Mechanism of Pore Formation BC12 Family Proteins

描述（由申请人提供）：程序性细胞死亡在多细胞生物体的生长、分化和稳态中至关重要。程序性细胞死亡（凋亡）的形态学表达具有明显依赖于BCL-2家族蛋白的遗传基础。这个蛋白质家族决定了程序性细胞死亡的早期共同决定点。对该家族的促凋亡和抗凋亡成员的结构研究表明，它们与白喉和大肠杆菌素毒素有关。这些蛋白质插入细胞膜，形成离子通道并参与蛋白质转运。我们和其他人已经表征了BCL-2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中发挥重要作用的可能性上。在该提议的初步研究中，我们已经证明BAX在人工脂质体中形成大的水填充孔。这些孔经历浓度依赖性的二聚体到四聚体的转变，增加它们的尺寸足以激活细胞色素c穿过脂质双层的转移。这种转变发生在纳摩尔BAX浓度下，在生理盐中和生理pH下，而不添加其他蛋白质。在该提议中，我们计划研究和表征大BAX和BID孔的形成和活化的分子基础，包括动力学、化学计量以及与抗凋亡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活化的影响。

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.