Dissecting the mechanisms of pro-apoptotic BAX modulation

Abstract

Apoptosis plays a critical role in maintaining normal tissue homoeostasis in multicellular organisms. Deregulation of apoptosis can contribute to diseases such as cancer. Proapoptotic BCL-2 associated X protein (BAX) represents the gateway to mitochondria) apoptosis. In healthy mammalian cells, BAX is found in the cytosol. Following stresses that induce apoptosis signaling, BAX translocates and inserts onto the mitochondria) outer membrane where it induces membrane permeabilization. Despite advances in understanding the structure, function, and interactions of BAX, the mechanism of inhibition of cytosolic BAX and/or the conformational changes associated with its activation remains elusive. Therefore, studies aimed at understanding how cytosolic BAX is modulated are necessary. We investigated the mechanism of BAX modulation using biochemical and biophysical structural studies. Firstly, we examined the inhibition mechanism of BAX using synthetic antibodies. Our biochemical studies identified Fabs that bind with high affinity to BAX and inhibit its activation. Also, the Fabs form stoichiometric and stable complexes without inducing a significant conformational change on BAX. Further structural analysis demonstrated that the Fab-binding surface overlaps with the N-terminal activation site on BAX, suggesting a novel mechanism of BAX inhibition that competes with the BAX activation process. Identified Fabs in this study provide insight into mechanisms for inhibiting monomeric BAX. Secondly, we investigated BAX activation via its interaction with BIM. Using full-length BIM, we sought to further analyze the mechanism of BAX-BIM interaction and provide mechanistic and structural insights that more closely resemble their physiological interactions. We successfully expressed and purified recombinant full-length BIM in a fusion protein construct. We confirmed BIM activity in BAX activation studies in the presence of liposomal membranes but not in liposome free solutions. Our results indicate that further investigations of full-length BIM-BAX interaction with membranes or in a membrane-like environment could be successful. Lastly, BAX has been shown to have two different activation sites depending on its cytosol- or mitochondrion-associated conformations: the N-terminal BH3 pocket and the C-terminal BH3 pocket. We demonstrate the use of computational analysis and mutagenesis studies of BAX mutants and mutant BIM BH3 peptides to study the contributions of the two sites on BAX activation.