Deubiquitinating enzymes (DUBs): Regulation, homeostasis, and oxidative stress response.

Ubiquitin signaling is a conserved, widespread, and dynamic process in which protein substrates are rapidly modified by ubiquitin to impact protein activity, localization, or stability. To regulate this process, deubiquitinating enzymes (DUBs) counter the signal induced by ubiquitin conjugases and ligases by removing ubiquitin from these substrates. Many DUBs selectively regulate physiological pathways employing conserved mechanisms of ubiquitin bond cleavage. DUB activity is highly regulated in dynamic environments through protein-protein interaction, posttranslational modification, and relocalization. The largest family of DUBs, cysteine proteases, are also sensitive to regulation by oxidative stress, as reactive oxygen species (ROS) directly modify the catalytic cysteine required for their enzymatic activity. Current research has implicated DUB activity in human diseases, including various cancers and neurodegenerative disorders. Due to their selectivity and functional roles, DUBs have become important targets for therapeutic development to treat these conditions. This review will discuss the main classes of DUBs and their regulatory mechanisms with a particular focus on DUB redox regulation and its physiological impact during oxidative stress.

The Epstein-Barr virus deubiquitinase BPLF1 targets SQSTM1/p62 to inhibit selective autophagy.

Macroautophagy/autophagy plays an important role in the control of viral infections and viruses have evolved multiple strategies to interfere with autophagy to avoid destruction and promote their own replication and spread. Here we report that the deubiquitinase encoded in the N-terminal domain of the Epstein-Barr virus (EBV) large tegument protein, BPLF1, regulates selective autophagy. Mass spectrometry analysis identified several vesicular traffic and autophagy related proteins as BPLF1 interactors and potential substrates, suggesting that the viral protein targets this cellular defense during productive infection. Direct binding of BPLF1 to the autophagy receptor SQSTM1/p62 (sequestosome 1) was confirmed by co-immunoprecipitation of transfected BPLF1 and by in vitro affinity isolation of bacterially expressed proteins. Expression of the catalytically active BPLF1 was associated with decreased SQSTM1/p62 ubiquitination and failure to recruit LC3 to SQSTM1/p62-positive aggregates. Selective autophagy was inhibited as illustrated by the accumulation of large protein aggregates in BPLF1-positive cells co-transfected with an aggregate-prone HTT (huntingtin)-Q109 construct, and by a slower autophagy-dependent clearance of protein aggregates upon transfection of BPLF1 in cells expressing a tetracycline-regulated HTT-Q103. The inhibition of aggregate clearance was restored by overexpression of a SQSTM1/p62[E409A,K420R] mutant that does not require ubiquitination of Lys420 for cargo loading. These findings highlight a previously unrecognized role of the viral deubiquitinase in the regulation of selective autophagy, which may promote infection and the production of infectious virus.Abbreviations: BPLF1, BamH1 fragment left open reading frame-1; EBV, Epstein-Barr virus; GFP, green fluorescent protein; HTT, huntingtin; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; PB1, Phox and Bem1 domain; PE, phosphatidylethanolamine; SQSTM1/p62, sequestosome 1; UBA, ubiquitin-associated domain.

Deubiquitylases in developmental ubiquitin signaling and congenital diseases.

Metazoan development from a one-cell zygote to a fully formed organism requires complex cellular differentiation and communication pathways. To coordinate these processes, embryos frequently encode signaling information with the small protein modifier ubiquitin, which is typically attached to lysine residues within substrates. During ubiquitin signaling, a three-step enzymatic cascade modifies specific substrates with topologically unique ubiquitin modifications, which mediate changes in the substrate's stability, activity, localization, or interacting proteins. Ubiquitin signaling is critically regulated by deubiquitylases (DUBs), a class of ~100 human enzymes that oppose the conjugation of ubiquitin. DUBs control many essential cellular functions and various aspects of human physiology and development. Recent genetic studies have identified mutations in several DUBs that cause developmental disorders. Here we review principles controlling DUB activity and substrate recruitment that allow these enzymes to regulate ubiquitin signaling during development. We summarize key mechanisms of how DUBs control embryonic and postnatal differentiation processes, highlight developmental disorders that are caused by mutations in particular DUB members, and describe our current understanding of how these mutations disrupt development. Finally, we discuss how emerging tools from human disease genetics will enable the identification and study of novel congenital disease-causing DUBs.

USP15 Deubiquitinase Safeguards Hematopoiesis and Genome Integrity in Hematopoietic Stem Cells and Leukemia Cells.

Altering ubiquitination by disruption of deubiquitinating enzymes (DUBs) affects hematopoietic stem cell (HSC) maintenance. However, comprehensive knowledge of DUB function during hematopoiesis in vivo is lacking. Here, we systematically inactivate DUBs in mouse hematopoietic progenitors using in vivo small hairpin RNA (shRNA) screens. We find that multiple DUBs may be individually required for hematopoiesis and identify ubiquitin-specific protease 15 (USP15) as essential for HSC maintenance in vitro and in transplantations and Usp15 knockout (KO) mice in vivo. USP15 is highly expressed in human hematopoietic tissues and leukemias. USP15 depletion in murine progenitors and leukemia cells impairs in vitro expansion and increases genotoxic stress. In leukemia cells, USP15 interacts with and stabilizes FUS (fused in sarcoma), a known DNA repair factor, directly linking USP15 to the DNA damage response (DDR). Our study underscores the importance of DUBs in preserving normal hematopoiesis and uncovers USP15 as a critical DUB in safeguarding genome integrity in HSCs and leukemia cells.

A Functional Genomic Screen Identifies the Deubiquitinase USP11 as a Novel Transcriptional Regulator of ERα in Breast Cancer.

Approximately 70% of breast cancers express estrogen receptor α (ERα) and depend on this key transcriptional regulator for proliferation and differentiation. While patients with this disease can be treated with targeted antiendocrine agents, drug resistance remains a significant issue, with almost half of patients ultimately relapsing. Elucidating the mechanisms that control ERα function may further our understanding of breast carcinogenesis and reveal new therapeutic opportunities. Here, we investigated the role of deubiquitinases (DUB) in regulating ERα in breast cancer. An RNAi loss-of-function screen in breast cancer cells targeting all DUBs identified USP11 as a regulator of ERα transcriptional activity, which was further validated by assessment of direct transcriptional targets of ERα. USP11 expression was induced by estradiol, an effect that was blocked by tamoxifen and not observed in ERα-negative cells. Mass spectrometry revealed a significant change to the proteome and ubiquitinome in USP11-knockdown (KD) cells in the presence of estradiol. RNA sequencing in LCC1 USP11-KD cells revealed significant suppression of cell-cycle-associated and ERα target genes, phenotypes that were not observed in LCC9 USP11-KD, antiendocrine-resistant cells. In a breast cancer patient cohort coupled with in silico analysis of publicly available cohorts, high expression of USP11 was significantly associated with poor survival in ERα-positive (ERα+) patients. Overall, this study highlights a novel role for USP11 in the regulation of ERα activity, where USP11 may represent a prognostic marker in ERα+ breast cancer. SIGNIFICANCE: A newly identified role for USP11 in ERα transcriptional activity represents a novel mechanism of ERα regulation and a pathway to be exploited for the management of ER-positive breast cancer.

Systematic analysis reveals a functional role for STAMBPL1 in the epithelial-mesenchymal transition process across multiple carcinomas.

BACKGROUND: Deubiquitinating enzymes (DUBs) are linked to cancer progression and dissemination, yet less is known about their regulation and impact on epithelial-mesenchymal transition (EMT). METHODS: An integrative translational approach combining systematic computational analyses of The Cancer Genome Atlas cancer cohorts with CRISPR genetics, biochemistry and immunohistochemistry methodologies to identify and assess the role of human DUBs in EMT. RESULTS: We identify a previously undiscovered biological function of STAM-binding protein like 1 (STAMBPL1) deubiquitinase in the EMT process in lung and breast carcinomas. We show that STAMBPL1 expression can be regulated by mutant p53 and that its catalytic activity is required to affect the transcription factor SNAI1. Accordingly, genetic depletion and CRISPR-mediated gene knockout of STAMBPL1 leads to marked recovery of epithelial markers, SNAI1 destabilisation and impaired migratory capacity of cancer cells. Reversely, STAMBPL1 expression reprogrammes cells towards a mesenchymal phenotype. A significant STAMBPL1-SNAI1 co-signature was observed across multiple tumour types. Importantly, STAMBPL1 is highly expressed in metastatic tissues compared to matched primary tumour of the same lung cancer patient and its expression predicts poor prognosis. CONCLUSIONS: Our study provides a novel concept of oncogenic regulation of a DUB and presents a new role and predictive value of STAMBPL1 in the EMT process across multiple carcinomas.

Independent inhibition of the polymerase and deubiquitinase activities of the Crimean-Congo Hemorrhagic Fever Virus full-length L-protein.

BACKGROUND: The Crimean-Congo hemorrhagic fever virus (CCHFV) is a segmented negative-sense RNA virus that can cause severe human disease. The World Health Organization (WHO) has listed CCHFVas a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. Here we adopted a biochemical approach that targets the viral RNA-dependent RNA polymerase (RdRp). The CCHFV RdRp activity is part of a multifunctional L protein that is unusually large with a molecular weight of ~450 kDa. The CCHFV L-protein also contains an ovarian tumor (OTU) domain that exhibits deubiquitinating (DUB) activity, which was shown to interfere with innate immune responses and viral replication. We report on the expression, characterization and inhibition of the CCHFV full-length L-protein and studied both RNA synthesis and DUB activity. METHODOLOGY/PRINCIPLE FINDINGS: Recombinant full-length CCHFV L protein was expressed in insect cells and purified to near homogeneity using affinity chromatography. RdRp activity was monitored with model primer/templates during elongation in the presence of divalent metal ions. We observed a 14-mer full length RNA product as well as the expected shorter products when omitting certain nucleotides from the reaction mixture. The D2517N mutation of the putative active site rendered the enzyme inactive. Inhibition of RNA synthesis was studies with the broad-spectrum antivirals ribavirin and favipiravir that mimic nucleotide substrates. The triphosphate form of these compounds act like ATP or GTP; however, incorporation of ATP or GTP is markedly favored over the inhibitors. We also studied the effects of bona fide nucleotide analogues 2'-deoxy-2'-fluoro-CTP (FdC) and 2'-deoxy-2'-amino-CTP and demonstrate increased inhibitory effects due to higher rates of incorporation. We further show that the CCHFV L full-length protein and the isolated OTU domain cleave Lys48- and Lys63-linked polyubiqutin chains. Moreover, the ubiquitin analogue CC.4 inhibits the CCHFV-associated DUB activity of the full-length L protein and the isolated DUB domain to a similar extent. Inhibition of DUB activity does not affect elongation of RNA synthesis, and inhibition of RNA synthesis does not affect DUB activity. Both domains are functionally independent under these conditions. CONCLUSIONS/SIGNIFICANCE: The requirements for high biosafety measures hamper drug discovery and development efforts with infectious CCHFV. The availability of full-length CCHFV L-protein provides an important tool in this regard. High-throughput screening (HTS) campaigns are now feasible. The same enzyme preparations can be employed to identify novel polymerase and DUB inhibitors.

The potential roles of deubiquitinating enzymes in brain diseases.

Most proteins undergo posttranslational modification such as acetylation, methylation, phosphorylation, biotinylation, and ubiquitination to regulate various cellular processes. Ubiquitin-targeted proteins from the ubiquitin-proteasome system (UPS) are degraded by 26S proteasome, along with this, deubiquitinating enzymes (DUBs) have specific activity against the UPS through detaching of ubiquitin on ubiquitin-targeted proteins. Balancing between protein expression and degradation through interplay between the UPS and DUBs is important to maintain cell homeostasis, and abnormal expression and elongation of proteins lead to diverse diseases such as cancer, diabetes, and autoimmune response. Therefore, development of DUB inhibitors as therapeutic targets has been challenging. In addition, understanding of the roles of DUBs in neurodegeneration, specifically brain diseases, has emerged gradually. This review highlights recent studies on the molecular mechanisms for DUBs, and discusses potential therapeutic targets for DUBs in cases of brain diseases.

Epigenomic regulation of OTU5 in Arabidopsis thaliana.

Epigenetic regulation by DNA methylation and histone marks is crucial to plant development. In Arabidopsis, the otu5 mutant exhibited altered root phenotypes resembling those of phosphate-deficient plants. In low phosphate (Pi) conditions, altered H3K4 and H3K27 trimethylation were associated with the expression of Pi homeostasis-related genes. However, the genetic effect of OTU5 on the epigenomes was left unexplored. We assessed genome-wide DNA methylation, gene expression and histone modifications of roots from both Col-0 and otu5 mutants. We found that OTU5 altered DNA methylation profile with a context-specific effect through targeting local genomic regions. Our analysis showed that in otu5 the abundance of H3K4me3 was clearly associated with the changes of DNA methylation, leading to the transcriptional difference from wildtype. We concluded that OTU5 induced cross-talks among epigenomes that altogether impacted the regulation of approximately 7060 genes. Of which 186 genes associated with root development were likely to be epigenetically regulated.

Effect of the deubiquitination enzyme gene UBP6 on the stress-responsive transcription factor Msn2-mediated control of the amino acid permease Gnp1 in yeast.

In the yeast Saccharomyces cerevisiae, the transcriptional factor Msn2 plays an essential role in response to a variety of environmental stresses by activating the transcription of many genes that contain the stress-responsive elements in the promoters. We previously reported that overexpression of the MSN2 gene confers tolerance to various stresses in industrial yeast strains. Recently, the overexpression of MSN2 was shown to increase the amount of the amino acid permease Gnp1 on the plasma membrane, leading to the increased uptake of proline into the cell, suggesting a novel link between the Msn2-mediated stress response and amino acid homeostasis in yeast. Here, we found that overexpression of MSN2 increased ubiquitinated protein levels with reduced free ubiquitin. Among deubiquitinating enzymes (DUBs), it was revealed that the loss of Ubp6 depleted the free ubiquitin level and decreased tolerance to the toxic amino acid analogues. The overexpression of UBP6 in MSN2-overexpressing cells clearly complemented the impaired tolerance towards the toxic amino acid analogues. Both the protein level and the plasma-membrane localization of Gnp1 were increased in ubp6-deleted cells, as shown in MSN2-overexpressing cells. These results suggest that an excess level of Msn2 impairs endocytic degradation of Gnp1 through dysfunction of Ubp6 and other DUBs.

Vitamin D Receptor Inhibits NLRP3 Activation by Impeding Its BRCC3-Mediated Deubiquitination.

The NLRP3 inflammasome is a multiprotein oligomer responsible for activation of the inflammatory response by promoting the maturation and secretion of the pro-inflammatory cytokines IL-1ß and IL-18. Dysregulation of this inflammasome has been linked to several autoimmune diseases, indicating that NLRP3 is tightly regulated to prevent aberrant activation. The regulation of NLRP3 activation remains unclear. Here, we report the identification of vitamin D receptor (VDR) as a negative regulator of NLRP3 oligomerization and activation. VDR can physically bind NLRP3 and block the association of NLRP3 with BRCC3. When BRCC3-mediated deubiquitination of NLRP3 is inhibited by VDR, NLRP3 activation is subsequently inhibited. In the absence of VDR, caspase-1 activation and IL-1ß release are increased in response to LPS-induced inflammation or alum-induced peritoneal inflammation, indicating that VDR is a negative regulator of NLRP3 inflammasome activation in vivo. In addition, vitamin D negatively regulates the NLRP3 inflammasome via VDR signaling to effectively inhibit IL-1ß secretion. These studies demonstrate that VDR signaling constrains NLRP3 inflammasome activation and might be a potential treatment target for NLRP3 inflammasome-related diseases.

ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination.

Deubiquitination of NLRP3 has been suggested to contribute to inflammasome activation, but the roles and molecular mechanisms are still unclear. We here demonstrate that ABRO1, a subunit of the BRISC deubiquitinase complex, is necessary for optimal NLRP3-ASC complex formation, ASC oligomerization, caspase-1 activation, and IL-1ß and IL-18 production upon treatment with NLRP3 ligands after the priming step, indicating that efficient NLRP3 activation requires ABRO1. Moreover, we report that ABRO1 deficiency results in a remarkable attenuation in the syndrome severity of NLRP3-associated inflammatory diseases, including MSU- and Alum-induced peritonitis and LPS-induced sepsis in mice. Mechanistic studies reveal that LPS priming induces ABRO1 binding to NLRP3 in an S194 phosphorylation-dependent manner, subsequently recruiting the BRISC to remove K63-linked ubiquitin chains of NLRP3 upon stimulation with activators. Furthermore, deficiency of BRCC3, the catalytically active component of BRISC, displays similar phenotypes to ABRO1 knockout mice. Our findings reveal an ABRO1-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome and provide novel potential targets for treating NLRP3-associated inflammatory diseases.

Deubiquitinating enzymes as cancer biomarkers: new therapeutic opportunities?

Cancer remains a life-threatening disease and accounts for the major mortality rates worldwide. The practice of using biomarkers for early detection, staging, and customized therapy may increase cancer patients' survival. Deubiquitinating enzymes (DUBs) are a family of proteases that remove ubiquitin tags from proteins of interest undergoing proteasomal degradation. DUBs play several functional roles other than deubiquitination. One of the important roles of DUBs is regulation of tumor progression. Several reports have suggested that the DUB family members were highly-elevated in various cancer cells and tissues in different stages of cancer. These findings suggest that the DUBs could be used as drug targets in cancer therapeutics. In this review, we recapitulate the role of the DUB family members, including ubiquitinspecific protease, otubain protease, and important candidates from other family members. Our aim was to better understand the connection between DUB expression profiles and cancers to allow researchers to design inhibitors or gene therapies to improve diagnosis and prognosis of cancers. [BMB Reports 2019; 52(3): 181-189].

The BRISC deubiquitinating enzyme complex limits hematopoietic stem cell expansion by regulating JAK2 K63-ubiquitination.

Hematopoietic stem cell (HSC) homeostasis is controlled by cytokine receptor-mediated Janus kinase 2 (JAK2) signaling. We previously found that JAK2 is promptly ubiquitinated upon cytokine stimulation. Whether a competing JAK2 deubiquitination activity exists is unknown. LNK is an essential adaptor protein that constrains HSC expansion through dampening thrombopoietin (TPO)-induced JAK2 signaling. We show here that a LNK-associated lysine-63 (K63)-deubiquitinating enzyme complex, Brcc36 isopeptidase complex (BRISC), attenuates HSC expansion through control of JAK2 signaling. We pinpoint a direct interaction between the LNK SH2 domain and a phosphorylated tyrosine residue in KIAA0157 (Abraxas2), a unique and defining BRISC component. Kiaa0157 deficiency in mice led to an expansion of phenotypic and functional HSCs. Endogenous JAK2 and phospho-JAK2 were rapidly K63-ubiquitinated upon TPO stimulation, and this action was augmented in cells depleted of the BRISC core components KIAA0157, MERIT40, or BRCC36. This increase in JAK2 ubiquitination after BRISC knockdown was associated with increased TPO-mediated JAK2 activation and protein levels, and increased MPL receptor presence at the cell surface. In addition, BRISC depletion promoted membrane proximal association between the MPL receptor and pJAK2/JAK2, thus enhancing activated JAK2/MPL at the cell membrane. These findings define a novel pathway by which K63-ubiquitination promotes JAK2 stability and activation in a proteasome-independent manner. Moreover, mutations in BRCC36 are found in clonal hematopoiesis in humans. This research may shed light on the mechanistic understanding of a potential role of BRCC36 in human HSCs.

E3 ubiquitin ligases and deubiquitinases as modulators of TRAIL-mediated extrinsic apoptotic signaling pathway.

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) initiates the extrinsic apoptotic pathway through formation of the death-inducing signaling complex (DISC), followed by activation of effector caspases. TRAIL receptors are composed of death receptors (DR4 and DR5), decoy receptors (DcR1 and DcR2), and osteoprotegerin. Among them, only DRs activate apoptotic signaling by TRAIL. Since the levels of DR expressions are higher in cancer cells than in normal cells, TRAIL selectively activates apoptotic signaling pathway in cancer cells. However, multiple mechanisms, including down-regulation of DR expression and pro-apoptotic proteins, and up-regulation of anti-apoptotic proteins, make cancer cells TRAIL-resistant. Therefore, many researchers have investigated strategies to overcome TRAIL resistance. In this review, we focus on protein regulation in relation to extrinsic apoptotic signaling pathways via ubiquitination. The ubiquitin proteasome system (UPS) is an important process in control of protein degradation and stabilization, and regulates proliferation and apoptosis in cancer cells. The level of ubiquitination of proteins is determined by the balance of E3 ubiquitin ligases and deubiquitinases (DUBs), which determine protein stability. Regulation of the UPS may be an attractive target for enhancement of TRAIL-induced apoptosis. Our review provides insight to increasing sensitivity to TRAIL-mediated apoptosis through control of post-translational protein expression. [BMB Reports 2019; 52(2): 119-126].

A balance of deubiquitinating enzymes controls cell cycle entry.

Protein degradation during the cell cycle is controlled by the opposing activities of ubiquitin ligases and deubiquitinating enzymes (DUBs). Although the functions of ubiquitin ligases in the cell cycle have been studied extensively, the roles of DUBs in this process are less well understood. Here, we used an overexpression screen to examine the specificities of each of the 21 DUBs in budding yeast for 37 cell cycle-regulated proteins. We find that DUBs up-regulate specific subsets of proteins, with five DUBs regulating the greatest number of targets. Overexpression of Ubp10 had the largest effect, stabilizing 15 targets and delaying cells in mitosis. Importantly, UBP10 deletion decreased the stability of the cell cycle regulator Dbf4, delayed the G1/S transition, and slowed proliferation. Remarkably, deletion of UBP10 together with deletion of four additional DUBs restored proliferation to near-wild-type levels. Among this group, deletion of the proteasome-associated DUB Ubp6 alone reversed the G1/S delay and restored the stability of Ubp10 targets in ubp10Δ cells. Similarly, deletion of UBP14, another DUB that promotes proteasomal activity, rescued the proliferation defect in ubp10Δ cells. Our results suggest that DUBs function through a complex genetic network in which their activities are coordinated to facilitate accurate cell cycle progression.

SHMT2 and the BRCC36/BRISC deubiquitinase regulate HIV-1 Tat K63-ubiquitylation and destruction by autophagy.

HIV-1 Tat is a key regulator of viral transcription, however little is known about the mechanisms that control its turnover in T cells. Here we use a novel proteomics technique, called DiffPOP, to identify the molecular target of JIB-04, a small molecule compound that potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines. Mass-spectrometry analysis of whole-cell extracts from 2D10 Jurkat T cells revealed that JIB-04 targets Serine Hydroxymethyltransferase 2 (SHMT2), a regulator of glycine biosynthesis and an adaptor for the BRCC36 K63Ub-specific deubiquitinase in the BRISC complex. Importantly, knockdown of SHMT1,2 or BRCC36, or exposure of cells to JIB-04, strongly increased Tat K63Ub-dependent destruction via autophagy. Moreover, point mutation of multiple lysines in Tat, or knockdown of BRCC36 or SHMT1,2, was sufficient to prevent destruction of Tat by JIB-04. We conclude that HIV-1 Tat levels are regulated through K63Ub-selective autophagy mediated through SHMT1,2 and the BRCC36 deubiquitinase.

OTUD7A Regulates Neurodevelopmental Phenotypes in the 15q13.3 Microdeletion Syndrome.

Copy-number variations (CNVs) are strong risk factors for neurodevelopmental and psychiatric disorders. The 15q13.3 microdeletion syndrome region contains up to ten genes and is associated with numerous conditions, including autism spectrum disorder (ASD), epilepsy, schizophrenia, and intellectual disability; however, the mechanisms underlying the pathogenesis of 15q13.3 microdeletion syndrome remain unknown. We combined whole-genome sequencing, human brain gene expression (proteome and transcriptome), and a mouse model with a syntenic heterozygous deletion (Df(h15q13)/+ mice) and determined that the microdeletion results in abnormal development of cortical dendritic spines and dendrite outgrowth. Analysis of large-scale genomic, transcriptomic, and proteomic data identified OTUD7A as a critical gene for brain function. OTUD7A was found to localize to dendritic and spine compartments in cortical neurons, and its reduced levels in Df(h15q13)/+ cortical neurons contributed to the dendritic spine and dendrite outgrowth deficits. Our results reveal OTUD7A as a major regulatory gene for 15q13.3 microdeletion syndrome phenotypes that contribute to the disease mechanism through abnormal cortical neuron morphological development.

Deubiquitylating enzymes as cancer stem cell therapeutics.

The focus of basic and applied research on core stem cell transcription factors has paved the way to initial delineation of their characteristics, their regulatory mechanisms, and the applicability of their regulatory proteins for protein-induced pluripotent stem cells (protein-IPSC) generation and in further clinical settings. Striking parallels have been observed between cancer stem cells (CSCs) and stem cells. For the maintenance of stem cells and CSC pluripotency and differentiation, post translational modifications (i.e., ubiquitylation and deubiquitylation) are tightly regulated, as these modifications result in a variety of stem cell fates. The identification of deubiquitylating enzymes (DUBs) involved in the regulation of core stem cell transcription factors and CSC-related proteins might contribute to providing novel insights into the implications of DUB regulatory mechanisms for governing cellular reprogramming and carcinogenesis. Moreover, we propose the novel possibility of applying DUBs coupled with core transcription factors to improve protein-iPSC generation efficiency. Additionally, this review article further illustrates the potential of applying DUB inhibitors as a novel therapeutic intervention for targeting CSCs. Thus, defining DUBs as core pharmacological targets implies that future endeavors to develop their inhibitors may revolutionize our ability to regulate stem cell maintenance and differentiation, somatic cell reprogramming, and cancer stem cells.

Loss of the deubiquitinase USP36 destabilizes the RNA helicase DHX33 and causes preimplantation lethality in mice.

Deubiquitinases are proteases with a wide functional diversity that profoundly impact multiple biological processes. Among them, the ubiquitin-specific protease 36 (USP36) has been implicated in the regulation of nucleolar activity. However, its functional relevance in vivo has not yet been fully described. Here, we report the generation of an Usp36-deficient mouse model to examine the function of this enzyme. We show that Usp36 depletion is lethal in preimplantation mouse embryos, where it blocks the transition from morula to blastocyst during embryonic development. USP36 reduces the ubiquitination levels and increases the stability of the DEAH-box RNA helicase DHX33, which is critically involved in ribosomal RNA synthesis and mRNA translation. In agreement with this finding, O-propargyl-puromycin incorporation experiments, Northern blot, and electron microscopy analyses demonstrated the role of USP36 in ribosomal RNA and protein synthesis. Finally, we show that USP36 down-regulation alters cell proliferation in human cancer cells by inducing both apoptosis and cell cycle arrest, and that reducing DHX33 levels through short hairpin RNA interference has the same effect. Collectively, these results support that Usp36 is essential for cell and organism viability because of its role in ribosomal RNA processing and protein synthesis, which is mediated, at least in part, by regulating DHX33 stability.