RESUMEN
Post-translational modifications including protein ubiquitination regulate a plethora of cellular processes in distinct manners. RNA N6-methyladenosine is the most abundant post-transcriptional modification on mammalian mRNAs and plays important roles in various physiological and pathological conditions including hematologic malignancies. We previously determined that the RNA N6-methyladenosine eraser ALKBH5 is necessary for the maintenance of acute myeloid leukemia (AML) stem cell function, but the post-translational modifications involved in ALKBH5 regulation remain elusive. Here, we show that deubiquitinase ubiquitin-specific peptidase 9X (USP9X) stabilizes ALKBH5 and promotes AML cell survival. Through the use of mass spectrometry as an unbiased approach, we identify USP9X and confirm that it directly binds to ALKBH5. USP9X stabilizes ALKBH5 by removing the K48-linked polyubiquitin chain at K57. Using human myeloid leukemia cells and a murine AML model, we find that genetic knockdown or pharmaceutical inhibition of USP9X inhibits leukemia cell proliferation, induces apoptosis, and delays AML development. Ectopic expression of ALKBH5 partially mediates the function of USP9X in AML. Overall, this study uncovers deubiquitinase USP9X as a key for stabilizing ALKBH5 expression and reveals the important role of USP9X in AML, which provides a promising therapeutic strategy for AML treatment in the clinic.
Asunto(s)
Desmetilasa de ARN, Homólogo 5 de AlkB , Leucemia Mieloide Aguda , Ubiquitina Tiolesterasa , Animales , Humanos , Ratones , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Línea Celular Tumoral , Supervivencia Celular , Leucemia Mieloide Aguda/genética , ARN , Ubiquitina Tiolesterasa/genética , UbiquitinaciónRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy and one of the most common childhood cancers. Immunosuppressed patients, including HIV-infected patients, are more prone to KSHV-associated disease. KSHV encodes a viral protein kinase (vPK) that is expressed from ORF36. KSHV vPK contributes to the optimal production of infectious viral progeny and upregulation of protein synthesis. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used a bottom-up proteomics approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Subsequently, we validated this interaction using a co-immunoprecipitation assay. We report that both the ubiquitin-like and the catalytic domains of USP9X are important for association with vPK. To uncover the biological relevance of the USP9X/vPK interaction, we investigated whether the knockdown of USP9X would modulate viral reactivation. Our data suggest that depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Understanding how USP9X influences the reactivation of KSHV will provide insights into how cellular deubiquitinases regulate viral kinase activity and how viruses co-opt cellular deubiquitinases to propagate infection. Hence, characterizing the roles of USP9X and vPK during KSHV infection constitutes a first step toward identifying a potentially critical interaction that could be targeted by future therapeutics. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy. KSHV encodes a viral protein kinase (vPK) that aids viral replication. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used an affinity purification approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Overall, our data suggest a proviral role for USP9X.
Asunto(s)
Herpesvirus Humano 8 , Sarcoma de Kaposi , Ubiquitina Tiolesterasa , Niño , Humanos , Enzimas Desubicuitinizantes , Herpesvirus Humano 8/fisiología , Infecciones por VIH/complicaciones , Linfoma de Efusión Primaria , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patología , Sarcoma de Kaposi/virología , Ubiquitina Tiolesterasa/genética , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Radioresistance is a key clinical constraint on the efficacy of radiotherapy in lung cancer patients. REV1 DNA directed polymerase (REV1) plays an important role in repairing DNA damage and maintaining genomic stability. However, its role in the resistance to radiotherapy in lung cancer is not clear. This study aims to clarify the role of REV1 in lung cancer radioresistance, identify the intrinsic mechanisms involved, and provide a theoretical basis for the clinical translation of this new target for lung cancer treatment. METHODS: The effect of targeting REV1 on the radiosensitivity was verified by in vivo and in vitro experiments. RNA sequencing (RNA-seq) combined with nontargeted metabolomics analysis was used to explore the downstream targets of REV1. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify the content of specific amino acids. The coimmunoprecipitation (co-IP) and GST pull-down assays were used to validate the interaction between proteins. A ubiquitination library screening system was constructed to investigate the regulatory proteins upstream of REV1. RESULTS: Targeting REV1 could enhance the radiosensitivity in vivo, while this effect was not obvious in vitro. RNA sequencing combined with nontargeted metabolomics revealed that the difference result was related to metabolism, and that the expression of glycine, serine, and threonine (Gly/Ser/Thr) metabolism signaling pathways was downregulated following REV1 knockdown. LC-MS/MS demonstrated that REV1 knockdown results in reduced levels of these three amino acids and that cystathionine γ-lyase (CTH) was the key to its function. REV1 enhances the interaction of CTH with the E3 ubiquitin ligase Rad18 and promotes ubiquitination degradation of CTH by Rad18. Screening of the ubiquitination compound library revealed that the ubiquitin-specific peptidase 9 X-linked (USP9X) is the upstream regulatory protein of REV1 by the ubiquitin-proteasome system, which remodels the intracellular Gly/Ser/Thr metabolism. CONCLUSION: USP9X mediates the deubiquitination of REV1, and aberrantly expressed REV1 acts as a scaffolding protein to assist Rad18 in interacting with CTH, promoting the ubiquitination and degradation of CTH and inducing remodeling of the Gly/Ser/Thr metabolism, which leads to radioresistance. A novel inhibitor of REV1, JH-RE-06, was shown to enhance lung cancer cell radiosensitivity, with good prospects for clinical translation.
Asunto(s)
Neoplasias Pulmonares , Nucleotidiltransferasas , Tolerancia a Radiación , Ubiquitina-Proteína Ligasas , Ubiquitinación , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Línea Celular Tumoral , Ratones , Animales , ADN Polimerasa Dirigida por ADNRESUMEN
BACKGROUND: MutT homolog 1 (MTH1) sanitizes oxidized dNTP pools to promote the survival of cancer cells and its expression is frequently upregulated in cancers. Polyubiquitination stabilizes MTH1 to facilitate the proliferation of melanoma cells, suggesting the ubiquitin system controls the stability and function of MTH1. However, whether ubiquitination regulates MTH1 in gastric cancers has not been well defined. This study aims to investigate the interaction between MTH1 and a deubiquitinase, USP9X, in regulating the proliferation, survival, migration, and invasion of gastric cancer cells. METHODS: The interaction between USP9X and MTH1 was evaluated by co-immunoprecipitation (co-IP) in HGC-27 gastric cancer cells. siRNAs were used to interfere with USP9X expression in gastric cancer cell lines HGC-27 and MKN-45. MTT assays were carried out to examine the proliferation, propidium iodide (PI) and 7-AAD staining assays were performed to assess the cell cycle, Annexin V/PI staining assays were conducted to examine the apoptosis, and transwell assays were used to determine the migration and invasion of control, USP9X-deficient, and USP9X-deficient plus MTH1-overexpressing HGC-27 and MKN-45 gastric cancer cells. RESULTS: Co-IP data show that USP9X interacts with and deubiquitinates MTH1. Overexpression of USP9X elevates MTH1 protein level by downregulating its ubiquitination, while knockdown of USP9X has the opposite effect on MTH1. USP9X deficiency in HGC-27 and MKN-45 cells causes decreased proliferation, cell cycle arrest, extra apoptosis, and defective migration and invasion, which could be rescued by excessive MTH1. CONCLUSION: USP9X interacts with and stabilizes MTH1 to promote the proliferation, survival, migration and invasion of gastric cancer cells.
Asunto(s)
Movimiento Celular , Proliferación Celular , Enzimas Reparadoras del ADN , Invasividad Neoplásica , Monoéster Fosfórico Hidrolasas , Neoplasias Gástricas , Ubiquitina Tiolesterasa , Humanos , Apoptosis , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , ARN Interferente Pequeño , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitinación , Hidrolasas Nudix/genética , Hidrolasas Nudix/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) is a breast cancer subtype that lacks targeted treatment options. The activation of the Notch developmental signaling pathway, which is a feature of TNBC, results in the secretion of proinflammatory cytokines and the recruitment of protumoral macrophages to the tumor microenvironment. While the Notch pathway is an obvious therapeutic target, its activity is ubiquitous, and predictably, anti-Notch therapies are burdened with significant on-target side effects. Previously, we discovered that, under conditions of cellular stress commonly found in the tumor microenvironment, the deubiquitinase USP9x forms a multiprotein complex with the pseudokinase tribbles homolog 3 (TRB3) that together activate the Notch pathway. Herein, we provide preclinical studies that support the potential of therapeutic USP9x inhibition to deactivate Notch. Using a murine TNBC model, we show that USP9x knockdown abrogates Notch activation, reducing the production of the proinflammatory cytokines, C-C motif chemokine ligand 2 (CCL2) and interleukin-1 beta (IL-1ß). Concomitant with these molecular changes, a reduction in tumor inflammation, the augmentation of antitumor immune response, and the suppression of tumor growth were observed. The pharmacological inhibition of USP9x using G9, a partially selective, small-molecule USP9x inhibitor, reduced Notch activity, remodeled the tumor immune landscape, and reduced tumor growth without associated toxicity. Proving the role of Notch, the ectopic expression of the activated Notch1 intracellular domain rescued G9-induced effects. This work supports the potential of USP9x inhibition to target Notch in metabolically vulnerable tissues like TNBC, while sparing normal Notch-dependent tissues.
Asunto(s)
Receptores Notch/genética , Transducción de Señal/genética , Neoplasias de la Mama Triple Negativas/genética , Ubiquitina Tiolesterasa/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Citocinas/genética , Regulación Neoplásica de la Expresión Génica/genética , Células HEK293 , Humanos , Interleucina-1beta/genética , Macrófagos/patología , Ratones , Neoplasias de la Mama Triple Negativas/patología , Microambiente Tumoral/genéticaRESUMEN
As a pattern recognition receptor, Toll-like receptor 4 (TLR4) is crucial for the development and progression of acute kidney injury (AKI). This study aims to explore whether the deubiquitinase Usp9x influences the TLR4/NF-B pathway to cause sepsis-induced acute kidney injury (S-AKI). The model of AKI was established in Sprague-Dawley rats using the cecal ligation and puncture (CLP) method, while renal tubular epithelial cell NRK-52E was stimulated with lipopolysaccharide (LPS) in vitro. All plasmids were transfected into NRK-52E cells according to the indicated group. The deubiquitinase of TLR4 was predicted by the online prediction software Ubibrowser. Subsequently, Western blot and Pearson correlation analysis identified Usp9x protein as a potential candidate. Co-IP analysis verified the interaction between TLR4 and Usp9x. Further research revealed that overexpression of Usp9x inhibited degradation of TLR4 protein by downregulating its ubiquitination modification levels. Both in vivo and in vitro experiments observed that interference with Usp9x effectively alleviated the inflammatory response and apoptosis of renal tubular epithelial cells (RTECs) induced by CLP or LPS, whereas overexpression of TLR4 reversed this situation. Transfection with sh-Usp9x in NRK-52E cells suppressed the expression of proteins associated with the TLR4/NF-κB pathway induced by LPS. Moreover, the overexpression of TLR4 reversed the effect of sh-Usp9x transfection. Therefore, the deubiquitinase Usp9x interacts with TLR4, leading to the upregulation of its expression through deubiquitination modification, and the activation of the TLR4/NF-κB signaling pathway, thereby promoting inflammation and apoptosis in renal tubular epithelial cells and contributing to sepsis-induced acute kidney injury.
Asunto(s)
Lesión Renal Aguda , Apoptosis , Células Epiteliales , Inflamación , Túbulos Renales , FN-kappa B , Ratas Sprague-Dawley , Sepsis , Transducción de Señal , Receptor Toll-Like 4 , Ubiquitina Tiolesterasa , Animales , Receptor Toll-Like 4/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/etiología , Lesión Renal Aguda/patología , Sepsis/complicaciones , Sepsis/metabolismo , FN-kappa B/metabolismo , Ratas , Células Epiteliales/metabolismo , Túbulos Renales/patología , Túbulos Renales/metabolismo , Túbulos Renales/citología , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Masculino , Inflamación/metabolismo , Modelos Animales de Enfermedad , Línea Celular , Lipopolisacáridos , UbiquitinaciónRESUMEN
Mammalian target of rapamycin (mTOR) is a central regulator of mammalian metabolism and physiology. Aberrant hyperactivation of the mTOR pathway promotes tumor growth and metastasis, and can also promote tumor resistance to chemotherapy and cancer drugs; this makes mTOR an attractive cancer therapeutic target. mTOR inhibitors have been approved to treat cancer; however, the mechanisms underlying drug sensitivity remain poorly understood. Here, whole exome sequencing of three chromophobe renal cell carcinoma (chRCC) patients with exceptional mTOR inhibitor sensitivity revealed that all three patients shared somatic mutations in the deubiquitinase gene USP9X. The clonal characteristics of the mutations, which were amassed by studying multiple patients' primary and metastatic samples from various years, together with the low USP9X mutation rate in unselected chRCC series, reinforced a causal link between USP9X and mTOR inhibitor sensitivity. Rapamycin treatment of USP9X-depleted HeLa and renal cancer 786-O cells, along with the pharmacological inhibition of USP9X, confirmed that this protein plays a role in patients' sensitivity to mTOR inhibitors. USP9X was not found to exert a direct effect on mTORC1, but subsequent ubiquitylome analyses identified p62 as a direct USP9X target. Increased p62 ubiquitination and the augmented rapamycin effect upon bortezomib treatment, together with the results of p62 and LC3 immunofluorescence assays, suggested that dysregulated autophagy in USP9X-depleted cells can have a synergistic effect with mTOR inhibitors. In summary, we show that USP9X constitutes a potential novel marker of sensitivity to mTOR inhibitors in chRCC patients, and represents a clinical strategy for increasing the sensitivity to these drugs.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Enzimas Desubicuitinizantes , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Inhibidores mTOR , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina Tiolesterasa/genéticaRESUMEN
Cell division cycle 123 (CDC123) has been implicated in a variety of human diseases. However, it remains unclear whether CDC123 plays a role in tumorigenesis and how its abundance is regulated. In this study, we found that CDC123 was highly expressed in breast cancer cells, and its high expression was positively correlated with a poor prognosis. Knowndown of CDC123 impaired the proliferation of breast cancer cells. Mechanistically, we identified a deubiquitinase, ubiquitin-specific peptidase 9, X-linked (USP9X), that could physically interact with and deubiquitinate K48-linked ubiquitinated CDC123 at the K308 site. Therefore, the expression of CDC123 was positively correlated with USP9X in breast cancer cells. In addition, we found that deletion of either USP9X or CDC123 led to altered expression of cell cycle-related genes and resulted in the accumulation of cells population in the G0/G1 phase, thereby suppressing cell proliferation. Treatment with the deubiquitinase inhibitor of USP9X, WP1130 (Degrasyn, a small molecule compound that USP9X deubiquitinase inhibitor), also led to the accumulation of breast cancer cells in the G0/G1 phase, but this effect could be rescued by overexpression of CDC123. Furthermore, our study revealed that the USP9X/CDC123 axis promotes the occurrence and development of breast cancer through regulating the cell cycle, and suggests that it may be a potential target for breast cancer intervention. In conclusion, our study demonstrates that USP9X is a key regulator of CDC123, providing a novel pathway for the maintenance of CDC123 abundance in cells, and supports USP9X/CDC123 as a potential target for breast cancer intervention through regulating the cell cycle.
Asunto(s)
Neoplasias de la Mama , Transformación Celular Neoplásica , Femenino , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , Ciclo Celular , Línea Celular Tumoral , Enzimas Desubicuitinizantes , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismoRESUMEN
The ubiquitin-specific protease USP9X has been found to play a role in multiple aspects of neural development including processes of neuronal migrations. In males, hemizygous partial loss of function variants in USP9X lead to a clinical phenotype primarily characterized by intellectual disability, hypotonia, speech and language impairment, behavioral disturbances accompanied by additional clinical features with variable expressivity. Structural brain abnormalities are reported in all cases where neuro-imaging was performed. The most common radiological features described include hypoplasia/agenesis of the corpus callosum, widened ventricles, white matter disturbances, and cerebellar hypoplasia. Here we report a child harboring a missense variant in USP9X presenting with the classical neurodevelopmental phenotype and a previously unreported radiological picture of periventricular heterotopia. This case expands the phenotypic landscape of this emergent condition and supports the critical role of USP9X in neuronal migration processes.
Asunto(s)
Discapacidad Intelectual , Heterotopia Nodular Periventricular , Humanos , Niño , Masculino , Heterotopia Nodular Periventricular/diagnóstico por imagen , Heterotopia Nodular Periventricular/genética , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Discapacidad Intelectual/complicaciones , Mutación Missense , Discapacidades del Desarrollo/genética , Radiografía , Ubiquitina Tiolesterasa/genéticaRESUMEN
Alveolar epithelial cells (AECs) function as a vital defense barrier avoiding the invasion of exogenous agents and preserving the functional and structural integrity of lung tissues, while damage/breakdown of this airway epithelial barrier is frequently associated with the pathogenesis of acute lung injury (ALI). NOD-like receptor family, pyrindomain-containing 3 (NLRP3) inflammasome activation-associated pyroptosis is involved in the development of ALI. Yet, how the activity of NLRP3 inflammasome is regulated in the context of ALI remains unknown. Herein we hypothesized that USP9X, an important deubiquitinase, participates in modulating the activation of NLRP3 inflammasome, thereby affecting the phenotypes in a lipopolysaccharide (LPS)-stimulated AEC model. Human pulmonary AECs were subjected to LPS/adenosine triphosphate (ATP) treatment to induce NLRP3 inflammasome activation and cell pyroptosis. Knockdown and overexpression of USP9X were applied to validate the function of USP9X. Inhibitors of proteinase and protein synthesis, as well as approach of co-immunoprecipitation coupled with Western blot, were utilized to explore the molecular mechanism. LPS/ATP challenge resulted in pronouncedly increased pyroptosis of AECs, activation of NLRP3 inflammasome and release of interleukin (IL)-1ß and IL-18 cytokines, while downregulation of USP9X could reverse these alterations. USP9X was found to have marked impact on NLRP3 protein instead of mRNA level. Furthermore, increased ubiquitination of NLRP3 was observed upon downregulating USP9X. Additionally, the inhibitory effect of USP9X downregulation was reversed by NLRP3 overexpression, while the promoting impact of USP9X overexpression was dampened by NLRP3 inhibitor in terms of cell pyroptosis and cytokine secretion. USP9X modulated the activity of NLRP3 inflammasome and pyroptosis of AECs via its deubiquitination function.
Asunto(s)
Lesión Pulmonar Aguda , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , Lipopolisacáridos/farmacología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Adenosina Trifosfato , Ubiquitina TiolesterasaRESUMEN
Endothelial pyroptosis is a pathological mechanism of atherosclerosis (AS). Circular RNAs (circRNAs) are vital in AS progression by regulating endothelial cell functions. The study aimed to explore whether circ-USP9× regulated pyroptosis of endothelial cell to involve in AS development and the molecular mechanism. Pyroptosis was determined using lactate dehydrogenase (LDH) assay, enzyme linked immunosorbent assay (ELISA), flow cytometry, propidium iodide (PI) staining assay, and western blot. The mechanism of circ-USP9× was determined using RNA pull-down and RNA binding protein immunoprecipitation (RIP) assays. Results showed that circ-USP9× was upregulated in AS and oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). Knockdown of circ-USP9× suppressed ox-LDL induced pyroptosis of HUVECs. Mechanically, circ-USP9× could bind to EIF4A3 in the cytoplasm. Moreover, EIF4A3 was bound to GSDMD and further affects GSDMD stability. Overexpression of EIF4A3 rescued cell pyroptosis induced by circ-USP9× depletion. In short, circ-USP9× interacted with EIF4A3 to enhance GSDMD stability, thus further promoting ox-LDL-induced pyroptosis of HUVECs. These findings suggested that circ-USP9× participates in AS progression and may be a potential therapeutic target for AS.
Asunto(s)
Aterosclerosis , MicroARNs , Humanos , Apoptosis , Aterosclerosis/genética , Proliferación Celular , ARN Helicasas DEAD-box , Ensayo de Inmunoadsorción Enzimática , Factor 4A Eucariótico de Iniciación , Células Endoteliales de la Vena Umbilical Humana , L-Lactato Deshidrogenasa , Lipoproteínas LDL/farmacología , Proteínas de Unión a Fosfato/genética , Proteínas Citotóxicas Formadoras de Poros , PiroptosisRESUMEN
Breast cancer is one of the most common malignancies in women worldwide. Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic subtype that has the characteristics of easy recurrence, poor prognosis as well as lack of targeted therapeutics. Snail1, a key factor regulating epithelial-mesenchymal transition (EMT) process, contributing to metastasis and chemoresistance in human cancers. However, the molecular mechanism of Snail1 stabilization in cancers is not fully understood. Here, we demonstrate that the deubiquitinating enzyme USP9X deubiquitinates and stabilizes Snail1, thereby promoting metastasis and chemoresistance. The depletion and pharmacological inhibition of USP9X by WP1130, an inhibitor of USP9X, downregulate endogenous Snail1 protein, inhibit cell migration, invasion, metastasis, and increase cellular sensitivity to cisplatin and paclitaxel both in vitro and in vivo, whereas the reconstitution of Snail1 in cells with USP9X depletion at least partially reverses these phenotypes. Overall, our study establishes the USP9X-Snail1 axis as an important regulatory mechanism of breast cancer metastasis and chemoresistance and provides a rationale for potential therapeutic interventions in the treatment of TNBC.
Asunto(s)
Resistencia a Antineoplásicos , Metástasis de la Neoplasia , Factores de Transcripción de la Familia Snail/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Ubiquitina Tiolesterasa/metabolismo , Línea Celular Tumoral , Movimiento Celular , Enzimas Desubicuitinizantes/metabolismo , Transición Epitelial-Mesenquimal , Femenino , Humanos , Factores de Transcripción de la Familia Snail/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
The ubiquitin-specific peptidase 9 X-linked (USP9X) is one of the highly conserved members belonging to the ubiquitin-specific proteases (USPs) family, which has been reported to control substrates-mediated biological functions through deubiquitinating and stabilizing substrates. Here, we have found that TGFBR2, the type II receptor of the transforming growth factor beta (TGF-ß) signaling pathway, is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells (GCs). Mechanically, USP9X positively influences the expression of TGFBR2 at different levels through two independent ways: (i) directly targets and deubiquitinates TGFBR2, which maintains the protein stability of TGFBR2 through avoiding degradation mediated by ubiquitin-proteasome system; (ii) indirectly maintains TGFBR2 messenger RNA (mRNA) expression via SMAD4/miR-143 axis. Specifically, SMAD4, another substrate of USP9X, acts as a transcription factor and suppresses miR-143 which inhibits the mRNA level of TGFBR2 by directly binding to its 3'-untranslated region. Functionally, the maintenance of TGFBR2 by USP9X activates the TGF-ß signaling pathway, which further represses GC apoptosis. Our study highlights a functional micro-regulatory network composed of deubiquitinase (USP9X), small noncoding RNA (miR-143) and the TGF-ß signaling pathway, which plays a crucial role in the regulation of GC apoptosis and female fertility.
Asunto(s)
Células de la Granulosa/metabolismo , MicroARNs , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Transducción de Señal , Ubiquitina Tiolesterasa/metabolismo , Regiones no Traducidas 3' , Animales , Apoptosis , Femenino , Células de la Granulosa/citología , MicroARNs/genética , ARN Mensajero/genética , Sus scrofa , PorcinosRESUMEN
In order to prevent the deleterious effects of genotoxic agents, cells have developed complex surveillance mechanisms and DNA repair pathways that allow them to maintain genome integrity. The ubiquitin-specific protease 9X (USP9X) contributes to genome stability during DNA replication and chromosome segregation. Depletion of USP9X leads to DNA double-strand breaks, some of which are triggered by replication fork collapse. Here, we identify USP9X as a novel regulator of homologous recombination (HR) DNA repair in human cells. By performing cellular HR reporter, irradiation-induced focus formation and colony formation assays, we show that USP9X is required for efficient HR. Mechanistically, we show USP9X is important to sustain the expression levels of key HR factors, namely BRCA1 and RAD51 through a non-canonical regulation of their mRNA abundance. Intriguingly, we find that the contribution of USP9X to BRCA1 and RAD51 expression is independent of its known catalytic activity. Thus, this work identifies USP9X as a regulator of HR, demonstrates a novel mechanism by which USP9X can regulate protein levels, and provides insights in to the regulation of BRCA1 and RAD51 mRNA.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Recombinasa Rad51 , Reparación del ADN por Recombinación , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Daño del ADN , Reparación del ADN/genética , Replicación del ADN , Recombinación Homóloga/genética , Humanos , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Ubiquitina Tiolesterasa/genética , Proteasas Ubiquitina-Específicas/genéticaRESUMEN
Pathogenic variants in USP9X, on X chromosome, have been implicated in syndromic intellectual disability (ID) in both males and females with distinct craniofacial features. We report a truncating variant, c.885_889delAAAAG, p.(Lys296Serfs*4), in the USP9X gene with incomplete penetrance in two nontwin female siblings with phenotypic resemblance to female-specific syndromic ID (MIM 300969, also known as MRX99F). To investigate the possible genetic etiology of the reduced penetrance, X-inactivation, RNA-Seq, and full quad exome analyses were attempted, but failed to identify a promising candidate modifier. While the penetrance of pathogenic variants in USP9X in female appears to be high (95%) and the variants frequently occur de novo, incomplete penetrance should be considered.
Asunto(s)
Discapacidad Intelectual , Ubiquitina Tiolesterasa , Exoma , Femenino , Humanos , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Masculino , Penetrancia , RNA-Seq , Ubiquitina Tiolesterasa/genética , Secuenciación del ExomaRESUMEN
Congenital diaphragmatic hernia (CDH) can occur in isolation or in conjunction with other birth defects (CDH+). A molecular etiology can only be identified in a subset of CDH cases. This is due, in part, to an incomplete understanding of the genes that contribute to diaphragm development. Here, we used clinical and molecular data from 36 individuals with CDH+ who are cataloged in the DECIPHER database to identify genes that may play a role in diaphragm development and to discover new phenotypic expansions. Among this group, we identified individuals who carried putatively deleterious sequence or copy number variants affecting CREBBP, SMARCA4, UBA2, and USP9X. The role of these genes in diaphragm development was supported by their expression in the developing mouse diaphragm, their similarity to known CDH genes using data from a previously published and validated machine learning algorithm, and/or the presence of CDH in other individuals with their associated genetic disorders. Our results demonstrate how data from DECIPHER, and other public databases, can be used to identify new phenotypic expansions and suggest that CREBBP, SMARCA4, UBA2, and USP9X play a role in diaphragm development.
Asunto(s)
Hernias Diafragmáticas Congénitas , Animales , Variaciones en el Número de Copia de ADN , Diafragma , Hernias Diafragmáticas Congénitas/genética , RatonesRESUMEN
Only a few patients with deletions or duplications at Xp11.4, bridging USP9X, DDX3X, and CASK genes, have been described so far. Here, we report on a female harboring a de novo Xp11.4p11.3 deletion and a male with an overlapping duplication inherited from an unaffected mother, presenting with syndromic intellectual disability. We discuss the role of USP9X, DDX3X, and CASK genes in human development and describe the effects of Xp11.4 deletion and duplications in female and male patients, respectively.
Asunto(s)
Discapacidad Intelectual , Cromosomas Humanos X , ARN Helicasas DEAD-box/genética , Femenino , Humanos , Discapacidad Intelectual/genética , Masculino , Fenotipo , Ubiquitina Tiolesterasa/genéticaRESUMEN
Genetic association studies have identified many factors associated with neurodevelopmental disorders such as autism spectrum disorder (ASD). However, the way these genes shape neuroanatomical structure and connectivity is poorly understood. Recent research has focused on proteins that act as points of convergence for multiple factors, as these may provide greater insight into understanding the biology of neurodevelopmental disorders. USP9X, a deubiquitylating enzyme that regulates the stability of many ASD-related proteins, is one such point of convergence. Loss of function variants in human USP9X lead to brain malformations, which manifest as a neurodevelopmental syndrome that frequently includes ASD, but the underlying structural and connectomic abnormalities giving rise to patient symptoms is unknown. Here, we analyzed forebrain-specific Usp9x knockout mice (Usp9x-/y) to address this knowledge gap. Usp9x-/y mice displayed abnormal communication and social interaction behaviors. Moreover, the absence of Usp9x culminated in reductions to the size of multiple brain regions. Diffusion tensor magnetic resonance imaging revealed deficits in all three major forebrain commissures, as well as long-range hypoconnectivity between cortical and subcortical regions. These data identify USP9X as a key regulator of brain formation and function, and provide insights into the neurodevelopmental syndrome arising as a consequence of USP9X mutations in patients.
Asunto(s)
Corteza Cerebral/fisiopatología , Vías Nerviosas/fisiopatología , Neurogénesis/fisiología , Ubiquitina Tiolesterasa/metabolismo , Animales , Conducta Animal , Masculino , Ratones , Ratones NoqueadosRESUMEN
Sepsis is a life-threatening condition manifested by concurrent inflammation and immunosuppression. Ubiquitin-specific peptidase 9, X-linked (USP9x), is a USP domain-containing deubiquitinase which is required in T-cell development. In the present study, we investigate whether USP9x plays a role in hepatic CD8 + T-cell dysfunction in septic mice. We find that CD8 + T cells are decreased in the blood of septic patients with liver injury compared with those without liver injury, the CD4/CD8 ratio is increased, and the levels of cytolytic factors, granzyme B and perforin are downregulated. The number of hepatic CD8 + T cells and USP9x expression are both increased 24â h after cecal ligation and puncture-induced sepsis in a mouse model, a pattern similar to liver injury. The mechanism involves promotion of CD8 + T-cell dysfunction by USP9x associated with suppression of cell cytolytic activity via autophagy inhibition, which is reversed by the USP9x inhibitor WP1130. In the in vivo studies, autophagy is significantly increased in hepatic CD8 + T cells of septic mice with conditional knockout of mammalian target of rapamycin. This study shows that USP9x has the potential to be used as a therapeutic target in septic liver injury.
Asunto(s)
Sepsis , Proteasas Ubiquitina-Específicas , Animales , Ratones , Autofagia , Linfocitos T CD8-positivos , Hígado , Mamíferos , Sepsis/complicacionesRESUMEN
USP9X is a conserved deubiquitinase (DUB) that regulates multiple cellular processes. Dysregulation of USP9X has been linked to cancers and X-linked intellectual disability. Here, we report the crystal structure of the USP9X catalytic domain at 2.5-Å resolution. The structure reveals a canonical USP-fold comprised of fingers, palm, and thumb subdomains, as well as an unusual ß-hairpin insertion. The catalytic triad of USP9X is aligned in an active configuration. USP9X is exclusively active against ubiquitin (Ub) but not Ub-like modifiers. Cleavage assays with di-, tri-, and tetraUb chains show that the USP9X catalytic domain has a clear preference for K11-, followed by K63-, K48-, and K6-linked polyUb chains. Using a set of activity-based diUb and triUb probes (ABPs), we demonstrate that the USP9X catalytic domain has an exo-cleavage preference for K48- and endo-cleavage preference for K11-linked polyUb chains. The structure model and biochemical data suggest that the USP9X catalytic domain harbors three Ub binding sites, and a zinc finger in the fingers subdomain and the ß-hairpin insertion both play important roles in polyUb chain processing and linkage specificity. Furthermore, unexpected labeling of a secondary, noncatalytic cysteine located on a blocking loop adjacent to the catalytic site by K11-diUb ABP implicates a previously unreported mechanism of polyUb chain recognition. The structural features of USP9X revealed in our study are critical for understanding its DUB activity. The new Ub-based ABPs form a set of valuable tools to understand polyUb chain processing by the cysteine protease class of DUBs.