Mechanism of Pore Formation BC12 Family Proteins

描述(由申请人提供)：程序性细胞死亡对多细胞生物体的生长、分化和动态平衡至关重要。程序性细胞死亡的形态表达，即细胞凋亡，具有明显依赖于bcl2家族蛋白的遗传学基础。这一蛋白质家族决定了细胞程序性死亡的早期共同决定点。对该家族促凋亡和抗凋亡成员的结构研究表明，它们与白喉和结肠素毒素有关。这些蛋白质插入细胞膜，形成离子通道，并参与蛋白质的转运。我们和其他人已经表征了bcl2家族成员的通道形成活动。关于这些蛋白质的研究集中在它们在增加线粒体通透性和释放细胞色素c方面发挥重要作用的可能性。在这一提议的前期研究中，我们证明了Bax在人工脂质体中形成了巨大的充满水的孔。这些孔经历了浓度依赖的二聚体到四聚体的转变，增加了它们的大小，足以激活细胞色素c在脂双层上的转移。这种转变发生在纳米分子Bax浓度、生理盐分和生理pH而不添加其他蛋白质的情况下，在这个建议中，我们计划研究和表征大孔Bax和Bid孔形成和激活的分子基础，包括动力学、化学计量以及与抗和促凋亡家族成员相互作用的影响。我们还将确定线粒体膜环境对孔激活的影响。

DESCRIPTION (provided by applicant): Programmed cell death is critical in growth, differentiation and homeostasis of multicellular organisms. The morphologic expression of programmed cell death, apoptosis, has a genetic basis that is clearly dependent on the BCL-2-family proteins. This family of proteins determines an early common decision point in programmed cell death. Structural studies on pro- and anti-apoptotic members of this family indicate they are related to diphtheria and colicin toxins. These proteins insert into membranes, form ion channels and participate in protein translocation. We and others have characterized the channel forming activities of the BCL-2-family members. Studies concerning these proteins have focused on the possibility that they play an important role in increased mitochondrial permeability and in release of cytochrome c. In studies preliminary to this proposal we have demonstrated that BAX forms large water filled pores in artificial liposomes. These pores undergo a concentration dependent dimer to tetramer transition, increasing their size sufficiently to activate cytochrome c transfer across the lipid bilayer. This transition occurs at nanomolar BAX concentrations, in physiologic salt and at physiologic pH without the addition of other proteins, tn this proposal we plan to study and characterize the molecular basis for formation and activation of the large BAX and BID pores, including kinetics, stoichiometry, and the effect of interaction with anti-and pro-apoptotic family members. We will also determine the effect on pore activation of the mitochondrial membrane environment.