The objectives of this proposal are to perform structural and mechanistic analysis on P35 a viral protein that inhibits apoptosis. Apoptosis is a naturally occurring process of cellular suicide that is vital to normal organismal development and tissue homeostasis. Very little is known about the regulation of apoptosis and aberrations in the cell death program can commence the onset of many diseases. All the different signals that trigger the cell death program utilize a new class of cysteine proteases to transmit the apoptosis program. This common pathway makes it the most attractive target for regulation by therapeutic agents. Therefore, a better understanding on the structural mechanics of the cysteine protease function and inhibition is required. The baculovirus expresses a 35 kDa protein that inhibits the cysteine proteases involved in executing the death signal. Baculovirus P35 is a general and very effective suppressor of apoptosis and can block apoptosis in mammalian, murine, and insect cell lines. The ability of P35 to suppress apoptosis in such a diverse range of organisms induced by different signals suggests that it acts at an evolutionarily conserved step in the apoptotic pathway. Baculovirus P35 defines a new class of protease inhibitors for which there is no structural information. P35 is first cleaved by the cysteine protease then inhibits the enzyme by forming a tightly bound dead-end complex. X-ray quality crystals of P35 have been grown and one heavy atom derivative obtained. Detailed x-ray structural analysis of P35 augmented with loss-of-function mutant structures will reveal the mechanism by which P35 blocks apoptosis. Additional insights will be gained by analyzing the crystal structure of cleaved P35 complexed with a cysteine protease. This structure will furnish new information and clues on the mechanism of the proteases as well as new levels of apoptotic inhibition. The new fundamentals gained on the function of P35 could also lead to anti-viral compounds.
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