Proteomic and Biochemical Studies of Bax Regulatory Proteins in Apoptosis

描述（由申请人提供）：凋亡（或程序性细胞死亡）在多细胞生物的正常发育和组织稳态中起着关键作用。受损的细胞凋亡在各种人类疾病的发展中都是至关重要的，也是有效治疗的主要障碍。Bcl-2蛋白家族在细胞凋亡途径中是一个重要的检查点。Bax是Bcl-2家族的促凋亡成员，控制线粒体介导的细胞死亡途径。在正常的健康细胞中，Bax位于细胞质中并维持在非活性状态。在凋亡刺激下，Bax发生构象改变，并从细胞质迁移到线粒体。通过将其c端跨膜结构域插入线粒体外膜，Bax破坏线粒体膜电位，诱导促凋亡蛋白（如细胞色素c）从线粒体释放到细胞质溶胶中。释放的促凋亡蛋白触发细胞凋亡。是什么因素使Bax在健康细胞的细胞质中保持无活性构象尚不完全清楚。我们假设可能存在与Bax相互作用并在健康细胞的细胞质中控制Bax的新型细胞死亡调节蛋白。我们的初步研究表明，除了大部分Bax单体外，一小部分Bax与健康细胞细胞质中的高分子量蛋白复合物有关。我们建议使用新的蛋白质组学方法系统地鉴定健康细胞细胞质中与Bax相关的蛋白质（Specific Aim 1）。在验证Bax与鉴定蛋白之间的相互作用后，将通过RNAi或过表达操纵鉴定蛋白在细胞中的表达，并在细胞中检测其对Bax构象变化、Bax向线粒体易位和细胞凋亡的影响。鉴定新的Bax相互作用的死亡调节蛋白对于理解Bax在细胞凋亡中活化的分子机制至关重要。鉴于Bax在人类疾病的发展和治疗中的重要作用，该结果可能为设计预防和治疗包括癌症和神经退行性疾病在内的人类疾病的新策略提供有价值的信息。公共卫生相关性：本项目的目的是使用新的定量蛋白质组学方法系统地鉴定Bax相互作用蛋白，这些蛋白可能在保持健康细胞细胞质中Bax的检测中发挥关键作用。鉴定新的Bax相互作用的死亡调节蛋白对于理解Bax在细胞凋亡中活化的分子机制至关重要。分子和细胞生物学技术将用于评估鉴定的蛋白质的生物学功能。

DESCRIPTION (provided by applicant): Apoptosis (or programmed cell death) plays critical roles in normal development and tissue homeostasis in multi-cellular organisms. Impaired apoptosis is both critical in the development of a variety of human diseases and a major barrier to effective treatment. The Bcl-2 family of proteins constitutes a crucial checkpoint in the apoptosis pathway. Bax is a pro-apoptotic member of the Bcl-2 family that controls the mitochondrion-mediated cell death pathway. In normal healthy cells, Bax locates in the cytosol and is maintained in an inactive state. Upon apoptotic stimulation, Bax undergoes conformational changes, and migrates from the cytosol to mitochondria. With insertion of its C-terminal transmembrane domains into the outer membrane of mitochondria, Bax disrupts mitochondrial membrane potential, and induces release of pro-apoptotic proteins (such as cytochrome c) from mitochondria to cytosol. The released pro-apoptotic proteins trigger cellular apoptosis. What factors maintain Bax in its inactive conformation in the cytosol of healthy cells is not fully understood. We hypothesize that novel cell death regulatory proteins that interact with Bax and keep Bax in check in the cytosol of healthy cells may exist. Our preliminary studies showed that in addition to the majority of monomeric Bax, a small portion of Bax was associated with high molecular weight protein complexes in the cytosol of healthy cells. We propose to use novel proteomic method to systematically identify the proteins that associate with Bax in the cytosol of healthy cells (Specific Aim 1). After validation of the interaction between Bax and the identified proteins, the expression of the identified proteins in cells will be manipulated by RNAi or overexpression, and their effects on Bax conformational changes, Bax translocation to mitochondria, and apoptosis will be examined in cells. The identifications of novel Bax interacting, death regulatory proteins are critical for understanding the molecular mechanisms of Bax activation in apoptosis. Given the important roles of Bax in the development and treatment of human diseases, the results may provide valuable information for designing new strategies to prevent and treat human diseases including cancer an neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: The objective of this project is to use novel quantitative proteomic method to systematically identify Bax interacting proteins that may play critical roles in keeping Bax in check in the cytosol of healthy cells. The identifications of novel Bax interacting, death regulatory proteins are critical for understanding the molecular mechanisms of Bax activation in apoptosis. Molecular and cell biological techniques will be used to assess the biological functions of the identified proteins.