Analysis of Proteins that Modulate Apoptosis

描述(申请人提供)：细胞凋亡是在后生动物中发生的大多数程序性细胞死亡的形态定义和进化保守的过程。细胞凋亡对于后生动物的发育和正常生理是必不可少的。严格控制的细胞凋亡程序的放松导致了人类疾病的发生，如癌症、神经退行性疾病和自身免疫性疾病、中风和心脏骤停损伤。Bcl2蛋白是一种强大的细胞凋亡抑制因子，尽管进行了大量的研究，但关于其作用和调控的分子机制仍存在许多问题。为了进一步了解Bcl2的功能和调控，对Bcl2结合蛋白进行了搜索，最终确定BMRP是一种新的Bcl2相互作用蛋白。目前的研究表明BMRP是细胞凋亡机制中不可或缺的一部分。 我们的长期目标是加深我们对介导Bcl2抗细胞凋亡活性的分子机制及其调控的了解。我们的假设是BMRP是一种促凋亡蛋白，其刺激细胞凋亡的能力是通过与Bcl-2结合来调节的。深入了解BMRP和Bcl2的功能和调控机制将有助于我们对细胞凋亡途径的认识，这将有助于设计新的药物来预防和/或治疗以细胞凋亡异常为特征的人类疾病。为了检验我们的中心假设，本文提出了两个具体目标。首先，将进行缺失和丙氨酸扫描突变，以确定BMRP促凋亡活性所必需的结构域和氨基酸残基。这些研究将利用聚合酶链式反应定点突变，随后进行细胞凋亡检测。此外，这些突变体与Bcl-2结合的能力将通过酵母双杂交试验进行测试。其次，BMRP在细胞凋亡过程中的去向及其在凋亡信号通路中的作用将通过亚细胞分裂来分析，以及在没有或存在其他过度表达的凋亡调控因子的情况下，BMRP的过度表达或下调对细胞的影响。这项拟议的研究是创新的，因为它将建立BMRP功能和调控的未知方面，以及BMRP与Bcl-2抗凋亡活性的关系。这一知识与公共卫生相关，因为它应该为癌症等其发病机制涉及非调控细胞凋亡的疾病的治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): Apoptosis is the morphologically defined and evolutionary conserved process by which most programmed cell deaths occur in metazoans. Cell death by apoptosis is essential to the development and normal physiology of metazoans. Deregulation of the tightly controlled apoptotic program contributes to the pathogenesis of human diseases such as cancer, neurodegenerative and autoimmune diseases, stroke, and cardiac arrest damage. The Bcl-2 protein is a powerful repressor of apoptotic cell death and, despite intense study, many questions remain regarding its molecular mechanisms of action and regulation. To gain further insight into the function and modulation of Bcl-2, a search of Bcl-2 binding proteins was performed, which resulted in the identification of BMRP as a novel Bcl-2 interacting protein. Current knowledge suggests that BMRP is an integral part of the apoptotic machinery. Our long-term goal is to enhance our understanding of the molecular mechanisms that mediate Bcl-2 anti-apoptotic activity and its regulation. Our hypothesis is that BMRP is a pro-apoptotic protein whose ability to stimulate apoptosis is regulated by binding with Bcl-2. An increased understanding of the mechanistic basis of BMRP and Bcl-2 function and modulation will enhance our knowledge of apoptotic pathways, which will facilitate the design of novel drugs to prevent and/or treat human diseases characterized by abnormal levels of apoptosis. Two specific aims are proposed to test our central hypothesis. First, deletion and alanine scanning mutagenesis will be carried out to identify domains and amino acid residues that are essential to the pro-apoptotic activity of BMRP. These studies will utilize PCR site-directed mutagenesis, followed by apoptosis assays. Additionally, the ability of these mutants to bind to Bcl-2 will be tested by yeast two-hybrid assays. Second, the fate of BMRP during apoptosis and the role that it plays in apoptosis signaling pathways will be analyzed by subcellular fractionation, and studies of the cellular effects of over-expression or down-regulation of BMRP in the absence and presence of other over-expressed apoptosis regulators. The proposed research is innovative, as it will establish unknown aspects of BMRP function and regulation, and BMRP's relationship to the anti-apoptotic activity of Bcl-2. This knowledge is relevant to public health, since it should provide novel targets for therapeutic intervention for diseases whose pathogenesis involves deregulated apoptosis, such as cancer.