Ubiquitin Hydrolase Research Articles

Distinct Regions within SAP25 Recruit O-Linked Glycosylation, DNA Demethylation, and Ubiquitin Ligase and Hydrolase Activities to the Sin3/HDAC Complex.

Sin3 is an evolutionarily conserved repressor protein complex mainly associated with histone deacetylase (HDAC) activity. Many proteins are part of Sin3/HDAC complexes, and the function of most of these members remains poorly understood. SAP25, a previously identified Sin3A associated protein of 25 kDa, has been proposed to participate in regulating gene expression programs involved in the immune response but the exact mechanism of this regulation is unclear. SAP25 is not expressed in HEK293 cells, which hence serve as a natural knockout system to decipher the molecular functions uniquely carried out by this Sin3/HDAC subunit. Using molecular, proteomic, protein engineering, and interaction network approaches, we show that SAP25 interacts with distinct enzymatic and regulatory protein complexes in addition to Sin3/HDAC. Additional proteins uniquely recovered from the Halo-SAP25 pull-downs included the SCF E3 ubiquitin ligase complex SKP1/FBXO3/CUL1 and the ubiquitin carboxyl-terminal hydrolase 11 (USP11). Furthermore, mutational analysis demonstrates that distinct regions of SAP25 participate in its interaction with USP11, OGT/TETs, and SCF(FBXO3). These results suggest that SAP25 may function as an adaptor protein to coordinate the assembly of different enzymatic complexes to control Sin3/HDAC-mediated gene expression. The data were deposited with the MASSIVE repository with the identifiers MSV000093576 and MSV000093553.

Uncovering seed vigor responsive miRNA in hybrid wheat and its parents by deep sequencing

BackgroundTwo-line hybrid wheat technology system is one way to harness wheat heterosis both domestically and internationally. Seed vigor is a crucial parameter for assessing seed quality, as enhanced seed vigor can lead to yield increments of over 20% to a certain extent. MicroRNAs (miRNAs) were known to participate in the development and vigor of seed in plants, but its impact on seed vigor in two-line hybrid wheat remains poorly elucidated.ResultsThe hybrid (BS1453/11GF5135) wheat exhibited superiority in seed vigor and anti-aging capacity, compared to its male parent (11GF5135, MP) and female parent (BS1453, FP). We identified four miRNAs associated with seed vigor, all of which are novel miRNAs. The majority of targets of miRNAs were related to ubiquitin ligases, kinases, sucrose synthases and hydrolases, involving in starch and sucrose metabolism, hydrolysis, catalysis, plant hormone signal transduction, and other pathways, which played crucial roles in seed development. Additionally, we also found miR531 was differentially expressed in both male parent and hybrid, and its target gene was a component of the E1 subunit of α-ketoate dehydrogenase complex, which interacted with dihydrolipoamide acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3). Finally, We established a presumptive interaction model to speculate the relationship of miR531 and seed vigor.ConclusionsThis study analyzed the seed vigor of two-line hybrid wheat, and screened seed vigor-related miRNAs. Meanwhile speculated the genetic relationship of hybrid and parents, in terms of miRNAs. Consequently, the present study provides new insights into the miRNA-mediated gene and protein interaction network that regulates seed vigor. These findings hold significance for enhancing the yield and quality of two-line hybrid wheat, facilitating its future applications.

Identification of targetable epigenetic vulnerabilities in uveal melanoma.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, which preferentially metastasizes to the liver in approximately half of all cases. Metastatic UM is notoriously resistant to therapy and is almost uniformly fatal. UM metastasis is most strongly associated with mutational inactivation of the BAP1 tumor suppressor gene. Given the role of BAP1 in epigenetic regulation as the ubiquitin hydrolase subunit of the polycomb repressive deubiquitinase (PR-DUB) complex, we conducted high-throughput drug screening using a well-characterized epigenetic compound library to identify new therapeutic vulnerabilities. We identified several promising new lead compounds, in particular the extra-terminal domain protein (BET) inhibitor mivebresib (ABBV-075). Mivebresib significantly improved survival rates in a metastatic uveal melanoma xenograft mouse model and entirely prevented detectable metastases to the bones, spinal cord, and brain. RNA sequencing revealed a notable overlap between the genes and pathways affected by HDAC and BET inhibition, including the reversal of gene signatures linked to high metastatic risk and upregulation of genes associated with a neuronal phenotype. Together, we found that UM cells are particularly vulnerable to class I HDAC and BET inhibition, and highlight the BET inhibitor mivebresib as a promising candidate for further clinical evaluation.

Weighted gene co-expression network analysis reveals immune evasion related genes in Echinococcus granulosus sensu stricto.

Cystic echinococcosis (CE) is a zoonotic disease caused by the tapeworm Echinococcus granulosus sensu lato (s.l). In the intermediate host, this disease is characterized by the growth of cysts in viscera such as liver and lungs, inside of which the parasite develops to the next infective stage known as protoscoleces. There are records that the infected viscera affect the development and morphology of E. granulosus s.l. protoscolex in hosts such as buffalo or humans. However, the molecular mechanisms that drive these differences remains unknown. Weighted gene co-expression network analysis (WGCNA) using a set of RNAseq data obtained from E. granulosus sensu stricto (s.s.) protoscoleces found in liver and lung cysts reveals 34 modules in protoscoleces of liver origin, of which 12 have differential co-expression from protoscoleces of lung origin. Three of these twelve modules contain hub genes related to immune evasion: tegument antigen, tegumental protein, ubiquitin hydrolase isozyme L3, COP9 signalosome complex subunit 3, tetraspanin CD9 antigen, and the methyl-CpG-binding protein Mbd2. Also, two of the twelve modules contain only hypothetical proteins with unknown orthology, which means that there are a group of unknown function proteins co-expressed inside the protoscolex of liver CE cyst origin. This is the first evidence of gene expression differences in protoscoleces from CE cysts found in different viscera, with co-expression networks that are exclusive to protoscoleces from liver CE cyst samples. This should be considered in the control strategies of CE, as intermediate hosts can harbor CE cysts in liver, lungs, or both organs simultaneously.

The genetic architecture of the adaptive potential of Arabidopsis thaliana in response to Pseudomonas syringae strains isolated from south‐west France

AbstractPhytopathogens are a threat for global food production and security. Emergence or re‐emergence of plant pathogens is highly dependent on the environmental conditions affecting pathogen spread and survival. Under climate change, a geographic expansion of pathogen distribution poleward has been observed, potentially resulting in disease outbreaks on crops and wild plants. Therefore, estimating the adaptive potential of plants to novel epidemics and describing the underlying genetic architecture is a primary need to propose agricultural management strategies reducing pathogen outbreaks and to breed novel plant cultivars adapted to pathogens that might spread under climate change. To address this challenge, we inoculated Pseudomonas syringae strains isolated from Arabidopsis thaliana populations from south‐west of France on the highly genetically polymorphic TOU‐A A. thaliana population from north‐east France. While no adaptive potential was identified in response to most P. syringae strains, the TOU‐A population displayed a variable disease response to the JACO‐CL strain belonging to the P. syringae phylogroup 7 (PG7). This strain carried a reduced type III secretion system (T3SS) characteristic of the PG7 as well as flexible genomic traits and potential novel effectors. Genome‐wide association mapping on 192 TOU‐A accessions revealed a polygenic architecture of disease response to JACO‐CL. The main quantitative trait locus (QTL) region encompasses two R genes and the AT5G18310 gene encoding ubiquitin hydrolase, a target of the AvrRpt2 P. syringae effector. Altogether, our results pave the way for a better understanding of the genetic and molecular basis of the adaptive potential in an ecologically relevant A. thaliana–P. syringae pathosystem.

Spatial interplay between synaptic loss, UCH‐L1 deficiency, and pTau accumulation in the retina of MCI and AD patients

AbstractBackgroundSynaptic dysfunction occurs early during Alzheimer’s disease (AD) progression and is considered a strong predictor of cognitive decline. However, it has never been investigated in the AD retina. The ubiquitin‐proteasome system (UPS) is a major degradation pathway for damaged proteins and has been implicated in synaptic integrity and AD pathogenesis. Deficiency in ubiquitin hydrolase (UCH‐L1), a vital UPS component known for its neuroprotective and synaptoprotective properties, was previously linked to cerebral Aβ accumulation in AD, yet has never been explored in the AD retina.MethodWe employed a range of histological techniques to evaluate synaptic integrity and retinal pathology in postmortem retinas of patients with mild cognitive impairment (MCI) attributable to AD or AD dementia, compared to retinas from age‐matched individuals who displayed normal cognition (NC). We assessed the presence of amyloidosis, gliosis, tauopathy, apoptosis, in relation to UCH‐L1 levels, and visualized and quantified synaptic density through analysis of pre‐ and post‐synaptic markers while also assessing the spatial and cellular layer distribution of these markers.ResultWe detected extensive pre‐ and post‐synaptic losses in the IPL and OPL layers of MCI and AD patients, particularly in the far peripheral regions. This was accompanied by 2‐ to 4‐fold decrease in levels of UCH‐L1 in the retinal layers of MCI and AD patients, which directly correlated with pre‐synaptic loss (p<0.0001). These changes were inversely correlated with pTau accumulation (e.g., AT8, pS396), colocalizing at sites of synaptic loss in the IPL and OPL layers. Furthermore, there were significant (1.8‐4.2‐fold) increases in apoptotic cell death in the retinas of these patients (p<0.05–0.001). Remarkably, the pre‐synaptic losses significantly correlated with decreased cognitive scores.ConclusionOur results reveal a substantial retinal synaptic loss at sites of pTau accumulation in prodromal and clinical AD patients. Expression of retinal UCH‐L1 was significantly reduced in MCI and AD patients and strongly associated with synaptic loss and cognitive decline, signifying its potential role in protecting retinal neurons against AD‐related pathology.

The deubiquitinase OTUB1 governs lung cancer cell fitness by modulating proteostasis of OXPHOS proteins

Aerobic glycolysis is a hallmark of cancer development, but this dogma has been challenged by reports showing a key role of oxidative phosphorylation (OXPHOS) in cancer cell survival. It has been proposed that increased levels of intramitochondrial proteins in cancer cells are associated with high OXPHOS activity and increased sensitivity to OXPHOS inhibitors. However, the molecular mechanisms leading to the high expression of OXPHOS proteins in cancer cells remain unknown. Multiple proteomics studies have detected the ubiquitination of intramitochondrial proteins, suggesting the contribution of the ubiquitin system to the proteostatic regulation of OXPHOS proteins. Here, we identified the ubiquitin hydrolase OTUB1 as a regulator of the mitochondrial metabolic machinery essential for lung cancer cell survival. Mitochondria-localized OTUB1 modulates respiration by inhibiting K48-linked ubiquitination and turnover of OXPHOS proteins. An increase in OTUB1 expression is commonly observed in one-third of non-small-cell lung carcinomas and is associated with high OXPHOS signatures. Moreover, OTUB1 expression highly correlates with the sensitivity of lung cancer cells to mitochondrial inhibitors.

Ubiquitome profiling reveals a regulatory pattern of UPL3 with UBP12 on metabolic-leaf senescence.

The HECT-type UPL3 ligase plays critical roles in plant development and stress protection, but understanding of its regulation remains limited. Here, the multi-omics analyses of ubiquitinated proteins in <i>upl3</i> mutants were performed. A landscape of UPL3-dependent ubiquitinated proteins is constructed: Preferential ubiquitination of proteins related to carbon fixation represented the largest set of proteins with increased ubiquitination in the <i>upl3</i> plant, including most of carbohydrate metabolic enzymes, BRM, and variant histone, whereas a small set of proteins with reduced ubiquitination caused by the <i>upl3</i> mutation were linked to cysteine/methionine synthesis, as well as hexokinase 1 (HXK1) and phosphoenolpyruvate carboxylase 2 (PPC2). Notably, ubiquitin hydrolase 12 (UBP12), BRM, HXK1, and PPC2 were identified as the UPL3-interacting partners in vivo and in vitro. Characterization of <i>brm</i>, <i>upl3</i>, <i>ppc2</i>, <i>gin2</i>, and <i>ubp12</i> mutant plants and proteomic and transcriptomic analysis suggested that UPL3 fine-tunes carbohydrate metabolism, mediating cellular senescence by interacting with UBP12, BRM, HXK1, and PPC2. Our results highlight a regulatory pattern of UPL3 with UBP12 as a hub of regulator on proteolysis-independent regulation and proteolysis-dependent degradation.

Bro1 binds the Vps20 subunit of ESCRT-III and promotes ESCRT-III regulation by Doa4.

The budding of intralumenal vesicles (ILVs) at endosomes requires membrane scission by the ESCRT-III complex. This step is negatively regulated in yeast by Doa4, the ubiquitin hydrolase that deubiquitinates transmembrane proteins sorted as cargoes into ILVs. Doa4 acts non-enzymatically to inhibit ESCRT-III membrane scission activity by directly binding the Snf7 subunit of ESCRT-III. This interaction inhibits the remodeling/disassembly of Snf7 polymers required for the ILV membrane scission reaction. Thus, Doa4 is thought to have a structural role that delays ILV budding while it also functions enzymatically to deubiquitinate ILV cargoes. In this study, we show that Doa4 binding to Snf7 in vivo is antagonized by another ESCRT-III subunit, Vps20. Doa4 is restricted from interacting with Snf7 in yeast expressing a mutant Vps20 allele that constitutively binds Doa4. This inhibitory effect of Vps20 is suppressed by overexpression of another ESCRT-III-associated protein, Bro1. We show that Bro1 binds directly to Vps20, suggesting that Bro1 has a central role in relieving the antagonistic relationship that Vps20 has toward Doa4.

The ubiquitin hydrolase OTUB1 promotes glioma cell stemness via suppressing ferroptosis through stabilizing SLC7A11 protein

ABSTRACT The ubiquitin hydrolase OTUB1 has been elucidated to be highly expressed in tumors, however, its roles in glioma progression are still confusing. Here, via analyzing several online datasets, OTUB1 expression was shown to be remarkably increased in glioma tissues compared to that in the adjacent tissues, and predicted a poor overall survival of glioma patients. Then OTUB1 was knocked down in glioma cells and it was found that OTUB1 knockdown significantly reduced glioma cell stemness by detecting sphere-formation ability, stemness marker expression, and ALDH activity. Mechanistic experiments revealed that OTUB1 stabilized SLC7A11 protein via directly interacting with SLC7A11, which is a key suppressor of ferripotosis. Indeed, OTUB1 knockdown triggered ferroptosis dependent on SLC7A11 expression. Notably, ectopic expression of SLC7A11 attenuated the inhibition of OTUB1 knockdown on the stemenss of glioma cells. Finally, we found a positive correlation between OTUB1 and SLC7A11 expression in clinical samples. Taken together, this work identifies a novel OTUB1/SLC7A11 axis contributing to glioma cell stemness.

Ubiquitin C-terminal hydrolase-L1 expression in non-small-cell lung cancer and its association with clinicopathological features and prognosis.

UbiquitinC-terminal hydrolase-L1 (UCH-L1) is a cysteine hydrolase. It functions as a ubiquitin hydrolase, stabilizes the ubiquitin monomer, and affects cell division through cell cycle protein deubiquitination. Abnormal UCH-L1 expression is closely related to the occurrence and development of several tumors. Although some in vitro studies have demonstrated the significance of UCH-L1 in non-small-cell lung cancer (NSCLC), only few clinical studies have focused on the UCH-L1 expression in NSCLC, and the results are controversial and non-uniform. We investigated the UCH-L1 expression in 401 cases of surgically resected NSCLC, including 286 cases of adenocarcinoma (ADC) and 65 cases of squamous cell carcinoma. The associations between the UCH-L1 expression and clinicopathological features, programmed cell death-ligand 1 (PD-L1) expression, and prognostic significance were analyzed. For NSCLC, the UCH-L1 expression is associated with sex, smoking history, tumor size (>3 cm), lymphocyte infiltration, advanced pathological stages, and shortened overall survival (OS; 89.72 vs. 114.55 months; P = 0.005), but not PD-L1 expression. The UCH-L1 expression in ADC is associated with advanced pathological stages, pleural invasion, and shortened OS (90.38 vs. 118.55 months; P = 0.010). Multivariate analysis confirmed that UCH-L1 expression was an independent poor prognostic factor for NSCLC (OS: hazard ratio [HR], 1.854; 95% confidence interval [CI], 1.132-3.038; P = 0.014). Our results suggest that the UCH-L1 expression differs across tumors with different clinicopathological features, and it is related to poor prognosis.

Neurodevelopmental Disorders (NDD) Caused by Genomic Alterations of the Ubiquitin-Proteasome System (UPS): the Possible Contribution of Immune Dysregulation to Disease Pathogenesis.

Over thirty years have passed since the first description of ubiquitin-positive structures in the brain of patients suffering from Alzheimer’s disease. Meanwhile, the intracellular accumulation of ubiquitin-modified insoluble protein aggregates has become an indisputable hallmark of neurodegeneration. However, the role of ubiquitin and a fortiori the ubiquitin-proteasome system (UPS) in the pathogenesis of neurodevelopmental disorders (NDD) is much less described. In this article, we review all reported monogenic forms of NDD caused by lesions in genes coding for any component of the UPS including ubiquitin-activating (E1), -conjugating (E2) enzymes, ubiquitin ligases (E3), ubiquitin hydrolases, and ubiquitin-like modifiers as well as proteasome subunits. Strikingly, our analysis revealed that a vast majority of these proteins have a described function in the negative regulation of the innate immune response. In this work, we hypothesize a possible involvement of autoinflammation in NDD pathogenesis. Herein, we discuss the parallels between immune dysregulation and neurodevelopment with the aim at improving our understanding the biology of NDD and providing knowledge required for the design of novel therapeutic strategies.

Vacuolar H+-ATPase dysfunction rescues intralumenal vesicle cargo sorting in yeast lacking PI(3,5)P2 or Doa4.

Endosomes undergo a maturation process highlighted by a reduction in lumenal pH, a conversion of surface markers that prime endosome-lysosome fusion and the sequestration of ubiquitylated transmembrane protein cargos within intralumenal vesicles (ILVs). We investigated ILV cargo sorting in mutant strains of the budding yeast Saccharomyces cerevisiae that are deficient for either the lysosomal/vacuolar signaling lipid PI(3,5)P2 or the Doa4 ubiquitin hydrolase that deubiquitylates ILV cargos. Disruption of PI(3,5)P2 synthesis or Doa4 function causes a defect in sorting of a subset of ILV cargos. We show that these cargo-sorting defects are suppressed by mutations that disrupt Vph1, a subunit of vacuolar H+-ATPase (V-ATPase) complexes that acidify late endosomes and vacuoles. We further show that Vph1 dysfunction increases endosome abundance, and disrupts vacuolar localization of Ypt7 and Vps41, two crucial mediators of endosome-vacuole fusion. Because V-ATPase inhibition attenuates this fusion and rescues the ILV cargo-sorting defects in yeast that lack PI(3,5)P2 or Doa4 activity, our results suggest that the V-ATPase has a role in coordinating ILV cargo sorting with the membrane fusion machinery. This article has an associated First Person interview with the first author of the paper.

The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22

The SAGA coactivator complex is essential for eukaryotic transcription and comprises four distinct modules, one of which contains the ubiquitin hydrolase USP22. In yeast, the USP22 ortholog deubiquitylates H2B, resulting in Pol II Ser2 phosphorylation and subsequent transcriptional elongation. In contrast to this H2B‐associated role in transcription, we report here that human USP22 contributes to the early stages of stimulus‐responsive transcription, where USP22 is required for pre‐initiation complex (PIC) stability. Specifically, USP22 maintains long‐range enhancer–promoter contacts and controls loading of Mediator tail and general transcription factors (GTFs) onto promoters, with Mediator core recruitment being USP22‐independent. In addition, we identify Mediator tail subunits MED16 and MED24 and the Pol II subunit RBP1 as potential non‐histone substrates of USP22. Overall, these findings define a role for human SAGA within the earliest steps of transcription.

USP31 acetylation at Lys1264 is essential for its activity and cervical cancer cell growth.

Ubiquitin-specific protease 31 (USP31) is a member of deubiquitinase family that is involved in nuclear factor-κB activation and sarcomagenesis. However, little is known about posttranslational modification in the regulation of its activity and cervical cancer cell growth. In our study, we found that the Lys1264 residue of USP31 can be modified with an acetyl group by high-resolution mass spectrometry in HeLa cell line, and site-specific mutagenesis can significantly increase USP31 ubiquitin hydrolase activity and decrease the expression of p65. When being transfected with a plasmid expressing mutated USP31, the number of cancer cells was significantly decreased. We also observed that mutated USP31 could promote apoptosis but not cell cycle by flow cytometer analysis. Overexpression of mutated USP31 could reverse the effect in USP31 knockdown cell line. To further investigate its activity in tumorigenesis, deacetylase sirtuin 1 (Sirt1) was shown to interact with USP31 by co-immunoprecipitation and blocking the function of Sirt1 by knockdown or the inhibitor nicotinamide could increase the acetylation of USP31. When Lys1264 of USP31 mutated, Sirt1 could not remove its acetylation and alter the expression level of p65. Finally, inhibition or knockdown of Sirt1 suppressed USP31 activity in HeLa cell line, leading to cisplatin-induced apoptosis resistance. Therefore, acetylation at Lys1264 suppresses USP31 activity and plays a protective role in cancer cell growth. Our study contributes to understanding the mechanism of USP31 activity regulation and its role in tumorigenesis.

Variants in USP48 encoding ubiquitin hydrolase are associated with autosomal dominant non-syndromic hereditary hearing loss.

Non-Syndromic Hereditary Hearing Loss (NSHHL) is a genetically heterogeneous sensory disorder with about 120 genes already associated. Through exome sequencing (ES) and data aggregation, we identified a family with six affected individuals and one unrelated NSHHL patient with predicted-to-be deleterious missense variants in USP48. We also uncovered an eighth patient presenting unilateral cochlear nerve aplasia and a de novo splice variant in the same gene. USP48 encodes a ubiquitin carboxyl-terminal hydrolase under evolutionary constraint. Pathogenicity of the variants is supported by in vitro assays that showed that the mutated proteins are unable to hydrolyze tetra-ubiquitin. Correspondingly, three-dimensional representation of the protein containing the familial missense variant is situated in a loop that might influence the binding to ubiquitin. Consistent with a contribution of USP48 to auditory function, immunohistology showed that the encoded protein is expressed in the developing human inner ear, specifically in the spiral ganglion neurons, outer sulcus, interdental cells of the spiral limbus, stria vascularis, Reissner's membrane and in the transient Kolliker's organ that is essential for auditory development. Engineered zebrafish knocked-down for usp48, the USP48 ortholog, presented with a delayed development of primary motor neurons, less developed statoacoustic neurons innervating the ears, decreased swimming velocity and circling swimming behavior indicative of vestibular dysfunction and hearing impairment. Corroboratingly, acoustic startle response assays revealed a significant decrease of auditory response of zebrafish lacking usp48 at 600 and 800Hz wavelengths. In conclusion, we describe a novel autosomal dominant NSHHL gene through a multipronged approach combining ES, animal modeling, immunohistology and molecular assays.

Kinetic Characterization of ASXL1/2-Mediated Allosteric Regulation of the BAP1 Deubiquitinase

Abstract BAP1 is an ubiquitin hydrolase whose deubiquitinase activity is mediated by polycomb group-like protein ASXL2. Cancer-related BAP1 mutations/deletions lead to loss-of-function by targeting the catalytic ubiquitin C-terminal hydrolase (UCH) or UCH37-like domain (ULD) domains of BAP1, and the latter disrupts binding to ASXL2, an obligate partner for BAP1 enzymatic activity. However, the biochemical and biophysical properties of domains involved in forming the enzymatically active complex are unknown. Here, we report the molecular dynamics, kinetics, and stoichiometry of these interactions. We demonstrate that interactions between BAP1 and ASXL2 are direct, specific, and stable to biochemical and biophysical manipulations as detected by isothermal titration calorimetry (ITC), GST association, and optical biosensor assays. Association of the ASXL2-AB box greatly stimulates BAP1 activity. A stable ternary complex is formed, comprised of the BAP1-UCH, BAP1-ULD, and ASXL2-AB domains. Stoichiometric analysis revealed that one molecule of the ULD domain directly interacts with one molecule of the AB box. Real-time kinetic analysis of the ULD/AB protein complex to the BAP1-UCH domain, based on surface plasmon resonance, indicated that formation of the ULD/AB complex with the UCH domain is a single-step event with fast association and slow dissociation rates. In vitro experiments validated in cells that the ASXL-AB box directly regulates BAP1 activity. Implications: Collectively, these data elucidate molecular interactions between specific protein domains regulating BAP1 deubiquitinase activity, thus establishing a foundation for small-molecule approaches to reactivate latent wild-type BAP1 catalytic activity in BAP1-mutant cancers.

Histone H2Bub1 deubiquitylation is essential for mouse development, but does not regulate global RNA polymerase II transcription

Co-activator complexes dynamically deposit post-translational modifications (PTMs) on histones, or remove them, to regulate chromatin accessibility and/or to create/erase docking surfaces for proteins that recognize histone PTMs. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved multisubunit co-activator complex with modular organization. The deubiquitylation module (DUB) of mammalian SAGA complex is composed of the ubiquitin-specific protease 22 (USP22) and three adaptor proteins, ATXN7, ATXN7L3 and ENY2, which are all needed for the full activity of the USP22 enzyme to remove monoubiquitin (ub1) from histone H2B. Two additional USP22-related ubiquitin hydrolases (called USP27X or USP51) have been described to form alternative DUBs with ATXN7L3 and ENY2, which can also deubiquitylate H2Bub1. Here we report that USP22 and ATXN7L3 are essential for normal embryonic development of mice, however their requirements are not identical during this process, as Atxn7l3−/− embryos show developmental delay already at embryonic day (E) 7.5, while Usp22−/− embryos are normal at this stage, but die at E14.5. Global histone H2Bub1 levels were only slightly affected in Usp22 null embryos, in contrast H2Bub1 levels were strongly increased in Atxn7l3 null embryos and derived cell lines. Our transcriptomic analyses carried out from wild type and Atxn7l3−/− mouse embryonic stem cells (mESCs), or primary mouse embryonic fibroblasts (MEFs) suggest that the ATXN7L3-related DUB activity regulates only a subset of genes in both cell types. However, the gene sets and the extent of their deregulation were different in mESCs and MEFs. Interestingly, the strong increase of H2Bub1 levels observed in the Atxn7l3−/− mESCs, or Atxn7l3−/− MEFs, does not correlate with the modest changes in RNA Polymerase II (Pol II) occupancy and lack of changes in Pol II elongation observed in the two Atxn7l3−/− cellular systems. These observations together indicate that deubiquitylation of histone H2Bub1 does not directly regulate global Pol II transcription elongation.

USP24 Is a Cancer-Associated Ubiquitin Hydrolase, Novel Tumor Suppressor, and Chromosome Instability Gene Deleted in Neuroblastoma.

Deubiquitinating enzymes are increasingly recognized to play important roles in cancer, with many acting as oncogenes or tumor suppressors. In this study, we employed a bioinformatics approach to screen for enzymes from this family involved in cancer and found USP24 as a potent predictor of poor outcomes in neuroblastoma, an aggressive childhood cancer. USP24 resides in a region commonly deleted in neuroblastoma, yet was independently associated with poor outcomes in this disease. Deletion of Usp24 in a murine model resulted in degradation of collapsin response mediator protein 2 (CRMP2), a regulator of axon growth, guidance, and neuronal polarity. Cells lacking USP24 had significant increases in spindle defects, chromosome missegregation, and aneuploidy, phenotypes that were rescued by the restoration of CRMP2. USP24 prevented aneuploidy by maintaining spindle-associated CRMP2, which is required for mitotic accuracy. Our findings further indicate that USP24 is a tumor suppressor that may play an important role in the pathogenesis of neuroblastoma. SIGNIFICANCE: This study identifies the chromosome instability gene USP24 as frequently deleted in neuroblastoma and provides important insight into the pathogenesis of this aggressive childhood cancer.

Artemisinin susceptibility in the malaria parasite Plasmodium falciparum: propellers, adaptor proteins and the need for cellular healing.

ABSTRACTStudies of the susceptibility of Plasmodium falciparum to the artemisinin family of antimalarial drugs provide a complex picture of partial resistance (tolerance) associated with increased parasite survival in vitro and in vivo. We present an overview of the genetic loci that, in mutant form, can independently elicit parasite tolerance. These encode Kelch propeller domain protein PfK13, ubiquitin hydrolase UBP-1, actin filament-organising protein Coronin, also carrying a propeller domain, and the trafficking adaptor subunit AP-2μ. Detailed studies of these proteins and the functional basis of artemisinin tolerance in blood-stage parasites are enabling a new synthesis of our understanding to date. To guide further experimental work, we present two major conclusions. First, we propose a dual-component model of artemisinin tolerance in P. falciparum comprising suppression of artemisinin activation in early ring stage by reducing endocytic haemoglobin capture from host cytosol, coupled with enhancement of cellular healing mechanisms in surviving cells. Second, these two independent requirements limit the likelihood of development of complete artemisinin resistance by P. falciparum, favouring deployment of existing drugs in new schedules designed to exploit these biological limits, thus extending the useful life of current combination therapies.