Inhibiting the Calcineurin-NFAT (Nuclear Factor of Activated T Cells) Signaling Pathway with a Regulator of Calcineurin-derived Peptide without Affecting General Calcineurin Phosphatase Activity

Abstract

Calcineurin phosphatase plays a crucial role in T cell activation. Dephosphorylation of the nuclear factors of activated T cells (NFATs) by calcineurin is essential for activating cytokine gene expression and, consequently, the immune response. Current immunosuppressive protocols are based mainly on calcineurin inhibitors, cyclosporine A and FK506. Unfortunately, these drugs are associated with severe side effects. Therefore, immunosuppressive agents with higher selectivity and lower toxicity must be identified. The immunosuppressive role of the family of proteins regulators of calcineurin (RCAN, formerly known as DSCR1) which regulate the calcineurin-NFAT signaling pathway, has been described recently. Here, we identify and characterize the minimal RCAN sequence responsible for the inhibition of calcineurin-NFAT signaling in vivo. The RCAN-derived peptide spanning this sequence binds to calcineurin with high affinity. This interaction is competed by a peptide spanning the NFAT PXIXIT sequence, which binds to calcineurin and facilitates NFAT dephosphorylation and activation. Interestingly, the RCAN-derived peptide does not inhibit general calcineurin phosphatase activity, which suggests that it may have a specific immunosuppressive effect on the calcineurin-NFAT signaling pathway. As such, the RCAN-derived peptide could either be considered a highly selective immunosuppressive compound by itself or be used as a new tool for identifying innovative immunosuppressive agents. We developed a low throughput assay, based on the RCAN1-calcineurin interaction, which identifies dipyridamole as an efficient in vivo inhibitor of the calcineurin-NFAT pathway that does not affect calcineurin phosphatase activity. Calcineurin (Cn)3 is a calcium/calmodulin-dependent serine/threonine protein phosphatase and a key enzyme in many cellular processes, such as T cell activation (1). Activated Cn dephosphorylates many substrates, including the nuclear factor of activated T cells (NFAT) 1-4 transcription factors, thereby inducing their translocation to the cell nucleus. Nuclear NFAT is a key component of the cytokine gene expression stimulation that triggers T cell activation (2). Cn-induced NFAT dephosphorylation is regulated by a Cn-NFAT protein-protein interaction that involves the NFAT PXIXIT motif, which is crucial for NFAT activation and signaling (3).Current immunosuppressive protocols in transplantation therapies and in the treatment of autoimmune diseases include the administration of cyclosporine A (CsA) or FK506. These drugs are potent inhibitors of Cn phosphatase activity (4), and their continued administration has been associated with severe side effects. The binding of these drugs to intracellular immunophilin receptors, before the interaction with calcineurin, induces complete suppression of Cn phosphatase activity (5). Therefore, a deeper characterization of the molecular mechanisms that govern the interaction between endogenous Cn regulators and Cn in T cell activation would provide useful information for the developme...

Regulation of RCAN1 Protein Activity by Dyrk1A Protein-mediated Phosphorylation

“…S4). A Val-rich region in the C-terminal part of RCAN1 is involved in the interaction (36,37,48,49), and most C-terminal amino acids (where Thr 192 is located) are not required (48). In contrast, a 38-residue fragment of RCAN1-1 without the Val-rich region (residues 215-252) was able to bind Caln when overexpressed in a mammalian cell line (50).…”

Pathogenesis and targeted treatment of skin injury in SLE

“…Dipyridamole is another calcineurin-NFAT pathway inhibitor 95 that blocks the interaction between calcineurin and NFAT, thereby preventing dephosphorylation and activation of NFAT. 98 Treatment of MRL/lpr mice with dipyridamole suppressed lupus nephritis and prevented skin injury. 95 Activation of T cells from MRL/lpr mice, especially CD3 + CD4 -CD8 -cells, is associated with calcium influx and increased levels of the cytoplasmic form of NFAT, which provides B cell help to produce IgG in a calcineurindependent manner.…”

Epigenetics and Vasculitis: a Comprehensive Review

“…There is a risk of renal damage in patients taking CsA and FK506, possibly due to their inhibition of CaN dephosphorylation activity beyond just NFAT molecules [85]. Dipyridamole is an anti-platelet peptide compound that was found to interact with the PXIXIT-recognition site of the CaN protein but does not restrict the phosphatase activity of CaN on other proteins, making dipyridamole a highly specific inhibitor of the CaN/NFAT dephosphorylation activity with better tolerability and less side effects [86,87]. Dipyridamole was found to significantly reduce the expression of CD40L by activated CD4+ T cells to a degree approaching the suppressive effect of FK506, and significantly reduce the expression of IFN-γ, IL-2, and TNF-α [88].…”