RESUMEN
Coordination of DNA replication and cellular redox homeostasis mechanisms is essential for the sustained genome stability due to the sensitivity of replicating DNA to oxidation. However, substantial gaps remain in our knowledge of underlying molecular pathways. In this study, we characterise the interaction of Keap1, a central antioxidant response regulator in Metazoa, with the replicative helicase subunit protein MCM3. Our analysis suggests that structural determinants of the interaction of Keap1 with its critical downstream target - Nrf2 master transactivator of oxidative stress response genes - may have evolved in evolution to mimic the conserved helix-2-insert motif of MCM3. We show that this has led to a competition between MCM3 and Nrf2 proteins for Keap1 binding, and likely recruited MCM3 for the competitive binding dependent modulation of Keap1 controlled Nrf2 activities. We hypothesise that such mechanism could help to adjust the Keap1-Nrf2 antioxidant response pathway according to the proliferative and replicative status of the cell, with possible reciprocal implications also for the regulation of cellular functions of MCM3. Altogether this suggests about important role of Keap1-MCM3 interaction in the cross-talk between replisome and redox homeostasis machineries in metazoan cells.
Asunto(s)
Replicación del ADN , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Componente 3 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Estrés Oxidativo/fisiología , Secuencias de Aminoácidos , Animales , Células CHO , Línea Celular Tumoral , Cricetulus , Evolución Molecular , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/química , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/aislamiento & purificación , Queratinocitos , Componente 3 del Complejo de Mantenimiento de Minicromosoma/química , Componente 3 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 3 del Complejo de Mantenimiento de Minicromosoma/aislamiento & purificación , Factor 2 Relacionado con NF-E2/metabolismo , Cultivo Primario de Células , Unión Proteica/fisiología , Conformación Proteica en Hélice alfa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Células Sf9 , Spodoptera , Transactivadores/metabolismoRESUMEN
Induced protein degradation by PROTACs has emerged as a promising strategy to target nonenzymatic proteins inside the cell. The aim of this study was to identify Keap1, a substrate adaptor protein for ubiquitin E3 ligase involved in oxidative stress regulation, as a novel candidate for PROTACs that can be applied in the degradation of the nonenzymatic protein Tau. A peptide PROTAC by recruiting Keap1-Cul3 ubiquitin E3 ligase was developed and applied in the degradation of intracellular Tau. Peptide 1 showed strong in vitro binding with Keap1 and Tau. With proper cell permeability, peptide 1 was found to colocalize with cellular Keap1 and resulted in the coimmunoprecipitation of Tau and Keap1. The results of flow cytometry and western blotting assays showed that peptide 1 can downregulate the intracellular Tau level in both time- and concentration-dependent manner. The application of Keap1 siRNA silencing and the proteasome inhibitor MG132 confirmed that peptide 1 could promote the Keap1-dependent poly-ubiquitination and proteasome-dependent degradation of Tau. The results suggested that using PROTACs to recruit Keap1 to induce the degradation of Tau may show promising character in the treatment of neurodegenerative disease. Besides, our research demonstrated that Keap1 should be a promising E3 ligase adaptor to be used in the design of novel PROTACs.
