Apoptosis plays a critical role in maintaining normal tissue homoeostasis in multicellular organisms. Dysregulation of apoptosis can result in an imbalance of normal homeostasis contributing to diseases such as cancer and neurodegenerative diseases. Biochemical and genetic studies have revealed a prominent role for the BCL-2 protein family in regulating the commitment of the cell to mitochondrial apoptosis. Pro-apoptotic BAX is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. In healthy mammalian cells, BAX is found in its inactive form in the cytosol. Following cellular stresses that induce apoptosis signaling, BAX translocates to the mitochondria where it inserts into the mitochondrial outer membrane and induces membrane permeabilization. Despite recent advances in understanding the structure, function and interactions of BAX, we do not fully understand precisely how cytosolic BAX is activated by BH3-only protein BIM or how it is kept inhibited in the cytosol. Here, we propose to intelligently investigate the mechanism of BAX modulation using an interdisciplinary approach including biochemical, biophysical, structural and cellular studies.
Our specific aims are: 1.) To investigat the mechanism of BAX interaction and activation by BIM. For this aim, we will produce functional recombinant BIM for biochemical and structural studies. Once produced, we will determine the functional activity of BIM in BAX in vitro and in-cell activation studies. Finally, w will investigate the structural mechanism of BIM-BAX interaction. 2.) To examine the inhibition mechanism of BAX using synthetic antibodies. To accomplish our goals for this aim, we will first screen and evaluate the ability of phage display selected antibodies to bind to BAX. We will then evaluate the function of the selected BAX specific antibodies in binding and activation studies of BAX. Finally, we will determine the interaction site on BAX and the structural basis of BAX inhibition by the selected BAX specific inhibitory antibodies. With the mentorship of Dr. Evripidis Gavathiotis and Dr. Steven Almo, I will be able to accomplish the goals for the proposed research training and provide important knowledge for understanding and targeting BAX regulation while acquiring new skills and knowledge in structural biology, biophysics, biochemistry and cell biology of cell death. Furthermore, I will expand my training in communication skills, grant writing, scientific manuscript writing, mentoring, teaching and job application process, to further enhance my future career goals as a physician-scientist.
To maintain normal tissue homeostasis, a balance between cell proliferation and cell death is essential. Deregulation of apoptosis can result in an imbalanc in normal homeostasis contributing to such disease states such as cancer and neurodegenerative diseases. This proposal focuses on pro-apoptotic protein BAX, which operates as gateway to cell death, and attempts to elucidate the mechanisms of BAX modulation to provide important knowledge for the regulation of the apoptosis pathway and pharmacological targeting of BAX in disease.
|Uchime, Onyinyechukwu; Dai, Zhou; Biris, Nikolaos et al. (2016) Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site. J Biol Chem 291:89-102|