RESUMEN
Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27kip1 protein levels was observed. The synergy was not dependent on apoptosis induction, and appeared to occur in cells with higher USP8 levels. In vivo antitumor activity studies supported the advantage of the combination of CAPE and cisplatin in the subcutaneous model of cisplatin-resistant IGROV-1/Pt1 ovarian carcinoma as well as CAPE activity on intraperitoneal disease. This study reveals the therapeutic potential of CAPE in cisplatin-resistant ovarian tumors as well as in tumors expressing USP8.
Asunto(s)
Antineoplásicos/administración & dosificación , Ácidos Cafeicos/administración & dosificación , Cisplatino/administración & dosificación , Endopeptidasas/biosíntesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Neoplasias Ováricas/enzimología , Alcohol Feniletílico/análogos & derivados , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/biosíntesis , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Femenino , Humanos , Ratones , Ratones Desnudos , Neoplasias Ováricas/tratamiento farmacológico , Alcohol Feniletílico/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Hepatocellular carcinoma (HCC) has high morbidity and mortality. MicroRNAs (miRNAs), which could be regulated by cancer-derived exosomes, play critical regulatory roles in the initiation and development of cancer. However, the expressions, effects, and mechanisms of abundant miRNAs regulated by HCC cancer-derived exosomes in HCC remain largely unclear. Exosomes of HepG2 cells under heat shock, TGF-ß1, doxorubicin, acid and hypoxia/reoxygenation (H/R) conditions, and exosomes were successfully identified by transmission electron microscopy and Western blot analysis. The identified exosomes were then applied to evaluate the miRNA expression profiles by RNA sequencing. Mechanically, we discovered that doxorubicin was upregulated, TGF-ß1 downregulated the expressions of Vps4A, Rab27A, Alix, and Hrs in HepG2 cells and exosomes, and Vps4A and Rab27A, as target genes for miR-4454, could also be downregulated by miR-4454. Functionally, we revealed that miR-4454 inhibitor and miR-4454 inhibitor-mediated exosomes could markedly suppress proliferation, migration, invasion, and vascularization and accelerate cycle arrest, apoptosis, and ROS of HepG2 cells. This study provided many potential HCC cancer-derived exosome-mediated miRNAs in HCC under 5 different stimulus conditions. Meanwhile, we certified that miR-4454 in exosomes could provide a novel and effective mechanism for HCC function.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/patología , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , MicroARNs/genética , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Proteínas rab27 de Unión a GTP/antagonistas & inhibidores , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimiento Celular , Proliferación Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Células Tumorales Cultivadas , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteínas rab27 de Unión a GTP/genética , Proteínas rab27 de Unión a GTP/metabolismoRESUMEN
Ubiquitination is a crucial posttranslational protein modification involved in a myriad of biological pathways. This modification is reversed by deubiquitinases (DUBs) that deconjugate the single ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors, which was previously successfully applied to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to selectively target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbVSP.1 and UbVSP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbVSP.1 complex structure by X-ray crystallography, revealing hotspots of the JAMM-UbV interaction. Finally, we show that UbVSP.1 and UbVSP.3 are potent inhibitors of STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates that UbV technology is suitable to develop molecules as tools to target metalloproteases, which can be used to further understand the cellular function of JAMM family DUBs.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte , Péptido Hidrolasas , Biblioteca de Péptidos , Inhibidores de Proteasas/química , Ubiquitina Tiolesterasa , Ubiquitina , Cristalografía por Rayos X , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Humanos , Péptido Hidrolasas/química , Estructura Cuaternaria de Proteína , Ubiquitina/química , Ubiquitina/genética , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/químicaRESUMEN
The ubiquitin-specific protease 8 (USP8) is a prototypic multidomain deubiquitinating enzyme with pleiotropic functions. We investigated the role of USP8 in hepatocellular carcinoma (HCC) by analyzing expression patterns of USP8 in HCC patients, and evaluating its functions and underlying signaling. Among 20 HCC patients investigated, we found that USP8 protein upregulation was a common phenomenon (17 out of 20) in HCC compared to normal liver tissue. Furthermore, the upregulation of USP8 was not associated with any clinicopathology. USP8 inhibition via genetic and pharmacological approaches resulted in growth inhibition and apoptosis induction in both sensitive and doxorubicin-resistant HCC cells. Of note, USP8 inhibition significantly enhanced doxorubicin or sorafenib's efficacy in HCC cells and mouse models. We further found that USP8 inhibition decreased levels of multiple receptor tyrosine kinases (RTKs) by ~90%, such as epidermal growth factor receptor (EGFR) and c-Met. Consistently, the downstream signaling regulated by RTKs was disrupted in HCC cells after USP8 inhibition, as shown by the decreased p-Akt, p-STAT3 and p-Raf. Our findings demonstrate that USP8 is a novel therapeutic target in HCC. Inhibiting USP8 has potential to overcome current drug resistance, particularly on HCC patients with high USP8 expression.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Resistencia a Antineoplásicos , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Neoplasias Hepáticas/tratamiento farmacológico , Proteínas Tirosina Quinasas Receptoras/metabolismo , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Regulación hacia Abajo/genética , Endopeptidasas/genética , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones SCID , Transducción de Señal , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Regulación hacia Arriba/genéticaAsunto(s)
Proteínas AAA/genética , ATPasas Asociadas con Actividades Celulares Diversas/ultraestructura , Complejos de Clasificación Endosomal Requeridos para el Transporte/ultraestructura , Inhibidores Enzimáticos/química , Conformación Proteica/efectos de los fármacos , Proteínas AAA/antagonistas & inhibidores , Proteínas AAA/ultraestructura , ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , Adenosina Trifosfato/genética , Cristalografía por Rayos X , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Humanos , HidrólisisRESUMEN
USP8 is a deubiquitinating enzyme in the family of ubiquitin-specific proteases (USPs) which can remove ubiquitin from the substrate and protect the substrate from degradation. The upregulated or mutated USP8 becomes hyperactivated and stabilizes numerous oncogenes or proto-oncogenes leading to cancer progression and survival by activating multiple signaling pathways. Moreover, USP8 inhibition is also important to overcome anticancer drug-resistant. This review is the first study to find, combine, analyze, and represent the multiple oncogenic signaling pathways with their downstream and upstream regulation activated or enhanced by USP8, which will help the researchers to find any therapeutic strategy for drug discovery by inhibiting or suppressing the multi-targeted USP8.
Asunto(s)
Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Endopeptidasas , Complejos de Clasificación Endosomal Requeridos para el Transporte , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Ubiquitina Tiolesterasa , Descubrimiento de Drogas , Resistencia a Antineoplásicos/genética , Endopeptidasas/genética , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Neoplasias/genética , Neoplasias/patología , Proteínas Proto-Oncogénicas/genética , Transducción de Señal/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, caused by mutation in the gene encoding lamin A/C, which produces a truncated protein called progerin. In cells from HGPS patients, progerin accumulates at the nuclear membrane (NM), where it causes NM deformations. In this study, we investigated whether progerin-induced NM deformation involved ESCRT-III, a protein complex that remodels nuclear and cytoplasmic membranes. The ESCRT-III protein CHMP4B was recruited to sites of aberrant NM proliferation in human cells ectopically expressing progerin and in patient-derived HGPS fibroblasts. Derepression of NM deformation in these cells was observed following depletion of CHMP4B or an ESCRT-III adaptor, ALIX. Treatment with rapamycin (which induce autophagic clearance of progerin and reverse progerin-induced cellular phenotypes) down-regulated progerin-induced NM deformation, whereas treatment with bafilomycin A1 (an inhibitor of autophagy and lysosome-based degradation) or CHMP4B depletion antagonized the effects of rapamycin. These results indicate that the ALIX-mediated ESCRT-III pathway plays a suppressive role in progerin-induced NM deformation and suggest that autophagy down-regulates progerin-induced NM deformation in a manner dependent on ESCRT-III machinery.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Lamina Tipo A/genética , Progeria/genética , Envejecimiento/efectos de los fármacos , Envejecimiento/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Núcleo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Fibroblastos/efectos de los fármacos , Humanos , Lamina Tipo A/metabolismo , Macrólidos/farmacología , Mutación/genética , Membrana Nuclear/metabolismo , Membrana Nuclear/patología , Progeria/metabolismo , Progeria/patología , Sirolimus/farmacologíaRESUMEN
The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein-coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule-binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Endosomas/metabolismo , Receptores de Glucagón/metabolismo , Proteínas de Unión al GTP rab4/metabolismo , Línea Celular , Regulación hacia Abajo , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Glucagón/farmacología , Humanos , Monensina/farmacología , Mutagénesis , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores de Glucagón/agonistas , Receptores de Glucagón/genética , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación/efectos de los fármacos , Proteínas de Unión al GTP rab4/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
USP8, one member of deubiquitinating enzymes (DUBs) families, maintains the ubiquitination level of EGFR and regulates the downstream signaling pathways. The deregulation of USP8 has been implicated in many human diseases, especially in cancer. Therefore, USP8 has been identified as a promising target for drug design. Herein, via high throughput screening based on Ubiquitin-rhodamine-110 (Ubiquitin-Rho-110) fluorometric activity assay, we discovered a novel inhibitor DC-U43. By structure optimization, DC-U43-10 reached a half-maximal inhibitory concentration (IC50) value of 2.6 ± 1.1 µM and exhibited 10-fold selectivity against USP7. The binding between DC-U43-10 and USP8 was validated by surface plasmon resonance (SPR) assay with a KD value of 10.5 ± 3.7 µM. It also inhibited the colony formation of H1975 cells. Hence, DC-U43-10 represents a kind of USP8 inhibitors with novel scaffold and has broad prospects for being a probe for USP8-related academic and clinical research.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Endopeptidasas , Humanos , Simulación del Acoplamiento Molecular , Transducción de Señal , Espectrometría de Fluorescencia/métodos , Resonancia por Plasmón de Superficie , UbiquitinaciónRESUMEN
We performed a small interfering RNA screen to identify targets for cutaneous squamous cell carcinoma (cSCC) therapy in the ubiquitin/ubiquitin-like system. We provide evidence for selective anti-cSCC activity of knockdown of the E3 ubiquitin ligase MARCH4, the ATPase p97/VCP, the deubiquitinating enzyme USP8, the cullin-RING ligase (CRL) 4 substrate receptor CDT2/DTL, and components of the anaphase-promoting complex/cyclosome (APC/C). Specifically attenuating CRL4CDT2 by CDT2 knockdown can be more potent in killing cSCC cells than targeting CRLs or CRL4s in general by RBX1 or DDB1 depletion. Suppression of the APC/C or forced APC/C activation by targeting its repressor EMI1 are both potential therapeutic approaches. We observed that cSCC cells can be selectively killed by small-molecule inhibitors of USP8 (DUBs-IN-3/compound 22c) and the NEDD8 E1 activating enzyme/CRLs (MLN4924/pevonedistat). A substantial proportion of cSCC cell lines are very highly MLN4924-sensitive. Pathways that respond to defects in proteostasis are involved in the anti-cSCC activity of p97 suppression. Targeting USP8 can reduce the expression of growth factor receptors that participate in cSCC development. EMI1 and CDT2 depletion can selectively cause DNA re-replication and DNA damage in cSCC cells.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Ciclopentanos/farmacología , Ciclopentanos/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Endopeptidasas/genética , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Terapia Molecular Dirigida/métodos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , ARN Interferente Pequeño/metabolismo , Neoplasias Cutáneas/patología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) is highly metastatic and frequently has a poor prognosis. The lack of comprehension of TNBC and gene therapy targets has led to limitedly effective treatment for TNBC. This study was conducted to better understand the molecular mechanism behind TNBC progression, and to find out promising gene therapy targets for TNBC. Herein the influence of miR-122-5p's binding charged multivesicular body protein 3 (CHMP3) 3'-untranslated region (3'-UTR) on in TNBC cells was investigated. in vitro experiments quantitative real-time polymerase chain reaction, immunoblot analysis, dual-luciferase reporter gene assay, cell counting assay, transwell invasion assay, and flow cytometry-determined cell apoptosis assay were employed. We also used TargetScan Human 7.2 database to find out the target relationship between miR-122-5p and CHMP3 3'-UTR. TImer algorithm was used to provide an overview of the expression of CHMP3 gene across human pan-cancer, to predict the survival outcome of breast cancer patients, and to predict the correlation between CHMP3 gene expression and epithelial-mesenchymal transition (EMT) and mitogen-activated protein kinase (MAPK)-related gene expression. CHMP3 gene was significantly downregulated across a wide range of human cancers including breast cancer (BRCA). A higher level of CHMP3 gene predicted a better 3- and 5-year survival outcome of patients with BRCA. In our experiments, miR-122-5p was significantly upregulated and CHMP3 gene was significantly downregulated in TNBC cells compared with normal cell line. miR-122-5p mimics enhanced TNBC cell viability, proliferation, and invasion whereas the upregulation of CHMP3 gene led to an opposite outcome. Forced expression of miR-122-5p suppressed cell apoptosis, compelled EMT and MAPK signaling whereas forced expression of CHMP3 did the opposite. We then conclude that miR-122-5p promotes aggression and EMT in TNBC by suppressing CHMP3 through MAPK signaling.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Transición Epitelial-Mesenquimal/genética , Sistema de Señalización de MAP Quinasas/fisiología , MicroARNs/genética , Neoplasias de la Mama Triple Negativas/genética , Regiones no Traducidas 3'/genética , Apoptosis/genética , Movimiento Celular/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Femenino , Terapia Genética/métodos , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Cushing's disease is primarily caused by autonomic hypersecretion of adrenocorticotropic hormone (ACTH) from a pituitary adenoma. In Cushing's disease, mutations in the ubiquitin-specific protease 8 (USP8) have been detected. These mutations are associated with hyperactivation of USP8 that prevent epidermal growth factor receptor (EGFR) degradation. This leads to increased EGFR stability and results in the maintenance of EGFR signaling in Cushing's disease. USP8 inhibitors can suppress the growth of various tumors. In this study, the effects of a potent USP8 inhibitor, DUBs-IN-2, on ACTH production and cell proliferation were examined in mouse corticotroph tumor (AtT-20) cells. Proopiomelanocortin (Pomc) mRNA levels and ACTH levels were decreased in AtT-20 cells by DUBs-IN-2. Further, cell proliferation was inhibited, and apoptosis was induced by DUBs-IN-2. Transcript levels of pituitary tumor-transforming gene 1 (Pttg1), a pituitary tumor growth marker, were increased; and transcript levels of stress response growth arrest and DNA damage-inducible 45 (Gadd45ß) and Cdk5 and ABL enzyme substrate 1 (Cables1) mRNA levels were increased in response to the drug. Gadd45ß or Cables1 knockdown partially inhibited the DUBs-IN-2-induced decrease in cell proliferation, but not Pomc mRNA levels. Both GADD45ß and CABLES1 may be responsible, at least in part, for the USP8-induced suppression of corticotroph tumor cell proliferation. USP-8 may be a new treatment target in Cushing's disease.
Asunto(s)
Adenoma Hipofisario Secretor de ACTH/metabolismo , Adenoma/metabolismo , Hormona Adrenocorticotrópica/efectos de los fármacos , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Indenos/farmacología , Pirazinas/farmacología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Hormona Adrenocorticotrópica/metabolismo , Animales , Antígenos de Diferenciación/efectos de los fármacos , Antígenos de Diferenciación/genética , Línea Celular Tumoral , Quinasa 5 Dependiente de la Ciclina/efectos de los fármacos , Quinasa 5 Dependiente de la Ciclina/genética , Ciclinas/efectos de los fármacos , Ciclinas/genética , Endopeptidasas , Receptores ErbB/efectos de los fármacos , Receptores ErbB/metabolismo , Técnicas de Silenciamiento del Gen , Ratones , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/metabolismo , Proopiomelanocortina/efectos de los fármacos , Proopiomelanocortina/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Securina/efectos de los fármacos , Securina/genéticaRESUMEN
The divergent clinical outcomes of human T cell leukemia virus type 1 (HTLV-1) and HTLV-2 infections have been attributed to functional differences in their antisense proteins. In contrast to HTLV-1 bZIP factor (HBZ), the role of the antisense protein of HTLV-2 (APH-2) in HTLV-2 infection is poorly understood. In previous studies, we identified the endosomal sorting complex required for transport 0 (ESCRT-0) subunit HRS as a novel interaction partner of APH-2 but not HBZ. HRS is a master regulator of endosomal protein sorting for lysosomal degradation and is hijacked by many viruses to promote replication. However, no studies to date have shown a link between HTLVs and HRS. In this study, we sought to characterize the interaction between HRS and APH-2 and to investigate the impact of HRS on the life cycle of HTLV-2. We confirmed a direct specific interaction between APH-2 and HRS and showed that the CC2 domain of HRS and the N-terminal domain of APH-2 mediate their interaction. We demonstrated that HRS recruits APH-2 to early endosomes, possibly furnishing an entry route into the endosomal/lysosomal pathway. We demonstrated that inhibition of this pathway using either bafilomycin or HRS overexpression substantially extends the half-life of APH-2 and stabilizes Tax2B expression levels. We found that HRS enhances Tax2B-mediated long terminal repeat (LTR) activation, while depletion of HRS enhances HTLV-2 production and release, indicating that HRS may have a negative impact on HTLV-2 replication. Overall, our study provides important new insights into the role of the ESCRT-0 HRS protein, and by extension the ESCRT machinery and the endosomal/lysosomal pathway, in HTLV-2 infection.IMPORTANCE While APH-2 is the only viral protein consistently expressed in infected carriers, its role in HTLV-2 infection is poorly understood. In this study, we characterized the interaction between the ESCRT-0 component HRS and APH-2 and explored the role of HRS in HTLV-2 replication. HRS is a master regulator of protein sorting for lysosomal degradation, a feature that is manipulated by several viruses to promote replication. Unexpectedly, we found that HRS targets APH-2 and possibly Tax2B for lysosomal degradation and has an overall negative impact on HTLV-2 replication and release. The negative impact of interactions between HTLV-2 regulatory proteins and HRS, and by extension the ESCRT machinery, may represent an important strategy used by HTLV-2 to limit virus production and to promote persistence, features that may contribute to the limited pathogenic potential of this infection.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Productos del Gen tax/genética , Virus Linfotrópico T Tipo 2 Humano/genética , Fosfoproteínas/genética , Proteínas de los Retroviridae/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Cicloheximida/farmacología , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Regulación de la Expresión Génica , Productos del Gen tax/metabolismo , Células HEK293 , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/efectos de los fármacos , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Virus Linfotrópico T Tipo 2 Humano/efectos de los fármacos , Virus Linfotrópico T Tipo 2 Humano/metabolismo , Virus Linfotrópico T Tipo 2 Humano/patogenicidad , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/virología , Macrólidos/farmacología , Fosfoproteínas/metabolismo , Proteínas de los Retroviridae/metabolismo , Transducción de Señal , Replicación Viral/efectos de los fármacosRESUMEN
Hepatocellular carcinoma-related protein-1 (HCRP-1), a subunit of mammalian endosomal sorting complex required for transport-I (ESCRT-I), is frequently downregulated in various kinds of malignant tumors. The role of HCRP-1 in colorectal cancer (CRC) remains unknown. We investigate the clinical value of HCRP-1 and its impact on anoikis in CRC. The negative expression of HCRP-1 was significantly correlated with tumor size (P = 0.033), PT status (P = 0.001), TNM stage (P = 0.039), and histological grade (P = 0.01). Univariate and multivariate analyses revealed that HCRP-1 was an independent prognostic factor for CRC (hazard ratio (HR) = 0.237, P < 0.001 for 5-year overall survival). In the in vitro assay, we found that HCRP-1 depletion resulted in cell anoikis resistance. Knockdown of HCRP-1 suppressed Bcl-2 interacting mediator of cell death (BIM) expression, with phosphorylation of AKT and p-FoxO3a, which was reversed by AKT siRNA or AKT inhibitor. Further analysis showed that loss of HCRP-1 obviously increased the activation of EGFR. Inhibition of EGFR blocked si-HCRP1-mediated phosphorylation of EGFR, AKT, FoxO3a, and BIM expression. Moreover, the in vivo results revealed that loss of HCRP-1 promoted cancer metastasis. Our findings implied that reduced HCRP-1 expression in CRC resulted in anoikis resistance and contributed to CRC metastasis and poor prognosis. These data may help design effective therapy targeting HCRP-1 pathway to control colon cancer growth and metastasis.
Asunto(s)
Proteína 11 Similar a Bcl2/genética , Biomarcadores de Tumor/genética , Neoplasias del Colon/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-akt/genética , Anciano , Animales , Anoicis/genética , Proteína 11 Similar a Bcl2/metabolismo , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Estudios de Cohortes , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/mortalidad , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Humanos , Metástasis Linfática , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Análisis de Supervivencia , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Intracellular pathogens have varied strategies to breach the endolysosomal barrier so that they can deliver effectors to the host cytosol, access nutrients, replicate in the cytoplasm, and avoid degradation in the lysosome. In the case of Mycobacterium tuberculosis, the bacterium perforates the phagosomal membrane shortly after being taken up by macrophages. Phagosomal damage depends upon the mycobacterial ESX-1 type VII secretion system (T7SS). Sterile insults, such as silica crystals or membranolytic peptides, can also disrupt phagosomal and endolysosomal membranes. Recent work revealed that the host endosomal sorting complex required for transport (ESCRT) machinery rapidly responds to sterile endolysosomal damage and promotes membrane repair. We hypothesized that ESCRTs might also respond to pathogen-induced phagosomal damage and that M. tuberculosis could impair this host response. Indeed, we found that ESCRT-III proteins were recruited to M. tuberculosis phagosomes in an ESX-1-dependent manner. We previously demonstrated that the mycobacterial effectors EsxG/TB9.8 and EsxH/TB10.4, both secreted by the ESX-3 T7SS, can inhibit ESCRT-dependent trafficking of receptors to the lysosome. Here, we additionally show that ESCRT-III recruitment to sites of endolysosomal damage is antagonized by EsxG and EsxH, both within the context of M. tuberculosis infection and sterile injury. Moreover, EsxG and EsxH themselves respond within minutes to membrane damage in a manner that is independent of calcium and ESCRT-III recruitment. Thus, our study reveals that T7SS effectors and ESCRT participate in a series of measures and countermeasures for control of phagosome integrity.IMPORTANCEMycobacterium tuberculosis causes tuberculosis, which kills more people than any other infection. M. tuberculosis grows in macrophages, cells that specialize in engulfing and degrading microorganisms. Like many intracellular pathogens, in order to cause disease, M. tuberculosis damages the membrane-bound compartment (phagosome) in which it is enclosed after macrophage uptake. Recent work showed that when chemicals damage this type of intracellular compartment, cells rapidly detect and repair the damage, using machinery called the endosomal sorting complex required for transport (ESCRT). Therefore, we hypothesized that ESCRT might also respond to pathogen-induced damage. At the same time, our previous work showed that the EsxG-EsxH heterodimer of M. tuberculosis can inhibit ESCRT, raising the possibility that M. tuberculosis impairs this host response. Here, we show that ESCRT is recruited to damaged M. tuberculosis phagosomes and that EsxG-EsxH undermines ESCRT-mediated endomembrane repair. Thus, our studies demonstrate a battle between host and pathogen over endomembrane integrity.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Interacciones Huésped-Patógeno , Mycobacterium tuberculosis/patogenicidad , Sistemas de Secreción Tipo VII/metabolismo , Factores de Virulencia/metabolismo , Animales , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Ratones , Mycobacterium tuberculosis/metabolismo , Fagosomas/metabolismo , Fagosomas/microbiología , Unión ProteicaRESUMEN
The ESCRT machinery mediates membrane fission in a variety of processes in cells. According to current models, ESCRT-III proteins drive membrane fission by assembling into helical filaments on membranes. Here, we used 3D STORM imaging of endogenous ESCRT-III component IST1 to reveal the evolution of the structural organization of ESCRT-III in mammalian cytokinetic abscission. Using this approach, ESCRT-III ring and spiral assemblies were resolved and characterized at different stages of abscission. Visualization of IST1 structures in cells lacking the microtubule-severing enzyme spastin and in cells depleted of specific ESCRT-III components or the ATPase VPS4 demonstrated the contribution of these components to the organization and function of ESCRTs in cells. This work provides direct evidence that ESCRT-III proteins form helical filaments to mediate their function in cells and raises new mechanistic scenarios for ESCRT-driven cytokinetic abscission.
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ATPasas Asociadas con Actividades Celulares Diversas/genética , Citocinesis/genética , Citosol/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Microtúbulos/metabolismo , Proteínas Oncogénicas/genética , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Citosol/ultraestructura , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Edición Génica , Regulación de la Expresión Génica , Células HeLa , Humanos , Microtúbulos/ultraestructura , Imagen Molecular , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Espastina/deficiencia , Espastina/genética , Imagen de Lapso de Tiempo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Hepatitis E virus (HEV) generally causes self-limiting acute viral hepatitis in normal individuals. It causes a more severe disease in immunocompromised persons and pregnant women. Due to the lack of an efficient cell culture system or animal model, the life cycle of the virus is understudied, few antiviral targets are known, and very few antiviral candidates against HEV infection have been identified. Inhibition of virus release is one possible antiviral development strategy, which limits the spread of the virus. Previous studies have demonstrated the essential role of the interaction between the PSAP motif of the viral open reading frame 3 protein (ORF3-PSAP) and the UEV domain of the host tumor susceptibility gene 101 (TSG101) protein (UEV-TSG101) in mediating the release of genotype 3 HEV. Cyclic peptide (CP) inhibitors of the interaction between the human immunodeficiency virus (HIV) gag-PTAP motif and UEV-TSG101 are known to block the release of HIV. Using a molecular dynamic simulation, we observed that both gag-PTAP and ORF3-PSAP motifs bind to the same site in UEV-TSG101 by hydrogen bonding. HIV-released inhibitory CPs also displayed binding to the same site in UEV-TSG101, indicating that they may compete with ORF3-PSAP or gag-PTAP for binding to UEV-TSG101. Two independent assays confirmed the ability of a cyclic peptide (CP11) to inhibit the ORF3-TSG101 interaction. CP11 treatment also reduced the release of both genotype 1 and genotype 3 HEV by approximately 90%, with a 50% inhibitory concentration (IC50) of 2 µM. Thus, CP11 appears to be an attractive candidate for further validation of its anti-HEV properties.IMPORTANCE There is no specific therapy against hepatitis E virus (HEV)-induced hepatic and nonhepatic health problems. Prevention of the release of the progeny viruses from infected cells is an attractive strategy to limit the spread of the virus. Interactions between the viral open reading frame 3 and the host tumor susceptibility gene 101 proteins have been shown to be essential for the release of genotype 3 HEV from infected cells. In this study, we have identified a cyclic peptide inhibitor of the above-mentioned interaction and demonstrate the efficiency of the inhibitor in preventing virus release from infected cells. Thus, our findings uncover the possibility of developing a specific antiviral agent against HEV by blocking its release from infected cells.
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Antivirales/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Virus de la Hepatitis E/efectos de los fármacos , Virus de la Hepatitis E/fisiología , Péptidos Cíclicos/metabolismo , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismo , Liberación del Virus/efectos de los fármacos , Línea Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Hepatocitos/virología , Humanos , Concentración 50 Inhibidora , Unión Proteica/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidoresRESUMEN
Mitophagy is a cellular quality-control pathway, which is essential for elimination of unhealthy mitochondria. While mitophagy is critical to pancreatic ß-cell function, the posttranslational signals governing ß-cell mitochondrial turnover are unknown. Here, we report that ubiquitination is essential for the assembly of a mitophagy regulatory complex, comprised of the E3 ligase Nrdp1, the deubiquitinase enzyme USP8, and Clec16a, a mediator of ß-cell mitophagy with unclear function. We discover that the diabetes gene Clec16a encodes an E3 ligase, which promotes nondegradative ubiquitin conjugates to direct its mitophagy effectors and stabilize the Clec16a-Nrdp1-USP8 complex. Inhibition of the Clec16a pathway by the chemotherapeutic lenalidomide, a selective ubiquitin ligase inhibitor associated with new-onset diabetes, impairs ß-cell mitophagy, oxygen consumption, and insulin secretion. Indeed, patients treated with lenalidomide develop compromised ß-cell function. Moreover, the ß-cell Clec16a-Nrdp1-USP8 mitophagy complex is destabilized and dysfunctional after lenalidomide treatment as well as after glucolipotoxic stress. Thus, the Clec16a-Nrdp1-USP8 complex relies on ubiquitin signals to promote mitophagy and maintain mitochondrial quality control necessary for optimal ß-cell function.
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Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células Secretoras de Insulina/metabolismo , Lectinas Tipo C/metabolismo , Mitofagia , Proteínas de Transporte de Monosacáridos/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Línea Celular , Células Cultivadas , Cruzamientos Genéticos , Endopeptidasas/química , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Inhibidores Enzimáticos/farmacología , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Lectinas Tipo C/antagonistas & inhibidores , Lectinas Tipo C/química , Lectinas Tipo C/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitofagia/efectos de los fármacos , Proteínas de Transporte de Monosacáridos/antagonistas & inhibidores , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/genética , Multimerización de Proteína/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Bancos de Tejidos , Técnicas de Cultivo de Tejidos , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/genética , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/efectos de los fármacosRESUMEN
Lung cancer surfaces to be the predominant determinant of mortality worldwide constituting 13% and 19% of all new cancer cases and deaths related to cancer respectively. Molecular profiling has now become a regular trend in lung cancer to identify the driver mutations. Epidermal Growth Factor Receptor (EGFR) is the most regular driver mutation encountered in Non-Small Cell Lung Cancer (NSCLC). Targeted therapies are now available for the treatment of EGFR mutant NSCLC. EGFR mutation is more frequently expressed in adenocarcinoma than squamous cell carcinoma. This article presents a detailed molecular insight of the therapeutic approaches for the treatment of EGFR mutant lung cancer. The article delineates molecular mechanism of the drugs that are approved, the drugs that are in clinical trial and the drugs that have not entered a clinical trial but shows promising future in the treatment of EGFR mutant lung cancer. Furthermore, this article provides concise information on relevant combinational or monotherapy clinical trials that have been completed for various approaches.
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Antineoplásicos Inmunológicos/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Antineoplásicos/metabolismo , Antineoplásicos Inmunológicos/metabolismo , Resistencia a Antineoplásicos , Endopeptidasas , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/metabolismo , Humanos , Terapia Molecular Dirigida , Mutación , Inhibidores de Proteínas Quinasas/metabolismo , Ubiquitina Tiolesterasa/antagonistas & inhibidoresRESUMEN
AAA ATPases have pivotal functions in diverse cellular processes essential for survival and proliferation. Revealing strategies for chemical inhibition of this class of enzymes is therefore of great interest for the development of novel chemotherapies or chemical tools. Here, we characterize the compound MSC1094308 as a reversible, allosteric inhibitor of the type II AAA ATPase human ubiquitin-directed unfoldase (VCP)/p97 and the type I AAA ATPase VPS4B. Subsequent proteomic, genetic and biochemical studies indicate that MSC1094308 binds to a previously characterized drugable hotspot of p97, thereby inhibiting the D2 ATPase activity. Our results furthermore indicate that a similar allosteric site exists in VPS4B, suggesting conserved allosteric circuits and drugable sites in both type I and II AAA ATPases. Our results may thus guide future chemical tool and drug discovery efforts for the biomedically relevant AAA ATPases.