BAX/BAK-Mediated Mitochondrial Outer Membrane Permeabilization in Apoptosis

描述（由申请人提供）：细胞凋亡在许多疾病如中风、神经退行性疾病、自身免疫性疾病和癌症中起关键作用。在经历细胞凋亡的细胞中，多结构域促凋亡BCL-2家族蛋白如BAX或巴克被激活以通过形成大孔来损伤线粒体膜。这导致蛋白质因子从线粒体膜间隙释放到细胞质中，以放大细胞质中细胞凋亡的级联事件。本研究的目的是了解BAX或巴克在线粒体中形成孔的机制。假设是在凋亡细胞中，促凋亡BCL-2蛋白BAX和巴克形成结构域交换的二聚体，并且这些结构域交换的二聚体通过其BH 3结构域的相互交换进一步寡聚化，导致线粒体外膜的透化。本研究的具体目的是：1）研究BH 3形成的界面（BCL-2同源结构域3）通过电子顺磁共振（EPR）光谱的定点自旋标记（SDSL）方法检测寡聚巴克孔中的结构域，2）通过EPR的SDSL方法检测巴克寡聚孔中结构域交换的BAK二聚体的存在，和3）开发一种方法，使用定向膜和通过SDSL方法的偶极线增宽的角度依赖性来确定通道形成结构域中螺旋（螺旋5- 6）相对于膜法线的取向。对于前两个目标，建议存在于蛋白质-蛋白质接触界面中的残基之间的距离将通过连续波（CW）方法或通过双电子共振（DEER）方法测量。此外，与二聚和低聚过程相关的巴克的构象变化将通过使用功率饱和法的溶剂可及性测量来确定。膜中自旋标记残基的深度也将通过相同的方法测量。对于第三个目标，将在定向膜中制备双自旋标记的<$-螺旋。通过记录磁场和膜法线之间不同角度下的EPR谱，测量EPR信号对取向样品相对于磁场的相对取向的依赖性。将开发一种理论方法来解释EPR信号的角度依赖性。这种方法将提供一种新的手段，以确定其他膜插入螺旋（包括BAX或巴克的那些）相对于膜法线的取向。本研究的结果将提供详细的了解线粒体透化的分子机制巴克和BAX。这反过来将提供有价值的见解，在设计治疗手段，以控制细胞死亡过程中，无论是通过增强癌症细胞凋亡或通过抑制它在中风或神经退行性疾病。

DESCRIPTION (provided by applicant): Apoptosis plays critical roles in many diseases such as stroke, neurodegenerative diseases, autoimmune diseases and cancer. In the cells undergoing apoptosis, the multi-domain proapoptotic BCL-2 family proteins such as BAX or BAK are activated to damage the mitochondrial membrane by forming large pores. This results in the release of protein factors into the cytoplasm from the mitochondrial intermembrane space to amplify the cascade events of apoptosis in the cytoplasm. The goal of this research is to understand the mechanism of the pore formation by BAX or BAK in the mitochondria. The hypothesis is that in apoptotic cells the proapoptotic BCL-2 proteins BAX and BAK form domain-swapped dimers and these domain-swapped dimers further oligomerize via reciprocal exchange of their BH3 domains, leading to the permeabilization of the mitochondrial outer membrane. The specific aims of this research are 1) to investigate the interface formed by the BH3 (BCL-2 homology domain 3) domains in the oligomeric BAK pore by the site-directed spin labeling (SDSL) method of electron paramagnetic resonance (EPR) spectroscopy, 2) to test the existence of the domain-swapped dimer of BAK in the BAK oligomeric pore by the SDSL method of EPR, and 3) to develop a method to determine the orientation of the helices in the channel-forming domain (helices ¿5-¿6) relative to the membrane normal using an oriented membrane and the angular dependence of the dipolar line broadening by the SDSL methodology. For the first two aims, the distance between the residues proposed to exist in the protein-protein contact interfaces will be measured by the continuous wave (CW) method or by the double electron resonance (DEER) method. Additionally, the conformational changes in BAK associated with the dimerization and oligomerization processes will be determined by the solvent accessibility measurements using the power saturation method. The depths of the spin-labeled residues in the membrane will also be measured by the same method. For the third aim, a doubly spin labeled ¿-helix will be prepared in oriented membranes. The dependence of the EPR signal on the relative orientation of the aligned sample to the magnetic field will be measured by recording the EPR spectra at various angles between the magnetic field and the membrane normal. A theoretical method will be developed to explain the angular dependence of the EPR signal. This approach will provide a novel means to determine the orientation of other membrane- inserted helices (including those of BAX or BAK) relative to the membrane normal. The outcome of this research will provide detailed understanding of the molecular mechanism of mitochondrial permeabilization by BAK and BAX. This in turn will provide valuable insights in designing therapeutic means to control the cell death processes either by enhancing apoptosis in cancer or by suppressing it in strokes or neurodegenerative diseases.