The majority of cell deaths that occur in the animals do so via apoptosis, and in mammals, this happenspredominantly by the mitochondrial pathway. This involves the process of mitochondrial outer membranepermeabilization (MOMP), upon which cytochrome c diffuses to the cytosol, caspase proteases are activated,and apoptosis proceeds. Thus, MOMP is often considered the critical decision point for this active cell deathpathway. MOMP is both caused and regulated by proteins of the BCL-2 family, and this control of MOMP islikely to be the most important function of this protein family. This proposal addresses a new 'unified model'for the function of this protein. In this model, anti-apoptotic BCL-2 proteins act either to sequester the proteinsthat activate MOMP (by activating the effector proteins), or to sequester the active effector proteinsthemselves. The differences between these two modes of inhibition, and how they can be de-repressed topromote MOMP and apoptosis, are the bases for this application. The following aims will be addressed.1. Characterize the properties of the two modes of anti-apoptotic BCL-2 protein function and their de-repression. We will employ isolated mitochondria to probe the properties of anti-apoptotic BCL-2 proteinsunder conditions in which they function by blocking MOMP by sequestration of direct activator proteins (MODE1) or by sequestration of the effectors, BAX and BAK (MODE 2). The relative efficiencies of the anti-apoptoticproteins will be assessed in terms of BAX/BAK activation and cytochrome c release. We will further examinethe relative sensitivity of each MODE to de-repression to induce MOMP. We propose that each condition willdisplay fundamentally different properties, in a manner that cannot be predicted by other models. 2. Analyzethe discrete modes of MOMP inhibition by anti-apoptotic BCL-2 proteins in cells. We will then extendour studies into cells to determine if and when these two modes of anti-apoptotic function are engaged uponcell stress. Here, the proposed studies are designed to test and extend our model of BCL-2 family function incontrolling MOMP, and establishing conditions under which MODE 1 or MODE 2 predominate in living cells.We will take advantage of biochemical and live cell imaging approaches to follow the behavior of the pro-apoptotic BCL-2 effector proteins in the context of MOMP. 3. Determine the consequences of de-repression of each mode of anti-apoptotic BCL-2 protein function for cell death and survival in cells.Here we will seek to probe the consequences of this model for cell death by de-repression, leading toapoptosis, under defined situations in cells. We will test if MODE 1 versus MODE 2 inhibition is differentiallysensitive to de-repression in cells, and how this affects 'priming for death,' observed upon pharmacologic de-repression and/or changes in the functions of anti-apoptotic proteins. These studies provide a number of testsand explorations of the new model we propose, and hold the potential to greatly increase of understanding ofthis fundamental process controlling life cell and death.
/Relevance Statement Cell death by apoptosis is crucial for normal homeostasis, and defects in this process underlie many human diseases, including autoimmunity and cancer. This project explores how cell death is controlled at the level of precise molecular interactions, amenable to pharmacologic manipulation, testing a new model of this process.
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