| The Inhibitor of Apoptosis (IAP) protein family constitutes an important regulatory component of the apoptotic program. Apoptotic proteases, referred to as caspases, are the central mediators of apoptosis, and the mammalian and Drosophila apoptotic inhibitors XIAP and DIAP1, respectively, function in part by directly inhibiting caspases. Once the cell receives an apoptotic signal, pro-apoptotic molecules are activated which bind to IAPs and therefore promote caspase activity. Examples of such interactions include Smac binding to XIAP, and Reaper, Hid, and Grim binding to DIAP1.; Smac is targeted to the mitochondria where it undergoes maturation that exposes a four amino acid N-terminus required for binding to IAPs and promoting caspase activation. The Drosophila protein Hid also contains these four conserved amino acids. It has been hypothesized that Hid is the functional homolog of Smac but no in vivo processing of Hid had been shown. In Chapter 1, using structural and mutational analysis, it was determined that Hid binds the baculoviral IAP, Op-IAP, in a manner similar to Smac binding of XIAP. It was further shown that Hid must be processed by a methionine aminopeptidase before its amino terminus can bind Op-IAP. This conserved interaction underscores its importance in apoptotic regulation and suggests a favorable apoptotic control point for chemotherapeutic intervention.; Although Op-IAP could bind Hid-like proteins and inhibit their pro-apoptotic activity it was not known whether Op-IAP was able to directly inhibit active caspases like its cellular counterparts. Data in Chapter 2 indicate that Op-IAP is unable to inhibit or bind to the Drosophila caspases Dronc, Drice, or Dcp-1 in vitro. Surprisingly, Op-IAP could not block apoptosis in Drosophila cells but instead seemed to sensitize them to viral induced apoptosis. However, Op-IAP did not sensitize Drosophila cells to UV or actinomycin D-induced apoptosis. Taken together with previously published data, this suggests that Op-IAP inhibits apoptosis by a mechanism that is upstream of caspase activation. |
