Characterizaton of Mcl-1 in Regulating Different Forms of Cell Death

Abstract

Programmed Cell Deaths (apoptosis, autophagic cell death and necrosis) play essential roles in animal development and certain diseases. Autophagy is a cellular process that provides nutrients to starved cells by digesting cellular constituents. Defects in autophagy have been implicated in cancer and neurodegeneration, but how autophagy causes cell death is not understood. In mammals, there are two apoptotic pathways: the extrinsic one through death receptors in the cell membrane and the intrinsic one through mitochondria that release death proteins. The Bcl-2 family of proteins function upstream in the intrinsic pathway. They can be subdivided into anti-apoptotic and proapoptotic proteins. Mcl-1 has two different splice variants, Mcl-1L and Mcl-1S. Mcl-1L is an anti-apoptotic Bcl-2 family protein with a short half-life, which functions in the apical step of the intrinsic apoptotic pathway and can inhibit the release of death proteins from mitochondria. After genotoxic treatment, Mcl-1L is rapidly degraded, resulting in mitochondria damage and apoptosis. Prevention of Mcl-1L degradation with proteasome inhibitors blocks apoptosis. An Mcl-1L ubiquitin ligase was identified using biochemical purification method and named Mule, which has five recognizable domains including UBA, WWE, HECT and two ARM repeats like domains. Mule also contains a region similar to the Bcl-2 homology region 3 (BH3) that allows it to specifically interact with Mcl-1L. Depletion of Mule by RNA interference stabilizes Mcl-1L, resulting in an attenuation of apoptosis induced by DNA-damaging agents. To further understand Mcl-1 function, I generated inducible Mcl-1 knockdown stable U2OS cell lines. Depletion of Mcl-1L but not Mcl-1S causes the cells to commit autophagic cell death. The autophagic phenotype can be blocked by knocking down Beclin 1, which is a known upstream component of the autophagic pathway. Mcl-1L knockdown induced cell death is accompanied by and requires the upregulation of p53 and p21. Loss-of-function experiments confirmed the involvement of Mcl-1S in inducing autophagy. Taken together, my data showed that Mcl-1 genes function in the apical step of both apoptosis and autophagic cell death pathways.