Regulation of apoptosis by Bcl-XL, Bak and Bax

描述（由申请人提供）：癌症的发展涉及基因变化导致组织发育和稳态维持控制的改变，这是由细胞自杀程序细胞凋亡严格调节的。许多肿瘤细胞抵抗内源性和外源性凋亡刺激，可能是由于这些细胞的凋亡通路受损。Bcl-2家族蛋白是细胞对凋亡信号反应的主要调节因子。其中抗凋亡的Bcl-XLand、促凋亡的Bak和Bax已被证明在细胞凋亡过程中发挥重要作用。初步研究表明，除了在线粒体中发挥作用外，Bcl-2蛋白还定位于内质网（ER），并调节细胞凋亡的平行途径。本实验旨在阐明Bcl-2蛋白调控细胞凋亡的详细分子机制。具体目的如下：1)研究Bcl-XL、Bak和Bax在内质网膜上的电生理特性及其在细胞凋亡过程中诱导细胞膜透性的作用。一种记录单离子通道活性的新型膜片钳方法将用于表征内质网膜上的Bcl-2蛋白。研究纯化的重组Bcl-XL、Bak和Bax蛋白在内质网膜上的电生理特性。在细胞凋亡过程中，Bak和Bax在细胞膜上形成脂质孔的假说也将被检验；2)研究定位于不同细胞器上的Bcl-2家族蛋白介导的不同凋亡途径。我们将利用RNAi方法更好地表征由Bak或Bcl-XL介导的内质网或线粒体上的caspase通路。通过选择性地将促凋亡和抗凋亡的Bcl-2蛋白靶向特定细胞器，研究细胞内定位对细胞凋亡的重要性。将研究Ca2+在不同凋亡途径中的调节作用；3)确定Bak和Bax的表达是否影响细胞生理，从而影响细胞对死亡信号的反应。寡核苷酸微阵列分析将用于检测Bak或Bax表达对基因表达模式的影响。可能参与细胞凋亡调控的基因表达有待进一步研究。

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.