Title: Functional studies of the calcineurin-DSCR1 complex
Description: Calcineurin is a protein phosphatase that regulates the nuclear localization of the NFAT family of transcription factors during immune challenge. Cyclosporin A-cyclophilin A and FK506-FKBP12 inhibit calcineurin non-competitively. A family of calcineurin endogenous protein inhibitors was recently identified and shown to block calcineurin-mediated NFAT nuclear localization and transcriptional activation. Preliminary analyses of one of these inhibitory proteins, Down Syndrome Critical Region 1 (DSCR1), revealed that it inhibits calcineurin activity towards a small molecule substrate, pNPP, possibly functioning as a novel calcineurin inhibitor that contacts calcineurin at or near the active site. This dissertation describes an investigation of the mechanism by which calcineurin is regulated by DSCR1. Crystallization of the complex was attempted to reveal the physical basis of enzyme inhibition, but failure of the complex to crystallize led to the realization that DSCR1 is largely unstructured in complex with calcineurin. Biochemical methods were used to identify and characterize a short peptide segment derived from DSCR1 that mediates competitive inhibition of phosphatase activity in vitro and in vivo . Small molecule compounds were also identified that disrupt the interaction of DSCR1 with calcineurin and thereby relieve inhibition by DSCR1. The results described here could facilitate the development of new therapeutic agents while furthering our understanding of calcineurin regulation.