Dissecting the Execution Phase of BAK-Mediated Apoptosis in Cancer

点击翻译按钮获取中文摘要

PROJECT SUMMARY BCL-2 family proteins are critical regulators of apoptosis and deregulation of their protein interactions contributes to the development and chemoresistance of cancer. The cardinal executioners of cell death, BAX and BAK, respectively reside in the cytosol and mitochondria of the cell as monomers until activated by stress stimuli to self-associate and porate the mitochondrial outer membrane, leading to apoptotic cell death. To thwart chemotherapy-induced apoptosis and enforce cellular immortality, cancer usurps the cell survival arm of the BCL-2 pathway by overexpressing anti-apoptotic members, which can bind to BAX and BAK and prevent their transformation into toxic mitochondrial pores. The longstanding inability to generate stable and homogeneous oligomeric forms of full-length BAX and BAK has precluded the determination of their porating structures, which would inform both the execution phase of mitochondrial apoptosis and reveal novel surfaces for therapeutic activation of BAX and BAK in cancer. The Walensky laboratory recently reported a novel strategy for generating a BAX oligomer that was amenable to a battery of structure-function studies. In contrast to BAX, BAK constitutively residues at the mitochondria, is triggered by a distinct binding site, and exhibits differential expression patterns in mammalian tissues and in human cancers. Further, whereas full-length BAX has long been amenable to expression in recombinant monomeric form, production of BAK has been especially challenging. The Walensky lab developed a triple-mutant construct that allowed for the expression of full-length monomeric BAK that exhibited physiologic activation and membrane-porating activity. In preliminary studies, I have applied our learnings from the production of monomeric BAK and oligomeric BAX to produce a full-length BAK oligomer for the first time. Here, I propose to optimize and validate the stability and homogeneity of this species for rigorous biochemical and structural characterization (SA1). By mutating discrete functional regions of BAK, I further propose to identify the structural determinants of each step of the activation pathway, and evaluate the mechanistic findings and their functional implications in BAK-dependent leukemia cells (SA2). To achieve my goals, I will apply multidisciplinary approaches that include protein engineering, biochemical assays in model membranes and mitochondria, hydrogen-deuterium exchange mass spectrometry, cryo-electron microscopy, and cellular apoptosis analyses. I am eager to embark on the rigorous training program proposed for my graduate studies and look forward to developing as an independent and innovative physician-scientist at the interface of chemical biology, cancer biology, and clinical oncology.