Regulation of apoptosis by Bcl-XL, Bak and Bax

描述（由申请人提供）：癌症的发展涉及遗传变化，导致组织发育和稳态维持控制的改变，而组织发育和稳态维持受到细胞自杀程序（细胞凋亡）的严格调节。许多肿瘤细胞对内源性和外源性凋亡刺激具有抗性，可能是由于这些细胞中的凋亡途径受损。Bcl-2家族蛋白是细胞对凋亡信号应答的主要调节因子。其中，抗凋亡基因Bcl-X、促凋亡基因巴克和Bax在细胞凋亡过程中起重要作用。初步研究表明，Bcl-2蛋白除了在线粒体中发挥作用外，还定位于内质网（ER）上，并发挥调节细胞凋亡的平行途径的作用。本实验旨在阐明Bcl-2蛋白调控细胞凋亡的分子机制。1）研究细胞凋亡过程中Bcl-XL、巴克和Bax在内质网膜上的电生理特性及其在膜透化中的作用。一种新的膜片钳方法记录单离子通道活性将被用来表征ER膜上的Bcl-2蛋白。将研究在ER膜上纯化的重组以及再表达的Bcl-XL、巴克和Bax蛋白的电生理特性。研究细胞凋亡过程中巴克和Bax在细胞膜上形成介导孔的假说; 2）研究位于不同细胞器上的Bcl-2家族蛋白介导的不同凋亡途径。将努力使用RNAi方法更好地表征由位于ER或线粒体上的巴克或Bcl-XL介导的胱天蛋白酶途径。细胞内定位的促凋亡和抗凋亡Bcl-2蛋白的调节细胞凋亡的重要性将研究通过选择性地将这些蛋白质靶向特定的细胞器。将研究Ca 2+在不同凋亡途径调节中的可能作用;和3）确定巴克和Bax的表达是否影响细胞生理学，从而影响细胞对死亡信号的反应。将进行基于寡核苷酸的微阵列分析以检查响应于巴克或Bax表达的基因表达模式的变化。其表达可能参与细胞凋亡调控的基因将被进一步研究。

DESCRIPTION (provided by applicant): The development of cancer involves genetic changes leading to alterations in the control of tissue development and homeostatic maintenance, which is tightly regulated by the cellular suicide program, apoptosis. Many tumor cells are resistant to intrinsic and extrinsic apoptotic stimuli, probably due to impaired apoptotic pathways in these cells. Bcl-2 family proteins are major regulators of the cellular response to apoptotic signals. Among them, anti-apoptotic Bcl-XLand pro-apoptotic Bak and Bax have-been shown to play an essential role during apoptosis. Initial studies suggest that in addition to their functions at mitochondria, Bcl-2 proteins also localize on the endoplasmic reticulum (ER) and function to regulate a parallel pathway of apoptosis. Experiments in this proposal are designed to elucidate the detailed molecular mechanisms of Bcl-2 proteins regulating apoptosis. The following specific aims are proposed: 1) Investigate the electrophysiological properties of Bcl-XL, Bak and Bax on the ER membrane and their contribution to the induction of membrane permeabilization during apoptosis. A novel patch-clamp approach to record single ion channel activity will be used to characterize Bcl-2 proteins on the ER membrane. The electrophysiological properties of purified recombinant as well as reexpressed Bcl-XL, Bak and Bax proteins on the ER membrane will be studied. The hypothesis that Bak and Bax form lipidic pores on membranes during apoptosis will also be examined; 2) Study the distinct apoptotic pathways mediated by Bcl-2 family proteins localized on different organelles. Efforts will be made to better characterize caspase pathways mediated by Bak or Bcl-XL localized on the ER or mitochondria using a RNAi approach. The importance of intracellular localizations of pro- and anti-apoptotic Bcl-2 proteins for the regulation of apoptosis will be studied by selectively targeting these proteins to specific organelles. The possible role of Ca2+ in the regulation of distinct apoptotic pathways will be investigated; and 3) Determine whether the expression of Bak and Bax affects cellular physiology and thus cellular response to death signals. Oligonucleotide-based microarray analysis will be performed to examine the changes in gene expression patterns in response to Bak or Bax expression. Genes whose expression may be involved in apoptosis regulation will be further studied.