Clark Lab Projects

Projects in the Clark Lab

Research in my lab focuses on the broad topics of caspase assembly and cell death. Caspases are integral proteases in programmed cell death (apoptosis), and the dysregulation of apoptosis is observed in a number of human diseases, from autoimmune diseases (rheumatoid arthritis, diabetes), to neurodegenerative diseases, to cancer.

Conservation of amino acids in caspases

Site-specific conservation levels

Caspases are also an attractive system for examining protein evolution. Caspases evolved from an ancestral protease into two subfamilies – inflammatory and apoptotic – more than 700 million years ago. Gene duplications followed by neofunctionalization resulted in two apoptotic caspase subfamilies – the initiators and the effectors. All caspases recognize an aspartate residue at the P1 position of the substrate, so discrete cellular functions of caspases result from evolution of substrate specificity and allosteric regulation. We examine the evolutionary pathways from the ancestral scaffold that provide specific functions to modern caspases.

We use biochemical, biophysical, and structural studies to understand the changes in evolutionary networks  that result in unique allosteric regulation, enzyme specificity, and oligomerization. We have developed an extensive library of allosteric mutants of caspase-3 as well as a large curated database of caspase sequences, the CaspBase, which provides tools for reconstructing ancestral proteins.

Workflow for Ancestral Protein Reconstruction

Workflow for Ancestral Protein Reconstruction

We characterize ancestral and extant caspases using X-ray crystallography, spectroscopy and other biophysical techniques. We use phage display and other high through-put screening assays to determine enzyme specificity. Together the data describe evolutionary changes leading to unique characteristics of modern enzymes.