Asunto(s)
Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Quinonas/aislamiento & purificación , Quinonas/farmacología , Sumoilación/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Concentración 50 Inhibidora , Estructura MolecularAsunto(s)
Antineoplásicos Fitogénicos/farmacología , Cornaceae/química , Descubrimiento de Drogas , Neoplasias/tratamiento farmacológico , Hojas de la Planta/química , Sumoilación/efectos de los fármacos , Taninos/farmacología , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Taninos Hidrolizables/química , Taninos Hidrolizables/aislamiento & purificación , Taninos Hidrolizables/metabolismo , Taninos Hidrolizables/farmacología , Japón , Estructura Molecular , Concentración Osmolar , Extractos Vegetales/química , Extractos Vegetales/farmacología , Taninos/química , Taninos/aislamiento & purificación , Taninos/metabolismoRESUMEN
We found that the small ubiquitin-related modifier (SUMO) conjugation reaction can be visualized simply by incubating green fluorescent protein (GFP)-SUMO-1 with permeabilized cells in the presence of ATP for 15 min. Neither special equipment for protein purification nor highly developed skills for recombinant technologies is required, making the assay potentially applicable to large-scale drug-screening strategies for the identification of drugs that can inhibit or enhance SUMOylation.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteína SUMO-1/metabolismo , Células HeLa , HumanosRESUMEN
Protein modification by small ubiquitin-related modifier proteins (SUMOs) controls diverse cellular functions. Dysregulation of SUMOylation or deSUMOylation processes has been implicated in the development of cancer and neurodegenerative diseases. However, no small-molecule inhibiting protein SUMOylation has been reported so far. Here, we report inhibition of SUMOylation by ginkgolic acid and its analog, anacardic acid. Ginkgolic acid and anacardic acid inhibit protein SUMOylation both in vitro and in vivo without affecting in vivo ubiquitination. Binding assays with a fluorescently labeled probe showed that ginkgolic acid directly binds E1 and inhibits the formation of the E1-SUMO intermediate. These studies will provide not only a useful tool for investigating the roles of SUMO conjugations in a variety of pathways in cells, but also a basis for the development of drugs targeted against diseases involving aberrant SUMOylation.
Asunto(s)
Ácidos Anacárdicos/metabolismo , Ginkgo biloba/metabolismo , Salicilatos/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/antagonistas & inhibidores , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ácidos Anacárdicos/química , Ginkgo biloba/química , Extractos Vegetales/química , Extractos Vegetales/metabolismo , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Unión Proteica , Salicilatos/química , Bibliotecas de Moléculas Pequeñas , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Relación Estructura-Actividad , UbiquitinaciónRESUMEN
Serum stimulation leads to activation of the serum response factor (SRF)-mediated transcription of immediate-early genes such as c-fos via various signal transduction pathways. We have previously reported that promyelocytic leukemia protein (PML) is involved in the transcriptional regulation by SRF. PML is one of the well-known substrates for modification by small ubiquitin-related modifier-1 (SUMO-1) and several SUMO-1-modified proteins associate with PML. Here, we report that SRF is modified by SUMO-1 chiefly at lysine(147) within the DNA-binding domain. Substitution of this target lysine for alanine did not affect the translocation of SRF to PML-nuclear bodies. The SRF mutant augmented the transcriptional activity under Rho A-stimulated condition but not under serum-starved condition, suggesting that activated SRF is suppressed by its sumoylation. These data support the transcriptional role of SUMO-1 conjugating system in cellular serum response.