Biochemical mechanisms of caspase regulation in apoptosis.

摘要

Apoptosis (programmed cell death) is a highly regulated process required for proper development and tissue homeostasis. There are two major cell death pathways: the death receptor and the mitochondrial pathways. Both of these pathways converge at the activation of a cysteine protease termed caspase-3, establishing a major regulation point in apoptosis. Although activated, caspase-3 remains inhibited by an Inhibitor of Apoptosis (IAP) family of proteins. The caspase inhibition is removed when a pro-apoptotic protein (stimulator) binds to the IAPs releasing the caspase. Conserved throughout species, these proteins are potential targets for drug development; especially for the treatment of apoptosis-related diseases such as cancer and Alzheimer's disease. This regulation point and the proteins involved are the focus of my research. Throughout my dissertation I study protein-protein interactions between caspases, IAPs and stimulators using the Drosophila system. Chapter 1 is a general introduction of apoptosis. It describes the importance of this process for the proper development of organisms. Both of the major apoptotic pathways, the death receptor and the mitochondrial pathways, are described in detail. Some of what is known about caspase regulation is also explained in this chapter. In Chapter 2, I focus on the structure of the BIR domain, describing the role of a conserved arginine in the functional folding of the BIR domain and introducing our binding assay. This chapter was published in the journal Biochemistry and is included with the journal's permission. Chapter 3 deals with the characterization of the Drosophila effector caspase DCP-1. I developed a quantitative caspase activity assay and I obtained the kinetic parameters KM, Vmax and kcat for DCP-1. In Chapter 4, I make use of the caspase activity assay to quantify and describe the biochemical mechanism of caspase inhibition. Also, I determined the minimal region in DIAP1 required for DCP-1 inhibition and I established the regions in DIAP1 required for the removal of inhibition. Finally, I proposed a model for the biochemical mechanism of caspase regulation. In Chapter 5 I investigate Drosophila stimulator Grim and the possibility for this stimulator to regulate caspase activity in a manner different from the one described in Chapter 4.