| Summary: | 博士 === 國立臺灣大學 === 動物學研究研究所 === 95 === Our current understanding of apoptosis mainly comes from the studies of C. elegans, Drosophila and mammals. These studies have showed that this death program is evolutionarily conserved and that the participating core molecules in this conserved pathway are BCL-2 family proteins, APAF-1 homologues, and caspases. However, just like other signaling pathways or other regulatory networks, higher animals use more related proteins and have more complicated networks to regulate the apoptosis.
Caspases, a family of evolutionarily conserved intracellular cysteine proteases, are the executioners of apoptosis. They are activated through proteolytic cleavage cascades during apoptosis. These proteases are classified into either initiator or effector caspases. The initiator caspases receive and are activated by apoptotic signals. The activated initiators then activate downstream effector caspases, which subsequently digest vast arrays of cellular proteins, leading to the morphological and biochemical changes of apoptotic cells and ultimately to the demise of apoptotic cells.
In Drosophila and mammals, the activated caspases can be inhibited by members of inhibitor of apoptosis protein (IAP) family through direct interaction. The hallmark of IAP is the presence of one or more copies of baculoviral IAP repeat (BIR) domain at N-termini. The BIR domain is the structural unit for inhibiting caspase activity through direct binding. However, the caspase-inhibitory property of IAPs is in turn antagonized by another groups of proteins, the IAP binding motif (IBM)-containing proteins. Although these proteins show no relationship and sequence conservation to each other, their N-termini do share a conserved tetrapeptide motif, which is responsible for binding to BIR domain and releasing the suppression on caspases. These IBM-containing proteins are pro-apoptotic and when they were overexpressed in insect and mammalian cells, apoptosis is induced. The IBM-containing proteins, Rpr, Hid, Grim and Sickle, play very important roles in Drosophila embryogenesis and development.
In this PhD thesis study, two apoptosis-related genes were cloned from Penaeus monodon, IAP and caspase, and the corresponding proteins, PmIAP and Pm caspase, were further characterized. PmIAP has 3 BIR domains and a RING domain, and is structurally similar to Drosophila IAP2, mammalian c-IAP1, c-IAP2 and XIAP. The Pm caspase cloned in this thesis is highly similar to insect effector caspase. My studies revealed that PmIAP could block Rpr-induced apoptosis in SF9 cells through direct interaction. Deletion analysis revealed that PmIAP BIR2 and BIR3 domains were the minimal regions to interact with Rpr and to block the Rpr-induced apoptosis, and that although BIR1 domain does not bind to and inhibit Rpr’s activity, it seems to be able to enhance PmIAP’s inhibition to Rpr. The expression of Pm caspase induced apoptosis in SF9 cells. This apoptosis could be completely blocked by the anti-apoptosis protein WSSV449 encoded by white spot syndrome virus, but was only minimally inhibited by PmIAP, even though both WSSV449 and PmIAP could all bind to Pm caspase. Baculovirus P35 is a well-understood pan caspases inhibitor, and further studies showed that WSSV449 blocked that activity of Pm caspase through a mechanism similar to that used by P35.
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