Apoptosis plays a central role in the development and homeostasis of all multi-cellular organisms. Alterations in apoptotic pathways have been implicated in many debilitating human diseases including cancer. Genetic studies pioneered by Robert Horvitz have led to the identification of four genes that control the onset of apoptosis in the model organism Caenorhabditis elegans. The protein products of these four genes, Egl1, CED9, CED4, and CED3 act in a linear pathway to execute cell death and constitute a classic paradigm for the understanding of apoptosis. Systematic biochemical and X-ray crystallographic analyses of protein complexes involved in this paradigm have been initiated. Significant progress has been achieved; the work proposed here will build on the preliminary results with the following specific aims: (1) Structural and functional analysis of an Egl1-CED9 complex. Binding of Egl1 to CED9 triggers the release of CED4 from the anti-apoptotic protein CED9. Crystals of the Egl1-CED9 complex that diffract X-rays to 2.2 Angstroms resolution have been obtained. The structure was determined by molecular replacement; refinement is in progress. In collaboration with the laboratories of Robert Horvitz and Ding Xue, structure-based functional analysis is underway. (2) Determination of the structure of a CED9-CED4 complex. CED4 remains constitutively associated with CED9 as an inactive complex prior to apoptosis. A binary complex between the full-length CED4 and a large functional domain of CED9 has been characterized. Small crystals have been obtained most recently. The structure will be determined by multi-wavelength anomalous dispersion (MAD). (3) Biochemical characterization of CED4-mediated activation of CED3. (4) Determination of the structure of oligomeric CED4 in isolation. The disruption of a CED9-CED4 hetero-dimer by Egl1 results in the oligomerization of CED4, which is essential for the recruitment and activation of CED3. (5) Determination of the structure of a CED4-CED3 complex. CED3 forms a holoenzyme with oligomeric CED4. A CED4-CED3 complex has been reconstituted in vitro. The complex will be crystallized and its three-dimensional structure will be determined.
NIH ExPorter
NIH ExPorter