Junying Yuan IBN #9418785 The overall objective of this ambitious project is the molecular characterization of the vertebrate programmed cell death gene Ice-3, a homolog of the nematode C. elegans cell death gene ced-3 and mammalian cell death gene interleukin 1-beta converting enzyme (ICE). Naturally occurring cell death is an integral part of normal development and homeostasis. In the nematode C elegans, a genetic pathway of programmed cell death has been identified. Genetic mosaic analysis showed the ced-3 is most likely acting within dying cells to cause cell death. ICE is a vertebrate homolog of ced-3. Overexpression of ICE in mammalian fibroblasts causes cells to undergo programmed cell death. A family of vertebrate homologs of the C.elegans ced-3 gene has been identified by this laboratory. Ice- 3 is one of the most interesting homologs because it appears to encode both an activator and an inhibitor of cell death. The mRNA of Ice-3 is alternatively spliced into two different forms: Ice-3L, and Ice-3S. When expressed in Rat-1 cells, Ice-3L causes cells to die, while Ice-3S has the ability to prevent Rat-1 cell death induced by serum deprivation. A model is proposed in which ICE and ICE-3 act in parallel to control cell death. This model will be tested in a variety of experiments. Biochemical and enzymatic properties of the ICE-3 protein will be studied to reveal how ICE-3 acts to induce cell death. The developmental pattern of Ice-3 expression will be examined. Attempts will be made to isolate the activator, inhibitor and substrate of ICE-3 to identify upstream and downstream elements of Ice-3. Antibodies against ICE-3 will be generated and used to determine when and where the ICE-3 protein is made. ICE-3 protein made by E. coli or baculovirus will be used to study the enzymatic properties of ICE-3, with particular attention paid to the different potential roles of Ice-3L vs. Ice-3S. The yeast two hybrid system will also be used to isolate the genes that may encode proteins interacting with the ICE-3 protein. Through these studies, it is hoped to gain an understanding of the mechanism and function of Ice-3 in controlling programmed cell death. ***
