BAX/BAK-Mediated Mitochondrial Outer Membrane Permeabilization in Apoptosis

描述(申请人提供)：细胞凋亡在许多疾病中起着关键作用，如中风、神经退行性疾病、自身免疫性疾病和癌症。在发生凋亡的细胞中，多结构域促凋亡的bcl2家族蛋白如Bax或BAK被激活，通过形成大的孔洞来破坏线粒体膜。这导致蛋白质因子从线粒体膜间间隙释放到细胞质中，放大细胞质中的凋亡级联事件。本研究的目的是为了了解Bax或BAK在线粒体中形成孔洞的机制。假设在凋亡细胞中，促凋亡的bcl2蛋白Bax和BAK形成结构域交换的二聚体，这些结构域交换的二聚体通过它们的BH3结构域的相互交换进一步寡聚，导致线粒体外膜的通透性。本研究的具体目的是：1)用电子顺磁共振(EPR)的定点自旋标记(SDSL)方法研究BAK低聚孔中BH3(BCL-2同源结构域3)结构域形成的界面，2)用EPR的SDSL方法检测BAK低聚孔中BAK结构域交换二聚体的存在，3)建立一种方法来确定通道形成区域(螺旋5-6)中螺旋相对于膜正常的取向以及通过SDSL方法确定偶极线展宽的角度依赖性。对于前两个目标，建议存在于蛋白质-蛋白质接触界面的残基之间的距离将通过连续波(CW)方法或双电子共振(DER)方法来测量。此外，与二聚和齐聚过程相关的BAK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。自旋标记的残基在膜中的深度也将用同样的方法测量。对于第三个目的，将在取向膜中制备双自旋标记的β-螺旋。通过记录磁场和膜法向之间不同角度的EPR谱，可以测量EPR信号与取向样品相对于磁场的相对取向的依赖关系。将发展一种理论方法来解释EPR信号的角度相关性。这种方法将提供一种新的方法来确定其他膜插入螺旋(包括BAX或BAK的螺旋)相对于膜法线的取向。这项研究的结果将为BAK和Bax对线粒体通透性的分子机制提供详细的了解。这反过来将为设计控制细胞死亡过程的治疗方法提供有价值的见解，无论是通过增强癌症的细胞凋亡，还是通过抑制中风或神经退行性疾病的细胞死亡过程。

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.