UCSF

The caspases are unique proteases that mediate the major morphological changes of apoptosis and various other cellular remodeling processes. As biologists catalog and study the myriad proteins subject to cleavage by caspases, we are just beginning to appreciate the full functional repertoire of these enzymes. Part of my PhD work involved examining the literature about caspase cleavages: what kinds of proteins are cut, in what contexts, and to what end. I describe this work in Section I of this thesis, including an explanation of the technologies that have enabled high-throughput caspase substrate discovery, and the datasets they have yielded, a discussion of how caspases recognize their substrates, and a few vignettes about the functional impacts of specific caspase cuts. This section of my thesis is adapted from an article published in Annual Review of Biochemistry in 2011.

In Section II of this thesis, I describe the database I created for storage and analysis of mass spectrometry data generated by the many N-terminal labeling experiments conducted in the Wells lab. The database, built using FileMakerPro, allows all lab members to easily access the data, and has been essential for data analysis.

Some caspase substrates have been extensively characterized, revealing key functional nodes for apoptosis signaling. But the functional significance of most cuts remains mysterious. In the project described in Seciton III of this thesis, I addressed this issue by asking which cleavages are conserved across four metazoan model species. Using subtiligase N-terminal labeling, my colleagues and I identified 257 caspase cleavage sites in mouse, 130 in Drosophila, and 50 in C. elegans. Most of the short linear motifs recognized by the caspase active site are conserved from human to mouse, but not to Drosophila of C. elegans. Yet the mouse, Drosophila, and C. elegans substrate sets are all highly enriched with orthologs of known human caspase substrates. Furthermore, similar functional pathways are targeted by caspases in all four species. Our data suggest a model for the evolution of apoptotic caspase specificity that highlights the hierarchical importance of functional pathways over specific proteins, and proteins over their specific cleavage site motifs.