The laboratory's focus in programmed cell death is to understand the structure and function of regulatory proteins in TNF-receptor mediated apoptosis. The long term objective is to define the interactions between proteins comprising regulatory complexes. With a combination of biochemical and structural approaches, we aim to identify targets for the development of therapeutic agents which could be used to selectively induce cell death in tumors and downregulate apoptosis in peripheral T- cells in AIDS. Initially, our efforts will be to characterize the functional domains of two molecules, FADD and TRADD, which possess so- called death domains (DD) responsible for assembling regulatory complexes at the cytoplasmic domain surface of TNF receptors. This interaction initiates a process of recruitment of other apoptotic molecules to the complex, namely the collection of enzymes (caspases) which degrade specific metabolic pathways leading to apoptosis. Caspase activation by FADD and TRADD is achieved by distinct mechanisms which are to be characterized in this study. The functional domains of FADD and TRADD will be identified by limited proteolysis of these proteins coupled with mass spectrometry to identify putative structured regions in each molecule which can be correlated with specific functions. Following the analysis of this data, cloning strategies will be employed to overproduce these domains, form complexes with their cognate targets and elucidate their three-dimensional structures by multi-dimensional, multi-nuclear NMR.
