The calcineurin family is a group of ubiquitously expressed, calcium-dependent enzymes that are involved in a wide range of cellular functions in the brain, immune system, heart, kidney, and other organs. Inhibition of calcineurin with drugs including cyclosporine and FK506 are the cornerstone of post-transplant immunosuppression regiments and are also used to treat some autoimmune disorders. However, the effectiveness of calcineurin inhibitors (CNIs) for immune suppression is diminished by an array of detrimental side-effects that stem from inhibition of calcineurin in non-immune tissues. The longterm goal of our laboratory is to identify molecular mechanisms of specific calcineurin isoforms that can be exploited to selectively modulate enzyme activity and expand therapeutic applications. One strategy for increased specificity of calcineurin inhibition is rooted in the closely-related isofoms of the catalytic subunit. Calcineurin is a holoenzyme consisting of a catalytic subunit, a regulatory subunit (which binds calcium), and calmodulin. The two most widely expressed isoforms of the catalytic subunit - A?nd A? have distinct physiological functions as demonstrated by knockout mice. For example, mice lacking the A?soform (CnA?-) have impaired renal development and function, increased expression of TGF?nd fibrosis while CnA?- mice develop normally but have impaired cardiac and renal hypertrophic responses. Meanwhile, CnA?- mice have an immune suppressed-like phenotype and attenuated nuclear factor of activated T cell (NFAT) activity while T cells from CnA?- mice can still be inhibited b cyclosporine. Current CNIs inhibit both isoforms producing desired results such as immune suppression but also off-target effects including nephrotoxicity. These data clearly indicate that isoform-selective inhibition would be a superior therapeutic strategy for immune suppression with the advantages of fewer side-effects and expanded clinical applications. While calcineurin isoforms share 85% amino acid identity, there are small regions of divergence in the N and C termini. In particular, there is a proline-rich (PR) region that is present in the N terminus of Cn?but not CnA?In vitro, CnA?- cells fails to regulate NFAT nuclear translocation or transcriptional activity. Re-expression of CnA?estores NFAT regulation. However, expression of a mutated CnA?hat lacks the PR region does not indicating that the PR region is integral to CnA?ignaling specificity. We propose that the proline rich (PR) region mediates calcineurin A(CnA?regulation of NFAT. We will test the role of the PR region in CnA?ignaling specificity and we will determine if targeting this region produces isoform-selective inhibition.
This project will test the hypothesis that calcineurin isoform signaling-specificity is the result of a unique amino acid sequences at the N terminus of the beta isoform. We hypothesize that the proline-rich sequence mediates calcineurin Abeta's regulation of NFAT by influencing binding of the isoform to calmodulin and/or targeting the isoform for activation by specific pools of calcium. We will carefully examine the mechanism of the proline rich region and we will then test a novel competitive peptide based on the region to determine if it can selectively target calcineruin Abeta activity and regulation of NFAT. Results of these experiments may then lead to novel strategies to selectively target calcineurin isoforms and improve therapeutic efficacy of inhibitors.
| Francis, Cynthia E; Bai, Yun (2018) Differential expression of cyclosporine A-Induced calcineurin isoform-specific matrix metalloproteinase 9 (MMP-9) in renal fibroblasts. Biochem Biophys Res Commun 503:2549-2554 |
| Gooch, Jennifer L; King, Clayton; Francis, Cynthia E et al. (2017) Cyclosporine A alters expression of renal microRNAs: New insights into calcineurin inhibitor nephrotoxicity. PLoS One 12:e0175242 |
