Clark Lab

Department of Biology
University of Texas at Arlington

Allosteric Regulation of Caspases

We are interested in how cells fine-tune the activity of caspases in order to use the enzyme activity in development or differentiation processes without killing the cell. The threshold level of caspase activity required for cell development/differentiation versus cell death is not known, nor is it known how cells regulate caspase activity below the threshold. Caspases are produced in the cell as inactive zymogens, and the cell responds to intrinsic or extrinsic signals to initiate an activation program, producing the mature caspase.

We are examining the allosteric regulation of enzyme activity by conformational selection. In particular, we are examining the function and evolution of allosteric sites on the caspase enzymes and how the cell may regulate activity by influencing the conformational ensembles of active and inactive states through binding of allosteric modulators to the enzyme. For example, phosphorylation of caspases decreases enzyme activity and links caspase function to kinase signaling in the cell.

In these projects, we use phage display experiments to identify peptides that bind to hot-spots on the protein surface, and we use structure-based approaches to design small drug compounds that bind to the allosteric sites. We characterize the interactions using a variety of biochemical, biophysical, structural, and cell biological approaches.

Protein Design

We are re-designing the interface of procaspase-8 in order to understand why the protein is a stable monomer even though it is structurally homologous to procaspase-3. The procaspase-8 interface contains several "negative design" elements that protect an exposed β-strand. To this end, we have generated a number of caspase-8 interface mutants that demonstrate a shift to the dimeric state. In a related project, we are examining the binding of the intersubunit linker of procaspase-3 in the dimer interface in order to understand how the linker is involved in maintaining the inactive conformer of the protein.

In these projects, we examine the folding and assembly of the procaspase variants using equilibrium and kinetic techniques, enzymatic studies to assess changes in activity, x-ray crystallography, and cell biological approaches.

Cancer, Neurodegenerative and Autoimmune diseases

Understanding how caspase activation and activity are controlled is important for several human diseases, including cancer, arthritis, diabetes and neurodegenerative disorders. The inactive zymogen of procaspase-3 is prevalent in many cancer cells, and activation of the enzyme through changes in protein conformation could lead to exciting new therapies for the treatment of cancer. We are developing assays to examine the procaspase activation in animal cancer models.

In addition, selectively inhibiting caspases could lead to effective treatments for autoimmune and neurodegenerative disorders. Using phage display and other technologies, we are identifying and characterizing allosteric inhibitors of several caspases. Selective inhibition of caspases through binding in allosteric sites could provide inhibitors with high selectivity and lead to novel therapeutics for the treatment of diseases where excessive cell death is problematic.



Allostery, Apoptosis, Caspases, Cancer Biology, Protein Engineering, X-ray Crystallography, Spectroscopy (Equilibrium and Kinetic Studies), Isother mal Titration Calorimetry, Analytical Ultracentrifugation, Molecular Modeling, Molecular Dynamics, Site-Directed Mutagenesis, Assay Development, Transfection, Western Analysis, RT-PCR, Cell Sorting, Transgenic Animals, Phage Display