STRUCTURAL STUDIES OF PROTEINS THAT INHIBIT APOPTOSIS

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Apoptosis is a normal physiological cell suicide program highly conserved among vertebrates and invertebrates. This cell death program plays a critical role during normal development and tissue homeostasis, eliminating unwanted cells from the organism, including damaged and virus-infected cells. Disruptions of the apoptotic response are associated with cancer, where there is too little cell death, and with degenerative diseases, where is too much cell death. A family of cysteine proteases called caspases, related to the mammalian interleukin-1b converting enzyme (ICE/caspase-1) and to CED-3, the product of a suicidal gene from the nematode C. elegans, play a central role in the initiation and downstream execution of the apoptotic program. The gene p35 from the Autographa californica nucleopolyhedrovirus, a baculovirus, inhibits a broad spectrum of caspases, suppressing apoptosis. Structural studies are required to resolve the relationship between the inhibitory mechanism of P35 that involves binding to the caspase and P35-cleavage. Recently we have determined the crystal structure of P35 the first specific aim in the parent grant. Most recently we discovered the first homologue of p35 in the animal kingdom, slp49, a gene from the Spodoptera littoralis nucleopolyhedrovirus (SINPV). The slp49 sequence predicted a 49kDa polypeptide (446 amino acids) with 48.8 percent identity to P35 (of 299 amino acids). The availability of the first homologue of P35 for mutagenesis and functional analysis, together with the capability of determination of the structures of P35 and SLP49 provides the framework for this study that aims to define important motifs conserved in P35-like apoptotic suppressors (or modulators). This will pave the way to find out if a p35-like gene family exists in invertebrates and vertebrates through the isolation of p35-like genes from other baculoviruses and organisms, and establish the backbone to synthesize novel small peptide or peptide-like molecules with apoptotic suppressing/modulating activity.