RESUMEN
Ubiquitination and deubiquitination processes are widely involved in modulating the function, activity, localization, and stability of multiple cellular proteins regulating almost every aspect of cellular function. Several virus families have been shown to exploit the cellular ubiquitin-conjugating system to achieve a productive infection: enter the cell, promote genome replication, or assemble and release viral progeny. In this study, we analyzed the role of deubiquitinating enzymes (DUBs) during chikungunya virus (CHIKV) infection. HEK293T, Vero-E6, and Huh-7 cells were treated with two DUB inhibitors (PR619 or WP1130). Then, infected cells were evaluated by flow cytometry, and viral progeny was quantified using the plaque assay method. The changes in viral proteins and viral RNA were analyzed using Western blotting and RT-qPCR, respectively. Results indicate that treatment with DUB inhibitors impairs CHIKV replication due to significant protein and viral RNA synthesis deregulation. Therefore, DUB activity may be a pharmacological target for blocking CHIKV infection.
Asunto(s)
Fiebre Chikungunya , Virus Chikungunya , Enzimas Desubicuitinizantes , Inhibidores Enzimáticos , Replicación Viral , Humanos , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/efectos de los fármacos , Enzimas Desubicuitinizantes/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Células HEK293 , ARN Viral , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Deubiquitination is a posttranslational protein modification prevalent in mammalian cells. Deubiquitinases regulate the functions of the target protein by removing its ubiquitin chain. In this study, the effects of the deubiquitinase USP38's functions on the LSD1 protein and on cell physiology were investigated. MATERIALS AND METHODS: Western blotting, real-time quantitative PCR, immunoprecipitation, denaturing immunoprecipitation and luciferase reporter assays were used to analyze the protein stability, protein interactions and changes in the ubiquitin chain. Cell proliferation assays, colony formation assays, drug treatments and western blotting were used to explore the functions of USP38 in cells. RESULTS: The deubiquitinase USP38 stabilizes protein LSD1 in cells by binding LSD1 and cleaving its ubiquitin chain to prevent the degradation of LSD1 by the intracellular proteasome. USP38 enhances the ability of LSD1 to activate signaling pathways and hence promotes cellular abilities of proliferation and colony formation through interacting with LSD1. Furthermore, USP38 enhances the drug tolerance of human colon cancer cells. CONCLUSIONS: USP38 is an LSD1-specific deubiquitinase that affects cellular physiology through interacting with LSD1.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Histona Demetilasas/farmacología , Proteasas Ubiquitina-Específicas/farmacología , Western Blotting , Ensayo de Unidades Formadoras de Colonias , Humanos , Inmunoprecipitación , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
BACKGROUND: Deubiquitination is a posttranslational protein modification prevalent in mammalian cells. Deubiquitinases regulate the functions of the target protein by removing its ubiquitin chain. In this study, the effects of the deubiquitinase USP38's functions on the LSD1 protein and on cell physiology were investigated. MATERIALS AND METHODS: Western blotting, real-time quantitative PCR, immunoprecipitation, denaturing immunoprecipitation and luciferase reporter assays were used to analyze the protein stability, protein interactions and changes in the ubiquitin chain. Cell proliferation assays, colony formation assays, drug treatments and western blotting were used to explore the functions of USP38 in cells. RESULTS: The deubiquitinase USP38 stabilizes protein LSD1 in cells by binding LSD1 and cleaving its ubiquitin chain to prevent the degradation of LSD1 by the intracellular proteasome. USP38 enhances the ability of LSD1 to activate signaling pathways and hence promotes cellular abilities of proliferation and colony formation through interacting with LSD1. Furthermore, USP38 enhances the drug tolerance of human colon cancer cells. CONCLUSIONS: USP38 is an LSD1-specific deubiquitinase that affects cellular physiology through interacting with LSD1.