Regulation of apoptosis by Bcl-XL, Bak and Bax

描述(申请人提供)：癌症的发展涉及基因变化，导致组织发育和体内平衡维持的控制发生变化，这是由细胞自杀程序--细胞凋亡--严格控制的。许多肿瘤细胞对内源性和外源性凋亡刺激具有抵抗力，这可能是由于这些细胞中的凋亡通路受损所致。BCL-2家族蛋白是细胞对细胞凋亡信号反应的主要调节因子。其中，抗凋亡的BclXland、促凋亡的Bak和Bax在细胞凋亡过程中起着至关重要的作用。初步研究表明，除了线粒体的功能外，Bcl2蛋白还定位于内质网(ER)，并调节一条平行的凋亡途径。本方案中的实验旨在阐明Bcl-2蛋白调控细胞凋亡的详细分子机制。1)研究内质网膜上Bclxl、Bak和Bax的电生理特性及其在诱导细胞凋亡过程中膜通透性的作用。一种新的记录单个离子通道活性的膜片钳方法将被用来表征ER膜上的Bcl2蛋白。对纯化的重组和重组表达的Bclxl、Bak和Bax蛋白在ER膜上的电生理特性进行了研究。Bak和Bax在细胞凋亡过程中在膜上形成脂质孔的假说也将得到验证；2)研究不同细胞器上的Bcl2家族蛋白介导的不同的凋亡途径。将努力利用RNAi方法更好地表征定位于内质网或线粒体上的Bak或Bclxl介导的caspase通路。通过选择性地将促凋亡和抗凋亡的Bcl2蛋白定位于特定的细胞器，我们将研究这些蛋白在细胞内定位对调节细胞凋亡的重要性。将研究钙离子在不同的凋亡途径中的可能作用；以及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。将进行基于寡核苷酸的微阵列分析，以检查响应Bak或Bax表达的基因表达模式的变化。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.