Biochemical and structural dissection of the regulation of the apoptotic pathways in Drosophila and Caenorhabditis elegans.

摘要

Apoptosis, or programmed cell death (PCD), is a fundamental process in all metazoans. Two decades of studies have revealed a conserved apoptotic pathway across species, from nematode worms to mammals. Compared to the well characterized molecular mechanisms in mammalian apoptosis, little mechanistic information is available on the cell death pathways in C. elegans and Drosophila, due to a lack of relevant biochemical and structural studies.; In this thesis, biochemical, structural and genetic approaches were combined to address the molecular mechanisms for the activation as well as inhibition of apoptosis in Drosophila and C. elegans. I have three major findings. (1) Dronc, an initiator caspase in Drosophila, is activated by auto-cleavage-induced dimerization, which is facilitated by Dark in a cytochrome c independent manner. In contrast to its mammalian homolog caspase-9, active Dronc functions as a free enzyme, without the requirement for apoptosome. I determined the crystal structure of a prodomain deleted Dronc zymogen at 2.5 A, the first crystal structure of an initiator caspase in its zymogen form and the first structure of a caspase in Drosophila. (2) DIAP-1, the inhibitor of apoptosis in Drosophila, inhibited Dronc, the initiator caspase, and DrICE, the effector caspase, in two different ways. The BIR-2 and Ring domain of DIAP-1 negatively regulated Dronc by ubiquitinating it, thus resulting in its degradation, whereas the BIR-1 domain directly inhibited the enzymatic activity of DrICE. Three crystal structures and associated biochemical studies revealed that pro-apoptotic proteins Reaper, Hid and Grim effectively relieved DIAP-1 mediated inhibition to Dronc or DACE by disrupting the interaction between BIR-2 and Dronc, or BIR-1 and DrICE, respectively. (3) In C. elegans, the proapoptotic protein EGL-1, a BH3-only family protein, disrupted the interaction between CED-9 and CED-4 by inducing dramatic conformational changes of CED-9. This conclusion is based on the crystal structure of an EGL-I-CED-9 complex and associated biochemical studies.; These studies show that, despite a general conservation of the apoptotic pathways across species, distinct molecular mechanisms are employed to control the initiation and execution of apoptosis. My research findings deepen our understanding of apoptotic regulation.