The research in our lab has two long term objectives which, although distinct and separate, focus on a common enzyme, calcineurin. On one hand we would like to understand the enzymatic mechanism of calcineurin with emphasis on how metal ions promote phosphate ester hydrolysis. From a biomedical perspective, we are interested in the role of calcineurin in T-cells where it is known to be an important component between antigenic stimulation and transcription of the IL-2 gene. Further elucidation of the role of calcineurin in Ca2+-dependent signal transduction may provide a better understanding of the regulation of cell growth and differentiation. As a member of a family of phosphatases which hydrolyze protein serine/threonine phosphate esters calcineurin is distinguished by its Ca2+ and calmodulin dependence. In addition, calcineurin is a metalloprotein; tightly bound Fe and Zn are present in stoichiometric amounts and a divalent metal ion such as Mn2+, or Ni2+ are required for maximal activity. We are interested in understanding how these metals function int he catalytic mechanism and are using electron paramagnetic resonance spectroscopy to investigate the electronic and magnetic properties of these paramagnetic centers. These studies will provide a framework by which to understand the details of how metal ions catalyze phosphate ester hydrolysis in this class of protein phosphatases. The other objective of research in our group is a study of calcineurin in T-cell signal transduction pathways. Until recently, the role of calcineurin in vivo has been poorly understood, primarily because it is ubiquitously distributed and specific inhibitors for it that could be used to selectively study it in vivo were not available. Recently, it has been shown that calcineurin is the target of the powerful immunosuppressant drugs, cyclosporin A (CsA) and FK506, drugs given to transplant patients to prevent organ rejection. These drugs function by preventing the activation of T-cells which secrete IL-2 and cause cells of the immune system to proliferate and become activated. In the T-cell, these drugs bind to receptors and it is the complex between immunosuppressant and receptor which binds to and inhibits calcineurin. We are using both native and recombinant enzyme to understand the mechanisms by which the immunosuppressants inhibit calcineurin. Native enzyme is available in abundant amounts (20-30 mg per prep) for spectroscopic studies and we now have in hand a source of recombinant calcineurin which models the native enzyme from bovine brain in most respects. Most importantly, the recombinant enzyme is sensitive to CsA/cyclophilin and can be used to study the molecular details of the interaction between calcineurin and this drug/receptor complex. Studies are in progress using NMR spectroscopy, to map the surface of the CsA/cyclophilin complex which interacts with calcineurin. These experiments will hopefully provide information about the molecular architecture at the surface of this inhibitory complex which interacts with calcineurin and will provide a blueprint for future immunosuppressant drug design.
