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Nicholas J Donato - One of the best experts on this subject based on the ideXlab platform.
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abstract 2881 deubiquitinase usp9x controls tumorigenicity through regulation of the ets 1 transcription factor in melanoma
Cancer Research, 2016Co-Authors: Harish Potu, Luke F Peterson, Moshe Talpaz, Malathi Kandarpa, Anupama Pal, Hanshi Sun Sun, Paul W Harms, Peter C Hollenhorst, Ugur Eskiocak, Nicholas J DonatoAbstract:Transcription factors are frequently deregulated in cancer cells and may be good therapeutic targets, but few successful targeting strategies have been reported. Deubiquitinases (DUBs) are specialized enzymes that regulate the ubiquitin (Ub) content on many proteins and DUB expression and activity are elevated in a number of cancers where they can act to alter tumor suppressor and/or oncoprotein levels. We previously described Usp9x activity and expression in melanoma; here we sought to investigate its role in primary melanoma and metastatic disease. Usp9x was upregulated in tumor cells compared to normal melanocytes and Usp9x expression and activity were found to be essential for 3D growth and melanoma tumor expansion in vivo. We defined the Usp9x ubiquitinated protein landscape and demonstrate that Usp9x regulates Ets-1, a cancer-promoting transcription factor. Usp9x binds, deubiquitinates and thereby stabilizes Ets-1 protein, and primary tissue and tumor analysis demonstrated elevated and coincident Usp9x/Ets-1 protein expression in melanoma compared to normal skin or benign nevi. Usp9x knockdown or Usp9x inhibition with small molecule G9 reduced Ets-1 protein levels and blocked tumor growth in vitro and in vivo. Conversely, Usp9x overexpression in melanoma cells increased Ets-1 protein levels and enhanced 3D tumor growth in vitro and in vivo, which were all reversible by treatment with G9. We conclude that Usp9x is essential for Ets-1 protein stability and may be therapeutically exploited with small molecule Usp9x inhibitors to reduce Ets-1-dependent gene expression and tumorigenicity. Citation Format: Harish Potu, Luke F. Peterson Peterson, Malathi Kandarpa Kandarpa, Anupama Pal Pal, Hanshi Sun Sun, Paul W. Harms Harms, Peter C. Hollenhorst Hollenhorst, Ugur Eskiocak Eskiocak, Moshe Talpaz Talpaz, Nicholas J. Donato Donato. Deubiquitinase Usp9x controls tumorigenicity through regulation of the Ets-1 transcription factor in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2881.
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abstract 3621 deubiquitination of axin1 by usp9x promotes apoptosis in melanoma in response to braf or mek inhibition
Cancer Research, 2015Co-Authors: Harish Potu, Luke F Peterson, Moshe Talpaz, Eric R. Fearon, Malathi Kandarpa, Nicholas J DonatoAbstract:The Wnt/β-catenin pathway is important in development, transformation and self-renewal of various tissues and tumors. In contrast, Wnt/β-catenin signaling appears to play a paradoxical role in melanoma where its activation is anti-oncogenic and a marker of good prognosis. Previous studies have shown that activation of the NRAS/BRAF/MEK pathway suppresses Wnt/β-catenin signaling and reduces apoptotic sensitivity to BRAF or MEK inhibitors in melanoma. MEK pathway activation stabilized AXIN1, a Wnt/β-catenin inhibitor, in some melanoma cell lines through a proteasome-dependent process and co-treatment with Wnt3A and BRAF/MEK kinase inhibitors resulted in a reduction of AXIN1 and full activation of apoptosis. Further, AXIN1 knockdown sensitized BRAF/MEK inhibitor insensitive cells to kinase inhibition, providing a prominent role for AXIN1 in determining melanoma cell apoptotic responsiveness and exposing a potential mechanism for improving kinase inhibitor clinical activity. While AXIN1 protein levels appear to be regulated by ubiquitination, the enzymes involved in control of its ubiquitination or deubiquitination are not fully defined. Here we show that ubiquitin-specific peptidase 9, X-linked (USP9X), a deubiquitinating enzyme whose expression is elevated in melanoma, binds and deubiquitinates AXIN1. USP9X knockdown resulted in increased ubiquitination and proteasome-mediated depletion of AXIN1 (but not AXIN2), resulting in a reduction in AXIN1 cellular half-life. USP9X knockdown elevated β-catenin response genes (>2-fold) in melanoma cells treated with Wnt3A. USP9X depletion also increased apoptotic responsiveness to Wnt3A or BRAF/MEK inhibitor and overcame BRAF inhibitor resistance in A375R cells. In contrast, USP34 which was previously shown to regulate AXIN1 (and AXIN2) deubiquitination, did not alter AXIN1 levels or apoptotic responsiveness in melanoma. A previously identified small molecule USP9X inhibitor (EOAI3402143 or G9) replicated the effects of USP9X knockdown on AXIN1 depletion in vitro and in vivo, suppressed melanoma tumor growth in animals and fully blocked in vivo tumor growth when combined with BRAF kinase inhibitor. Thus, we identify USP9X as a potential therapeutic target in melanoma and established G9 as a lead compound for the development of AXIN1 depleting agents that may add to the targeted therapies already used in the treatment of melanoma. Note: This abstract was not presented at the meeting. Citation Format: Harish Potu, Luke F. Peterson, Malathi Kandarpa, Eric Fearon, Moshe Talpaz, Nicholas J. Donato. Deubiquitination of AXIN1 by USP9X promotes apoptosis in melanoma in response to BRAF or MEK inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3621. doi:10.1158/1538-7445.AM2015-3621
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bcr abl ubiquitination and usp9x inhibition block kinase signaling and promote cml cell apoptosis
Blood, 2011Co-Authors: Vaibhav Kapuria, Luke F Peterson, Dexing Fang, Geoffrey Bartholomeusz, Moshe Talpaz, William G Bornmann, Nicholas J DonatoAbstract:Although chronic myelogenous leukemia (CML) is effectively controlled by Bcr-Abl kinase inhibitors, resistance to inhibitors, progressive disease, and incomplete eradication of Bcr-Abl–expressing cells are concerns for the long-term control and suppression of this disease. We describe a novel approach to targeting key proteins in CML cells with a ubiquitin-cycle inhibitor, WP1130. Bcr-Abl is rapidly modified with K63-linked ubiquitin polymers in WP1130-treated CML cells, resulting in its accumulation in aggresomes, where is it unable to conduct signal transduction. Induction of apoptosis because of aggresomal compartmentalization of Bcr-Abl was observed in both imatinib-sensitive and -resistant cells. WP1130, but not Bcr-Abl kinase inhibitors, directly inhibits Usp9x deubiquitinase activity, resulting in the down-regulation of the prosurvival protein Mcl-1 and facilitating apoptosis. These results demonstrate that ubiquitin-cycle inhibition represents a novel and effective approach to blocking Bcr-Abl kinase signaling and reducing Mcl-1 levels to engage CML cell apoptosis. This approach may be a therapeutic option for kinase inhibitor–resistant CML patients.
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inhibition of usp9x deubiquitinase activity by wp1130 reduces mcl 1 levels and induces apoptosis in cells from patients with plasma cell dyscrasia and drug refractory multiple myeloma
Blood, 2010Co-Authors: Vaibhav Kapuria, Luke F Peterson, Moshe Talpaz, Diane Giannola, Kandarpa Malathi, Andrzej J Jakubowiak, Nicholas J DonatoAbstract:Abstract 3005 Multiple myeloma (MM) patients are typically treated with a number of combinations of unconventional drugs that provide excellent and sometimes durable responses. However, most patients are expected to relapse or progress and relapsed disease is usually less sensitive to chemotherapy. Therefore, drug resistance remains a key concern in MM therapy. Some important clues regarding MM drug and apoptotic resistance have been obtained from biochemical and molecular studies of pro-survival genes. Mcl-1 is one member of the Bcl-2 family with strong anti-apoptotic activity. Mcl-1 binds pro-apoptotic proteins such as Bim and Bak, forming hetero-dimers to suppress apoptosis. Specific knockdown of Mcl-1 but not other anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL) induces apoptosis in MM cells and a critical level of Mcl-1 is essential to MM cell viability. Also, drug resistant MM patients are reported to express higher levels of Mcl-1 when compared to drug naive patients. These observations suggest that Mcl-1 plays a key role in MM drug and apoptotic resistance. Mcl-1 is unique among survival proteins as its cellular protein level is primarily controlled by its ubiquitination and proteasomal degradation. Recently Usp9x, a high MW de-ubiquitinase (DUB), was shown to bind and stabilize Mcl-1 by mediating its de-ubiquitination and loss of recognition by the proteasome. Usp9x was also shown to be highly expressed in MM patients with short term progression-free survival. Analysis of Usp9x gene expression in MM patients treated at our center supports a correlation between elevated Usp9x expression (>4-fold over normal PBMCs) and poor patient prognosis. These observations suggest that agents that inhibit Usp9x activity may be effective in controlling Mcl-1 levels and have therapeutic impact in MM patients. Here we describe a small molecule Usp9x inhibitor, WP1130, that causes a rapid reduction in Mcl-1 protein levels and stimulates apoptosis in MM cells. We further demonstrate that the anti-tumor activity of WP1130 is mediated through inhibition of Usp9x and other active DUBs in B-cell tumors. WP1130-mediated inhibition of Usp9x activity promotes Mcl-1 ubiquitination leading to its rapid (1-4 hours) proteasomal degradation with subsequent induction of apoptosis. Pretreatment of MM cells with proteasome inhibitors (MG-132 or bortezomib) prior to WP1130 treatment blocked the down-regulation of Mcl-1, demonstrating proteasome involvement in regulation of Mcl-1 levels by WP1130. Primary MM tumor cells (bone marrow aspirates) derived from newly diagnosed and advanced therapy refractory patients were also screened for their responsiveness to WP1130 by DUB activity assays. Samples were obtained from a newly diagnosed plasmacytoma/plasma cell leukemia patient and 4 MM patients refractory to the Velcade/Doxorubicin/Dexamethasone (VDD) regimen. Usp9x activity was detected in 4 of 5 patient specimens and WP1130 treatment reduced Mcl-1 protein levels and induced apoptosis only in samples with active Usp9x. Furthermore, WP1130 completely eliminated CD138+ cells after 24 hours in MM patient specimens with active Usp9x while only an 11% reduction was measured in MM cells that did not exhibit Usp9x activity. Cytogenetic prognostic predictors (del13) did not distinguish between WP1130-responsive and non-responsive cells. Our results suggest that inhibition of Usp9x activity by WP1130 is associated with Mcl-1 down-regulation in MM cells. These results also suggest that drug-refractory MM patients with elevated Usp9x activity would likely benefit from Usp9x inhibitor-based therapy. Preliminary MM tumor studies have shown that WP1130 suppresses tumor growth in vivo and can be administered safely. Together, these results suggest that WP1130-mediated Usp9x inhibition may be an effective and novel approach in treating progressive or drug-refractory MM patients. Disclosures: Jakubowiak:Millenium, Celgene, Bristol-Myers Squibb, Johnson & Johnson Ortho-Centocor: Honoraria; Millennium, Bristol-Myers Squibb: Membership on an entity9s Board of Directors or advisory committees; Millennium, Celgene, Centocor-Ortho Biotech: Speakers Bureau.
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deubiquitinase inhibition by small molecule wp1130 triggers aggresome formation and tumor cell apoptosis
Cancer Research, 2010Co-Authors: Vaibhav Kapuria, Luke F Peterson, Dexing Fang, Moshe Talpaz, William G Bornmann, Nicholas J DonatoAbstract:Recent evidence suggests that several deubiquitinases (DUB) are overexpressed or activated in tumor cells and many contribute to the transformed phenotype. Agents with DUB inhibitory activity may therefore have therapeutic value. In this study, we describe the mechanism of action of WP1130, a small molecule derived from a compound with Janus-activated kinase 2 (JAK2) kinase inhibitory activity. WP1130 induces rapid accumulation of polyubiquitinated (K48/K63-linked) proteins into juxtanuclear aggresomes, without affecting 20S proteasome activity. WP1130 acts as a partly selective DUB inhibitor, directly inhibiting DUB activity of USP9x, USP5, USP14, and UCH37, which are known to regulate survival protein stability and 26S proteasome function. WP1130-mediated inhibition of tumor-activated DUBs results in downregulation of antiapoptotic and upregulation of proapoptotic proteins, such as MCL-1 and p53. Our results show that chemical modification of a previously described JAK2 inhibitor results in the unexpected discovery of a novel DUB inhibitor with a unique antitumor mechanism.
Shangda Yang - One of the best experts on this subject based on the ideXlab platform.
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usp9x mediated deubiquitination of b cell cll lymphoma 9 potentiates wnt signaling and promotes breast carcinogenesis
Journal of Biological Chemistry, 2019Co-Authors: Zesen Shang, Shangda Yang, Na Yu, Shanshan Tian, Jiao Zhao, Kai Zhang, Qi ZhangAbstract:: Hyperactivation of the canonical Wnt-signaling pathway is a prominent feature of a number of human malignancies. Transcriptional activation of this signaling cascade depends on the formation of the β-catenin-B-cell CLL/lymphoma 9 (BCL9)-pygopus (PYGO) family plant homeodomain finger 1 complex, yet how the assembly of this complex is regulated remains to be investigated. Here, using MCF-7, HeLa, HEK293T, MDA-MB-231, and Sf9 cells, along with immunoblotting and immunofluorescence, nano-HPLC-MS/MS, deubiquitination, immunoprecipitation, and chromatin immunoprecipitation (ChIP) assays, we report that BCL9 physically associates with a protein deubiquitinase, ubiquitin-specific peptidase 9, X-linked (USP9X), and that USP9X removes Lys-63-linked polyubiquitin on Lys-212 of BCL9. Importantly, the USP9X-mediated BCL9 deubiquitination facilitated the formation of the β-catenin-BCL9-PYGO complex, thereby potentiating the transcriptional activation of Wnt/β-catenin target genes. We also show that USP9X-mediated BCL9 deubiquitination promotes the proliferation and invasion of breast cancer cells. Together, these results uncover USP9X as a deubiquitinase of BCL9, implicating USP9X in Wnt/β-catenin signaling and breast carcinogenesis.
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usp9x regulates centrosome duplication and promotes breast carcinogenesis
Nature Communications, 2017Co-Authors: Xin Li, Xiaodan Ding, Nan Song, Shangda Yang, Dongxue Su, Lin Shan, Yuejiao Wang, Xing Zhou, X. Song, Qi ZhangAbstract:Defective centrosome duplication is implicated in microcephaly and primordial dwarfism as well as various ciliopathies and cancers. Yet, how the centrosome biogenesis is regulated remains poorly understood. Here we report that the X-linked deubiquitinase USP9X is physically associated with centriolar satellite protein CEP131, thereby stabilizing CEP131 through its deubiquitinase activity. We demonstrate that USP9X is an integral component of centrosome and is required for centrosome biogenesis. Loss-of-function of USP9X impairs centrosome duplication and gain-of-function of USP9X promotes centrosome amplification and chromosome instability. Significantly, USP9X is overexpressed in breast carcinomas, and its level of expression is correlated with that of CEP131 and higher histologic grades of breast cancer. Indeed, USP9X, through regulation of CEP131 abundance, promotes breast carcinogenesis. Our experiments identify USP9X as an important regulator of centrosome biogenesis and uncover a critical role for USP9X/CEP131 in breast carcinogenesis, supporting the pursuit of USP9X/CEP131 as potential targets for breast cancer intervention. USP9X is a deubiquitinating enzyme with many known substrates and functions; it has been linked to cancer but the mechanisms remain unclear. Here Liet al. report that USP9X stabilizes the centrosomal protein CEP131 leading to centrosome amplification and breast cancer development.
Stephen A Wood - One of the best experts on this subject based on the ideXlab platform.
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the deubiquitinase usp9x regulates fbw7 stability and suppresses colorectal cancer
Journal of Clinical Investigation, 2018Co-Authors: Omar M Khan, Stephen A Wood, Joana Carvalho, Bradley Spencerdene, Richard Mitter, David Frith, Ambrosius P Snijders, Axel BehrensAbstract:The tumor suppressor FBW7 targets oncoproteins such as c-MYC for ubiquitylation and is mutated in several human cancers. We noted that in a substantial percentage of colon cancers, FBW7 protein is undetectable despite the presence of FBW7 mRNA. To understand the molecular mechanism of FBW7 regulation in these cancers, we employed proteomics and identified the deubiquitinase (DUB) USP9X as an FBW7 interactor. USP9X antagonized FBW7 ubiquitylation, and Usp9x deletion caused Fbw7 destabilization. Mice lacking Usp9x in the gut showed reduced secretory cell differentiation and increased progenitor proliferation, phenocopying Fbw7 loss. In addition, Usp9x inactivation impaired intestinal regeneration and increased tumor burden in colitis-associated intestinal cancer. c-Myc heterozygosity abrogated increased progenitor proliferation and tumor burden in Usp9x-deficient mice, suggesting that Usp9x suppresses tumor formation by regulating Fbw7 protein stability and thereby reducing c-Myc. Thus, we identify a tumor suppressor mechanism in the mammalian intestine that arises from the posttranslational regulation of FBW7 by USP9X independent of somatic FBW7 mutations.
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deubiquitinating enzyme usp9x suppresses tumor growth via lats kinase and core components of the hippo pathway
Cancer Research, 2017Co-Authors: Stephen A Wood, Aleksandra Toloczko, Fusheng Guo, Hiufung Yuen, Qing Wen, Yan Shan Ong, Pei Yi Chan, Asfa Alli Shaik, Jayantha GunaratneAbstract:The core LATS kinases of the Hippo tumor suppressor pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Recent studies have identified various E3 ubiquitin ligases that negatively regulate the Hippo pathway via ubiquitination, yet few deubiquitinating enzymes (DUB) have been implicated. In this study, we report the DUB USP9X is an important regulator of the core kinases of this pathway. USP9X interacted strongly with LATS kinase and to a lesser extent with WW45, KIBRA, and Angiomotin, and LATS co-migrated exclusively with USP9X during gel filtration chromatography analysis. Knockdown of USP9X significantly downregulated and destabilized LATS and resulted in enhanced nuclear translocation of YAP and TAZ, accompanied with activation of their target genes. In the absence of USP9X, cells exhibited an epithelial-to-mesenchymal transition phenotype, acquired anchorage-independent growth in soft agar, and led to enlarged, disorganized, three-dimensional acini. YAP/TAZ target gene activation in response to USP9X knockdown was suppressed by knockdown of YAP, TAZ, and TEAD2. Deletion of USP9X in mouse embryonic fibroblasts resulted in significant downregulation of LATS. Furthermore, USP9X protein expression correlated positively with LATS but negatively with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. Overall, these results strongly indicate that USP9X potentiates LATS kinase to suppress tumor growth.
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de novo loss of function mutations in usp9x cause a female specific recognizable syndrome with developmental delay and congenital malformations
American Journal of Human Genetics, 2016Co-Authors: Margot R F Reijnders, Vasilios Zachariadis, Brooke L Latour, Lachlan A Jolly, G M S Mancini, Rolph Pfundt, Conny M A Van Ravenswaaijarts, Hermine E Veenstraknol, Brittmarie Anderlid, Stephen A WoodAbstract:Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function.
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identification of ubiquitin specific protease 9x usp9x as a deubiquitinase acting on ubiquitin peroxin 5 pex5 thioester conjugate
Journal of Biological Chemistry, 2012Co-Authors: Claudia P Grou, Tânia Francisco, Tony A Rodrigues, Stephen A Wood, Marta O Freitas, Manuel P Pinto, Andreia F Carvalho, Pedro Domingues, Jose E Rodriguezborges, Clara SamirandaAbstract:Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates.
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the ubiquitin e3 ligase march7 is differentially regulated by the deubiquitylating enzymes usp7 and usp9x
Traffic, 2008Co-Authors: James A Nathan, Stephen A Wood, Soma Sengupta, Arie Admon, Gabriel Markson, Christopher M Sanderson, Paul J LehnerAbstract:Protein modification by one or more ubiquitin chains serves a critical signalling function across a wide range of cellular processes. Specificity within this system is conferred by ubiquitin E3 ligases, which target the substrates. Their activity is balanced by deubiquitylating enzymes (DUBs), which remove ubiquitin from both substrates and ligases. The RING-CH ligases were initially identified as viral immunoevasins involved in the downregulation of immunoreceptors. Their cellular orthologues, the Membrane-Associated RING-CH (MARCH) family represent a subgroup of the classical RING genes. Unlike their viral counterparts, the cellular RING-CH proteins appear highly regulated, and one of these in particular, MARCH7, was of interest because of a potential role in neuronal development and lymphocyte proliferation. Difficulties in detection and expression of this orphan ligase lead us to search for cellular cofactors involved in MARCH7 stability. In this study, we show that MARCH7 readily undergoes autoubiquitylation and associates with two deubiquitylating enzymes - ubiquitin-specific protease (USP)9X in the cytosol and USP7 in the nucleus. Exogenous expression and short interfering RNA depletion experiments demonstrate that MARCH7 can be stabilized by both USP9X and USP7, which deubiquitylate MARCH7 in the cytosol and nucleus, respectively. We therefore demonstrate compartment-specific regulation of this E3 ligase through recruitment of site-specific DUBs.
Miguel Ramalhosantos - One of the best experts on this subject based on the ideXlab platform.
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the deubiquitinase usp9x regulates prc2 mediated chromatin reprogramming during mouse development
Nature Communications, 2021Co-Authors: Trisha A Macrae, Miguel RamalhosantosAbstract:Pluripotent cells of the mammalian embryo undergo extensive chromatin rewiring to prepare for lineage commitment after implantation. Repressive H3K27me3, deposited by Polycomb Repressive Complex 2 (PRC2), is reallocated from large blankets in pre-implantation embryos to mark promoters of developmental genes. The regulation of this global redistribution of H3K27me3 is poorly understood. Here we report a post-translational mechanism that destabilizes PRC2 to constrict H3K27me3 during lineage commitment. Using an auxin-inducible degron system, we show that the deubiquitinase Usp9x is required for mouse embryonic stem (ES) cell self-renewal. Usp9x-high ES cells have high PRC2 levels and bear a chromatin and transcriptional signature of the pre-implantation embryo, whereas Usp9x-low ES cells resemble the post-implantation, gastrulating epiblast. We show that Usp9x interacts with, deubiquitinates and stabilizes PRC2. Deletion of Usp9x in post-implantation embryos results in the derepression of genes that normally gain H3K27me3 after gastrulation, followed by the appearance of morphological abnormalities at E9.5, pointing to a recurrent link between Usp9x and PRC2 during development. Usp9x is a marker of "stemness" and is mutated in various neurological disorders and cancers. Our results unveil a Usp9x-PRC2 regulatory axis that is critical at peri-implantation and may be redeployed in other stem cell fate transitions and disease states.
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the deubiquitinase usp9x regulates prc2 mediated chromatin reprogramming during mouse development
bioRxiv, 2020Co-Authors: Trisha A Macrae, Miguel RamalhosantosAbstract:Pluripotent cells of the mammalian embryo undergo extensive chromatin rewiring to prepare for lineage commitment after implantation. Repressive H3K27me3, deposited by Polycomb Repressive Complex 2 (PRC2), is reallocated from large gene-distal blankets in pre-implantation embryos to mark promoters of developmental genes. The factors that mediate this global redistribution of H3K27me3 are unknown. Here we report a post-translational mechanism that destabilizes PRC2 to constrict H3K27me3 during lineage commitment. Using an auxin-inducible degron system, we show that the deubiquitinase Usp9x is required for mouse embryonic stem (ES) cell self-renewal. Usp9x-high ES cells have high PRC2 levels and bear a chromatin and transcriptional signature of the pre-implantation embryo, whereas Usp9x-low ES cells resemble the post-implantation, gastrulating epiblast. We show that Usp9x interacts with, deubiquitinates and stabilizes PRC2. Deletion of Usp9x in post-implantation embryos results in the derepression of genes that normally gain H3K27me3 after gastrulation, followed by the appearance of morphological abnormalities at E9.5, pointing to a recurrent link between Usp9x and PRC2 during development. Usp9x is a marker of "stemness" and is mutated in various neurological disorders and cancers. Our results unveil a Usp9x-PRC2 regulatory axis that is critical at peri-implantation and may be redeployed in other stem cell fate transitions and disease states.
Chao Sun - One of the best experts on this subject based on the ideXlab platform.
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an azoospermic man with a de novo point mutation in the y chromosomal gene USP9Y
Nature Genetics, 1999Co-Authors: Chao Sun, Helen Skaletsky, Bruce W Birren, Keri Devon, Zhaolan Tang, Sherman J Silber, Robert D OatesAbstract:In humans, deletion of any one of three Y-chromosomal regions—AZFa, AZFb or AZFc—disrupts spermatogenesis, causing infertility in otherwise healthy men1,2,3,4,5. Although candidate genes have been identified in all three regions3,6,7,8, no case of spermatogenic failure has been traced to a point mutation in a Y-linked gene, or to a deletion of a single Y-linked gene. We sequenced the AZFa region of the Y chromosome and identified two functional genes previously described: USP9Y (also known as DFFRY) and DBY (refs 7,8). Screening of the two genes in 576 infertile and 96 fertile men revealed several sequence variants, most of which appear to be heritable and of little functional consequence. We found one de novo mutation in USP9Y: a 4-bp deletion in a splice-donor site, causing an exon to be skipped and protein truncation. This mutation was present in a man with nonobstructive azoospermia (that is, no sperm was detected in semen), but absent in his fertile brother, suggesting that the USP9Y mutation caused spermatogenic failure. We also identified a single-gene deletion associated with spermatogenic failure, again involving USP9Y, by re-analysing a published study.
