RESUMEN
Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.
Asunto(s)
ADN Polimerasa I/química , ADN Polimerasa I/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Secuencia de Aminoácidos , Aminopeptidasas/metabolismo , Animales , Especificidad de Anticuerpos/inmunología , Ciclo Celular , Bases de Datos Genéticas , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Genoma , Humanos , Ratones , Modelos Biológicos , Células 3T3 NIH , Unión Proteica , Homología de Secuencia de Aminoácido , Serina Proteasas/metabolismo , Complejo Shelterina , Relación Estructura-Actividad , Fracciones Subcelulares/metabolismoRESUMEN
Approximately one third of patients with depression remain treatment resistant with existing antidepressants, suggesting that the currently-available antidepressants cannot induce appropriate responses in the brains of all patients. Long-term exposure to adrenocorticotrophic hormone (ACTH) has been proposed as a model that mimics at least some aspects of clinical treatment-resistant depression in rodents. The purpose of this study was to explore potential causes of antidepressant treatment resistance using the chronic ACTH-treated mouse model. We subjected ACTH-treated mice to a rodent model of electroconvulsive therapy, i.e., electroconvulsive seizure (ECS), which induces various molecular and cellular changes, including in gene expression and adult neurogenesis in the hippocampus. First, behavioral effect of repeated ECS in the forced swim test (FST) was examined. In our experimental setting, ACTH-treated mice showed resistance to the antidepressant-like effect of ECS in the FST. We then examined which cellular and molecular changes induced by ECS were attenuated by ACTH administration. Chronic ACTH treatment suppressed the increase of gene expression such as of Bdnf, Npy, and Drd1 induced by ECS in the hippocampus. In contrast, there was no difference in ECS-induced promotion of the early neurogenetic process in the hippocampus between ACTH-treated and control mice. Our results suggest the possibility that impaired neuromodulation and monoamine signaling in the hippocampus are among the factors contributing to antidepressant treatment resistance.