Apoptosis, a form of programmed cell death, is a highly orchestrated mechanism for maintaining the critical balance between new and dying cells during development and throughout life. Disruption of this balance can lead to unwanted cell death, such as in neurodegeneration, stroke, heart attack and cytopenias, or pathologic cell survival, such as in cancer and autoimmune disease. The BCL-2 family is composed of guardian and executioner proteins that form a complex interaction network that regulates apoptosis. Several small molecule and peptide-based approaches have emerged that target BCL-2 family proteins and successfully modulate them for potential therapeutic benefit. Based on our identification of discrete trigger sites on BAX and BAK, two of the essential pro-apoptotic BCL-2 family proteins, their direct modulation may present an opportunity to activate or inhibit cell death in the appropriate disease context. The goal of this proposal is to define the biochemistry, structure, and function of the elusive pro-apoptotic BCL-2 family protein BOK. Determining the context-dependent roles of BOK will advance our understanding of cellular lifespan control points and inform the development of novel pharmacologic strategies to target them for clinical benefit. I propose a multidisciplinary approach that employs synthetic chemistry, structural biology, and biochemical and cellular studies to investigate the functional role of BOK in maintaining tissue homeostasis.
I aim to: (1) Define and structurally characterize the network of interactions between pro-apoptotic BOK and the diversity of BH3 domain helices; and (2) Determine the functional consequences of BH3 engagement of BOK in vitro and in cells. First, I will build upon my recent success in overcoming the longstanding challenge of generating full-length, recombinant BOK in monomeric form. To probe BOK binding activity and its impact on functional activation, I will generate and apply Stabilized Alpha-Helices of BCL-2 domains (SAHBs), unique research tools that recapitulate the natural structure and specificity of native BH3 death ligands. SAHBs that directly bind to BOK will serve as molecular probes and prototype therapeutics to investigate and modulate the biological activity of BOK in vitro and in cells. Based on this mechanistic dissection of BOK activity, I hope to uncover new pharmacologic approaches to regulate cell death in diseases of unwanted cell death or pathologic cell survival. I am committed to a scientific career focused on dissecting the protein interaction dynamics of the cell death pathway and harnessing the structural, biochemical, and cellular insights to advance novel therapeutic strategies for optimizing quantity and quality of lie during aging. To that end, I have developed a comprehensive training program that blends chemistry, structural biology, apoptosis biology, and developmental therapeutics, in the context of a dynamic biomedical environment, to maximize my development as an independent investigator with unique expertise at the interface of chemical biology and cellular longevity research.
The pro-apoptotic BCL-2 family proteins BAX and BAK are essential regulators of the critical balance between cellular life and death, transforming from inactive monomers to lethal mitochondrial pores in response to insurmountable stress. BOK is a BAX/BAK homologue whose structure, ligand interactions, and functional activities remain relatively unknown, largely due to the longstanding challenge of expressing and characterizing the full-length recombinant protein. I propose to interrogate the biochemistry of BOK and elucidate novel pharmacologic approaches for regulating its function in diseases of cellular deficiency or excess by advancing new methods to produce the protein, define its functional interactions, and validate the structure-activity relationships in cells.
