrombin‐mediated p38 signaling and might be altered in endothelial dysfunction. To systematically identify DUBs, endothelial cells were transfected with pools of 4 siRNAs targeting each of the 96 DUBs and thrombin‐stimulated p38 phosphorylation was assessed by immunoblotting. In parallel, a genome‐wide DUB siRNA library screen was conducted in HeLa cells. We identified 8 different DUBs that preferentially cleave K63‐linked ubiquitin and regulate thrombin‐p38 signaling in both endothelial and HeLa cells. A time‐course analysis of thrombin‐stimulated p38 signaling confirmed that depletion of each of the 8 DUBs alone caused an increase in the magnitude and/or duration of p38 signaling. Among the candidate DUBs, USP34, USP47 and CYLD exhibited the highest expression in both endothelial and HeLa cells detected by RT‐qPCR and were further examined. Using individual siRNAs targeting each of the 3 candidate DUBs, we validated that knockdown of either USP34, USP47 or CYLD alone was sufficient to markedly enhance thrombin‐induced p38 signaling in endothelial cells. Currently, I am examining the impact of USP34‐mediated modulation of thrombin‐p38 signaling on endothelial inflammatory responses. I will present new data examining the role of USP34 on thrombin‐induced endothelial barrier disruption and induction of cytokine expression. While ubiquitin‐dependent signaling is widespread, our knowledge of the processes that control ubiquitin‐mediated GPCR signaling is limited, especially related to the function of GPCR‐specific DUBs. Here, we report the first genome‐wide DUB siRNA library screen on GPCR signaling and identified a subset of DUBs that specifically regulate PAR1‐p38 inflammatory signaling that may serve as important therapeutic targets for vascular inflammation.

An siRNA library screen for endothelial PAR1‐specific deubiquitinases regulating p38 MAPK inflammatory signaling

Endothelial dysfunction is induced by inflammatory mediators that signal through GPCRs and a hallmark of inflammation. Protease‐activated receptor‐1 (PAR1), a GPCR for thrombin, induces endothelial dysfunction via multiple signal transduction pathways including p38 mitogen‐activated protein kinase (MAPK). We showed that thrombin‐stimulates K63‐linked ubiquitination of PAR1 that drives recruitment of transforming growth factor‐β‐activated kinase‐1 binding protein‐2 (TAB2), an adaptor protein that binds TAB1, which triggers p38 activation on endosomes and promotes endothelial barrier disruption. However, the regulatory processes that control thrombin‐stimulated PAR1 ubiquitin‐dependent p38 inflammatory signaling are not known. The objective of this study was to identify specific deubiquitinases (DUBs) that counter thrombin‐induced ubiquitin‐mediated p38 signaling by conducting an unbiased genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells. We hypothesize that a PAR1‐specific DUB is essential for controlling proper dynamics of thrombin‐mediated p38 signaling and might be altered in endothelial dysfunction. To systematically identify DUBs, endothelial cells were transfected with pools of 4 siRNAs targeting each of the 96 DUBs and thrombin‐stimulated p38 phosphorylation was assessed by immunoblotting. In parallel, a genome‐wide DUB siRNA library screen was conducted in HeLa cells. We identified 8 different DUBs that preferentially cleave K63‐linked ubiquitin and regulate thrombin‐p38 signaling in both endothelial and HeLa cells. A time‐course analysis of thrombin‐stimulated p38 signaling confirmed that depletion of each of the 8 DUBs alone caused an increase in the magnitude and/or duration of p38 signaling. Among the candidate DUBs, USP34, USP47 and CYLD exhibited the highest expression in both endothelial and HeLa cells detected by RT‐qPCR and were further examined. Using individual siRNAs targeting each of the 3 candidate DUBs, we validated that knockdown of either USP34, USP47 or CYLD alone was sufficient to markedly enhance thrombin‐induced p38 signaling in endothelial cells. New data examining the function of CYLD on thrombin‐induced inflammatory responses will be presented. While ubiquitin‐dependent signaling is widespread, our knowledge of the processes that control ubiquitin‐mediated GPCR signaling is limited, especially related to the function of GPCR‐specific DUBs. In this study, we report the first genome‐wide DUB siRNA library screen on GPCR signaling and identified a subset of DUBs that specifically regulate PAR1‐p38 inflammatory signaling that may serve as important therapeutic targets for vascular inflammation.

Roles and Mechanisms of Deubiquitinases (DUBs) in Breast Cancer Progression and Targeted Drug Discovery.

Deubiquitinase (DUB) is an essential component in the ubiquitin—proteasome system (UPS) by removing ubiquitin chains from substrates, thus modulating the expression, activity, and localization of many proteins that contribute to tumor development and progression. DUBs have emerged as promising prognostic indicators and drug targets. DUBs have shown significant roles in regulating breast cancer growth, metastasis, resistance to current therapies, and several canonical oncogenic signaling pathways. In addition, specific DUB inhibitors have been identified and are expected to benefit breast cancer patients in the future. Here, we review current knowledge about the effects and molecular mechanisms of DUBs in breast cancer, providing novel insight into treatments of breast cancer-targeting DUBs.

Uncovering the deubiquitinase activity landscape of breast cancer

Breast cancer is a highly heterogeneous disease with dynamic changes in the tumor microenvironment. Precision medicine will in the future provide the possibility to treat each individual cancer patient with the right (combination) therapy specifically tailored to personal needs. However, in order to accomplish this, more accurate biomarkers for precise diagnosis, prognosis, therapy response, and target-specific drugs are required. Although an increasing number of (epi)genetic driving alterations have been reported in breast cancer, the major stumbling block for clinical application of many of them is that they are difficult to therapeutically target. Deubiquitinases (DUBs) are emerging drug targets that play important roles in cancer progression. Hence, we devoted our efforts to uncover the global DUB activity landscape of breast cancer in order to discover potential novel biomarkers or therapeutic targets. We developed a specific DUB activity-based inhibitor and probe and applied it to obtain new insights into breast cancer.

An siRNA screen for endothelial PAR1‐specific deubiquitinases regulating p38 inflammatory signaling

Endothelial dysfunction is a hallmark of vascular disease and induced by various inflammatory mediators which signal through GPCRs pathways that are poorly understood. Thrombin induces endothelial inflammatory responses through the activation of protease‐activated receptor 1 (PAR1), a GPCR. Previously, we showed that thrombin‐activated PAR1 is modified by ubiquitin and drives recruitment of transforming growth factor‐β–activated kinase‐1–binding protein 2 (TAB2), an adaptor protein that binds TAB1 to trigger p38‐dependent endothelial barrier disruption, a hallmark of inflammation. However, the regulatory processes that control PAR1‐deubiquitination and associated p38 inflammatory signaling are not known. To fill this gap in knowledge, we sought to identify PAR1‐specific deubiquitinases (DUBs) that removes K63‐linked ubiquitin by conducting an unbiased comprehensive genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells using multiple PAR1 ubiquitin‐dependent functional readouts. We hypothesize that a PAR1‐specific DUB is essential for terminating thrombin‐induced p38 inflammatory signaling and might be altered in endothelial dysfunction. In an initial screen, endothelial cells were transfected with siRNA pools targeting specific DUBs and PAR1‐ubiquitin dependent responses were assessed. We specifically examined PAR1 cell surface expression, controlled by basal PAR1 ubiquitination, and thrombin‐stimulated p38 activation and downstream inflammatory signaling, which is dependent on agonist‐triggered PAR1 ubiquitination. In preliminary studies have identified several candidates PAR1‐specific DUBs. Currently, we are now investigating the function of top candidate DUBs in regulating the status of PAR1 ubiquitination, cell surface expression and p38 inflammatory signaling. The molecular mechanism responsible for PAR1 deubiquination and relation to controlling the spatio‐temporal dynamics of pro‐inflammatory signaling will be further investigated. Taken together, we anticipate that these studies using a comprehensive siRNA library screening approach will reveal the identity of several DUBS that have important functions in regulating PAR1‐induced ubiquitin driven p38 MAPK endothelial inflammatory signaling.Support or Funding InformationAuthors acknowledge NIH/NIGMS funding support.

Ubiquitin Linkage Specificity of Deubiquitinases Determines Cyclophilin Nuclear Localization and Degradation.

SummaryUbiquitin chain specificity has been described for some deubiquitinases (DUBs) but lacks a comprehensive profiling in vivo. We used quantitative proteomics to compare the seven lysine-linked ubiquitin chains between wild-type yeast and its 20 DUB-deletion strains, which may reflect the linkage specificity of DUBs in vivo. Utilizing the specificity and ubiquitination heterogeneity, we developed a method termed DUB-mediated identification of linkage-specific ubiquitinated substrates (DILUS) to screen the ubiquitinated lysine residues on substrates modified with certain chains and regulated by specific DUB. Then we were able to identify 166 Ubp2-regulating substrates with 244 sites potentially modified with K63-linked chains. Among these substrates, we further demonstrated that cyclophilin A (Cpr1) modified with K63-linked chain on K151 site was regulated by Ubp2 and mediated the nuclear translocation of zinc finger protein Zpr1. The K48-linked chains at non-K151 sites of Cpr1 were mainly regulated by Ubp3 and served as canonical signals for proteasome-mediated degradation.

Targeting the signalling pathways regulated by deubiquitinases for prostate cancer therapeutics.

Prostate cancer (PCa) is the most common cancer diagnosed and the second most common cause of cancer-related death in men worldwide. The current androgen deprivation therapy for PCa cannot fully cure this disease. Moreover, androgen receptor gene amplification and mutation are associated with PCa to develop castration-resistant prostate cancer (CRPC). This review focuses on the deubiquitinases (DUBs) involved in PCa development and progression. For PCa development and progression, several cellular pathways are regulated by specific DUBs which are also highlighted in here. The ubiquitin-specific proteases (USPs), a family member of DUBs mostly involved in the regulation of cellular pathways for PCa development, and the ubiquitin C-terminal hydrolases (UCHs), another family member of DUBs, are responsible for PCa metastasis. Small molecular inhibitors against DUBs can inhibit or reduce the level of specific DUBs through the regulation of cellular pathway to treat this disease. Some small molecular inhibitors are already identified against some of the DUBs, but very few of them are clinically proved in PCa. So, to find out other DUBs involving in the regulation of PCa-related pathways and to develop more effective small molecule inhibitors with greater potency would be a great idea to target PCa cells for future therapeutics and drug development with or without the combination of other anticancer drugs. SIGNIFICANCE OF THE STUDY: This review is targeting DUB proteins which are responsible for PCa induction, proliferation, and metastasis by highlighting their signalling pathway so that the readers can get information about other mechanisms for PCa besides androgen receptor pathway and helps to find other oncogenic DUBs involving in these signalling pathways. This review also hopes to find other oncogenic DUBs involving in PCa-related signalling pathways or to find the DUBs that can regulate multiple oncogenic signalling pathways which might be a good target for PCa therapeutics. In addition, there are some small molecule inhibitors that can inhibit the oncogenic DUBs and thus able to control the oncogenic pathways which would be a novel strategy to treat CRPC by using DUB inhibitor combined with or without other anticancer drugs.

TUBE and UbiCRest assays for elucidating polyubiquitin modifications in protein complexes.

Ubiquitination is a reversible posttranslational modification that regulates nearly all cellular processes. The ubiquitin polypeptide is conjugated via its C-terminus to amine groups of lysine residues on target protein. Additionally, ubiquitins moieties can be conjugated in tandem to the initial ubiquitin via any of its internal lysine residues or N terminal methionine residue, resulting in the formation of polyubiquitin chains with distinct biophysical properties and biological functions. Elucidating the types of polyubiquitin chains present in proteins is essential for understanding their function and mechanism of regulation. Traditionally, ubiqutin modifications have been elucidated by exogenously co-expressing proteins of interest with epitope-tagged ubiquitins mutated in specific lysine residues. However, this strategy is prone experimental artifacts. In this protocol, we describe how to elucidate endogenous ubiquitin modifications. This procedure combines TUBE (Tandem Ubiquitin Binding Entity)-based isolation of ubiquitin conjugates, digestion with linkage specific deubiquitinases and immunoblotting. This procedure is very robust can be applied to profile types and architectural organization polyubiquitin chains present on the any proteins of interest and has been instrumental in elucidating ubiquitin modifications in NOD2 signaling in our recent study (Panda & Gekara, 2018).

Diarylcarbonates are a new class of deubiquitinating enzyme inhibitor

Promiscuous inhibitors of tyrosine protein kinases, proteases and phosphatases are useful reagents for probing regulatory pathways and stabilizing lysates as well as starting points for the design of more selective agents. Ubiquitination regulates many critical cellular processes, and promiscuous inhibitors of deubiquitinases (DUBs) would be similarly valuable. The currently available promiscuous DUB inhibitors are highly reactive electrophilic compounds that can crosslink proteins. Herein we introduce diarylcarbonate esters as a novel class of promiscuous DUB inhibitors that do not have the liabilities associated with the previously reported compounds. Diarylcarbonates stabilize the high molecular weight ubiquitin pools in cells and lysates. They also elicit cellular phenotypes associated with DUB inhibition, demonstrating their utility in ubiquitin discovery. Diarylcarbonates may also be a useful scaffold for the development of specific DUB inhibitors.

Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells

Prostate cancer (PCa), a leading cause of cancer-related death in men, becomes resistant to androgen deprivation therapy by inducing androgen receptor (AR) activity, which is known as castration-resistant PCa (CRPC). Enzalutamide is an approved drug that inhibits AR activity and increases overall survival. However, resistance to enzalutamide develops rapidly often by increasing AR activity, suggesting that new therapies are required for CRPC. We investigated whether betulinic acid (BA), a small molecule from plants that inhibits multiple deubiquitinases (DUBs), reduces AR, and selectively kills PCa cells, can provide an adjuvant strategy for CRPC. Our data indicated that BA reduced AR protein stability and mRNA expression, making it an attractive agent for CRPC. BA decreased AR mRNA possibly by inhibiting a histone 2A DUB thereby increasing ubiquitinated histone 2A, a transcriptional repressor. We identified multiple and specific DUBs inhibited by BA either in PCa cells or using recombinant DUBs. Similar results were obtained using another multi-DUB inhibitor WP1130, suggesting that these DUB inhibitors can decrease AR expression and increase PCa-specific death. Our results also suggest that combining multi-DUB inhibitors BA or WP1130 with enzalutamide may provide a novel strategy for CRPC by further decreasing AR expression and increasing apoptotic cell death.

Inhibition of 19S proteasome-associated deubiquitinases by metal-containing compounds.

Copper and gold complexes have clinical activity in several diseases including cancer. Recently, we have reported that the anti-cancer activity of copper (II) pyrithione CuPT and gold (I) complex auranofin is associated with targeting the 19S proteasome-associated deubiquitinases (DUBs), UCHL5 and USP14. Here we discuss metal DUB inhibitors in treating cancer and other diseases. (from Editor). Several copper and gold complexes have clinical activity in treating some human diseases including cancer. Recently, we have reported that the anti-cancer activity of copper (II) pyrithione CuPT and gold (I) complex auranofin is tightly associated with their ability to target and inhibit the 19S proteasome-associated deubiquitinases (DUBs), UCHL5 and USP14. In this article we review small molecule inhibitors of DUBs and 19S proteasome-associated DUBs. We then describe and discuss the ubique nature of CuPT and auranofin, which is inhibition of 19S proteasome-associated UCHL5 and USP14. We finally suggest the potential to develop novel, specific metal-based DUB inhibitors for treating cancer and other diseases.

Chemical synthesis of phosphorylated ubiquitin and diubiquitin exposes positional sensitivities of e1-e2 enzymes and deubiquitinases.

Modification of ubiquitin by phosphorylation extends the signaling possibilities of this dynamic signal, as it could affect the activity of ligases and the processing of ubiquitin chains by deubiquitinases. The first chemical synthesis of phosphorylated ubiquitin and of Lys63-linked diubiquitin at the proximal, distal or both ubiquitins is reported. This enabled the examination of how such a modification alters E1-E2 activities of the ubiquitination machinery. It is found that E1 charging was not affected, while the assembly of phosphorylated ubiquitin chains was differentially inhibited with E2 enzymes tested. Moreover, this study shows that phosphorylation interferes with the recognition of linkage specific antibodies and the activities of several deubiquitinases. Notably, phosphorylation in the proximal or distal ubiquitin unit has differential effects on specific deubiquitinases. These results support a unique role of phosphorylation in the dynamics of the ubiquitin signal.

Molecular and cellular basis for immunomodulation with monoclonal antibodies

Immunotherapy with immunostimulatroy monoclonal antibodies is based on the ability of antibodies to act as antagonists of inhibitory receptors or to act as agonists of costimulatory receptors. Interference of ligand to receptor binding underlies the first function while crosslinking of receptors to enforce signaling involves the second mode of action. Signalling receptors on lymphocytes and antigen presenting cells are embedded in the plasma membrane and are functionally linked to adaptors whose function is conducive to intracelluar signaling events. In the case of antibodies blocking CTLA-4 functions multiple mechanism of action take place: (i) efficient competition for costimulatory interactions of CD28 with the very same ligands (CD80 and CD86), (ii) functional expression on the surface of regulatory T cells that can be depleted by ADCC, (iii) prevention of the denudation by cooption of CD80 molecules from the surface of antigen presenting cells, a(iv) interference with putative negative signalling mediated by phosphatases. The restricted pattern of expression of CTLA-4 on Tregs and activated T cells is critical to understand its function and exploitation in immunotherapy. PD-1 upon interaction with PD-L1 or PD-L2 drives to the immune synapse the tyrosine phosphatases SHP-1 and SHP-2. These phosphatases, that are recruited to the cytoplasmic tail of PD-1 through its phosphorylated ITIM motive, dephosphorylate critical tyrosine residues in signaling adaptors activated by the concerted action of the TCR and CD28. Disruption of signalling in the immune synapse is prevented by blocking antibodies that avoid the guidance of PD-1 moieties to the synapse guided by PD-L1. Avoiding disruption of productive immune synapses is probably the main mechanism of action of anti-PD-1 and anti-PD-L1 blocking mAb. The costimulatory members of the TNFR family transduce costimulatory signals by recruitment of the adptors TRAF-1, -2, -5. Signal transduction enhances NF-κB and MAPK signaling routes. In this pathway K-63 polyubiquitination reactions are critical for early signaling and are regulated by specific deubiquitinases. Receptors signal depending on crosslinking by the agonist antibody. The pattern of expression of these receptors on the different subset resting or primed/activated T cells determines function. Overall precise understanding of the pattern of distributions of the receptors on immune cells and the signaling events that these receptors prevent or elicit will be ultimately important not only to understand the function but also for both therapeutic manipulation and biomarker finding.

Molecular pathways: translational potential of deubiquitinases as drug targets.

The ubiquitin proteasome system (UPS) is the main system for controlled protein degradation and a key regulator of fundamental cellular processes. The dependency of cancer cells on a functioning UPS coupled with the clinical success of bortezomib for the treatment of multiple myeloma have made the UPS an obvious target for drug development. Deubiquitinases (DUB) are components of the UPS that encompass a diverse family of ubiquitin isopeptidases that catalyze the removal of ubiquitin moieties from target proteins or from polyubiquitin chains, resulting in altered signaling or changes in protein stability. Increasing evidence has implicated deregulation of DUB activity in the initiation and progression of cancer. The altered pattern of DUB expression observed in many tumors can potentially serve as a clinical marker for predicting disease outcome and therapy response. The finding of DUB overexpression in tumor cells suggests that they may serve as novel targets for the development of anticancer therapies. Several specific and broad-spectrum DUB inhibitors are shown to have antitumor activity in preclinical in vivo models with low levels of systemic toxicity. Future studies will hopefully establish the clinical potential for DUB inhibitors as a strategy to treat cancer.

Linear ubiquitination-mediated NF- B regulation and its related disorders

Ubiquitination is a post-translational modification involved in the regulation of a broad variety of cellular functions, such as protein degradation and signal transduction, including nuclear factor-κB (NF-κB) signalling. NF-κB is crucial for inflammatory and immune responses, and aberrant NF-κB signalling is implicated in multiple disorders. We found that linear ubiquitin chain assembly complex (LUBAC), composed of HOIL-1L, HOIP and SHARPIN, generates a novel type of Met1 (M1)-linked linear polyubiquitin chain and specifically regulates the canonical NF-κB pathway. Moreover, specific deubiquitinases, such as CYLD, A20 (TNFAIP3) and OTULIN/gumby, inhibit LUBAC-induced NF-κB activation by different molecular mechanisms, and several M1-linked ubiquitin-specific binding domains have been structurally defined. LUBAC and these linear ubiquitination-regulating factors contribute to immune and inflammatory processes and apoptosis. Functional impairments of these factors are correlated with multiple disorders, including autoinflammation, immunodeficiencies, dermatitis, B-cell lymphomas and Parkinson's disease. This review summarizes the molecular basis and the pathophysiological implications of the linear ubiquitination-mediated NF-κB activation pathway regulation by LUBAC.

Roles of p97-Associated Deubiquitinases in Protein Quality Control at the Endoplasmic Reticulum

To maintain protein homeostasis in the ER, an ER protein quality control system retains unfolded polypeptides and misassembled membrane proteins, allowing only properly folded proteins to exit the ER. Misfolded proteins held in the ER are retrotranslocated into the cytosol, ubiquitinated, and degraded by the proteasome through the ER-associated degradation pathway (ERAD). By timely eliminating misfolded proteins, the ERAD system alleviates cytotoxic stress imposed by protein misfolding. It is well established that ER-associated ubiquitin ligases play pivotal roles in ERAD by assembling ubiquitin conjugates on retrotranslocation substrates, which serve as degradation signals for the proteasome. Surprisingly, recent studies have revealed an equally important function for deubiquitinases (DUBs), enzymes that disassemble ubiquitin chains, in ERAD. Intriguingly, many ERAD specific DUBs are physically associated with the retrotranslocation- driving ATPase p97. Here we discuss the potential functions of p97-associated DUBs including ataxin-3 and YOD1. Our goal is to integrate the emerging evidence into models that may explain how protein quality control could benefit from deubiquitination, a process previously deemed destructive for proteasomal degradation.

Mechanism of Action of a Small Molecule Inhibitor That Targets Usp9x Deubiquitinase Activity in Multiple Myeloma and Mantle Cell Lymphoma

Abstract 1415A growing number of compounds with proteasome and E3-ligase inhibitory activity have demonstrated clinical activity against multiple myeloma (MM) and mantle cell lymphoma. Some of these compounds are now part of the therapeutic regimen. In addition, there is a growing body of evidence that specific deubiquitinases (DUBs) are overexpressed in hematologic malignancies and may be appropriate myeloma/lymphoma therapeutic targets. However, only a limited number of compounds have been described with cell-permeant and selective activity for DUBs. We previously reported the DUB inhibitory activity of small molecule WP1130. This compound caused rapid inactivation of Usp9x, a DUB reported to be overexpressed in MM and some lymphoma primary specimens. Usp9x associates with the anti-apoptotic protein Mcl-1 and protects it from proteasomal destruction by deubiquitination. We have shown that WP1130 inhibits Usp9x activity (and some additional DUBs) and stimulates proteasomal destruction of Mcl-1. However, the mechanism of Usp9x inhibition by WP1130 has not been described. To investigate this, we synthesized a biotinylated derivative of WP1130 that retains cell-penetrant Usp9x DUB inhibitory activity. Using this as a probe, we demonstrate that WP1130 directly and stably interacts with Usp9x in high salt and ionic detergent, suggesting high affinity complex formation. Proteomic assessment revealed that other previously defined cellular targets of WP1130 (Usp5, Usp14, UCH-L5) also formed high affinity complexes with WP1130-biotin. Additional studies suggested that WP1130-biotin covalently modifies Usp9x through a Michael addition reaction with critical Usp9x cysteine residues that result in the inhibition of DUB activity. To further map the sites of the Usp9x-WP1130 interaction, we produced a recombinant catalytic domain of Usp9x (Usp9xCD) and verified that it retained enzymatic activity that could be inhibited by WP1130, albeit at higher concentrations than that required to inhibit the enzyme in intact cells. Mass spectral analysis of Usp9xCD/WP1130 complexes demonstrated formation of covalent WP1130 adducts with a concentration and time dependent stoichiometry. The catalytic domain was also used to determine the affinity and reversibility of WP1130 binding to Usp9x by solution-phase analysis using Bioforte instrumentation. These studies suggested a slow but high-affinity (<1 μM), irreversible binding of WP1130 to the catalytic domain in addition to a second, low-affinity, reversible binding mode. Molecular simulation was used to identify accessible and energetically favorable Cys residues that could represent the location(s) of high-affinity WP1130 binding. These sites are being confirmed by proteomic assessment of WP1130-biotin-retaining peptides derived from trypsinization of Usp9x/WP1130 adducts. Further structural simulation of Usp9x using homology modeling suggested that the catalytic domain of Usp9x contains a unique lobe (aa1576–1642), which lies close to recently described sites of mTOR phosphorylation. Our initial studies suggest that inhibition of mTOR (with Torin-2) in MM cells results in activation of Usp9x activity. The role of phosphorylation in the regulation of Usp9x activity and stability is currently being investigated but our initial observations suggest that Usp9x plays an important regulatory role in the mTOR/5’-AMP kinase cascade. Collectively, our studies suggest that WP1130 inhibits Usp9x activity through covalent modification of critical Cys residues important for Usp9x catalytic activity. Identification of these sites and how phosphorylation influences Usp9x catalytic activity are critical for developing more potent and selective Usp9x inhibitors. Since Usp9x expression has been associated with short survival and poor prognosis in MM patients, potent and selective Usp9x inhibitors could show therapeutic promise for MM patients and other cancers with up-regulated Usp9x activity. Disclosures:Jakubowiak:Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria. Talpaz:ARIAD: Research Funding.

Regulation of TNF-induced NF-κB activation by different cytoplasmic ubiquitination events

TNF is a multifunctional cytokine that plays a key role in innate immunity by inducing the expression of a variety of genes that are involved in an inflammatory response. TNF-induced NF-κB activation is one of the best studied signaling pathways in mammalian cells and has recently led to a revival of research in the biology of ubiquitin. Many NF-κB signaling proteins are modified by specific ubiquitin ligases with different types of ubiquitin chains that are recognized by other proteins and which determine the outcome of ubiquitination. In addition, specific de-ubiquitinases make the whole process reversible. This review summarizes recent findings that have shaped our current understanding on the role of cytoplasmic ubiquitination events in the regulation of TNF-induced NF-κB signaling.

Managing the Quality of Life for Proteins: Focusing on the Exit Path

Managing the Quality of Life for Proteins: Focusing on the Exit Path