Abstract
Calcineurin inhibitors have been used for transplant therapy. However, the inhibition of calcineurin outside the immune system has a number of side effects. We previously developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide (11R-VIVIT) selectively interferes with calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase and provides immunosuppression for fully mismatched islet allografts in mice. However, our recent study showed that 11R-VIVIT affected cell viability in vitro when it was used at higher concentration because of the VIVIT sequence. The aim of this study is to develop a safer NFAT inhibitor (RCAN-11R) that does not affect cell viability, and which is less toxic than calcineurin inhibitors. The minimal sequence of the protein family of regulators of calcineurin (RCAN) that is responsible for the inhibition of calcineurin-NFAT signaling was recently characterized. The peptide could selectively interfere with the calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase, similar to 11R-VIVIT. RCAN-11R did not affect cell viability when it was used at a higher concentration than the toxic concentration of 11R-VIVIT. RCAN-11R could therefore be useful as a therapeutic agent that is less toxic than current drugs or 11R-VIVIT.
Highlights
An important mechanism whereby calcineurin promotes the activation of T cells and the induction of cytokine-related genes is largely attributed to a family of transcriptional regulators that are referred to as NFAT
To the VIVIT peptide, the regulators of calcineurin (RCAN)-derived peptide does not inhibit the general calcineurin phosphatase activity, suggesting that it may have a specific immunosuppressive effect on the calcineurin-NFAT signaling pathway (Supplemental Fig. 1). We developed another safer NFAT inhibitor (RCAN-11R) which was less toxic than calcineurin inhibitors or 11R-VIVIT
We demonstrated that the RCAN-11R peptide provides immunosuppression for fully mismatched islet allografts in mice by inhibiting calcineurin-NFAT signaling
Summary
The immunosuppressants cyclosporine A and FK506, which are used clinically to prevent transplant rejection, inhibit the activity of calcineurin phosphatase on all its protein substrates, including NFAT1, 2. We synthesized VIVIT peptide as a C-terminal fusion protein with 11-arginine (11R) to facilitate the efficient in vivo delivery of the NFAT inhibitor peptide into T cells. Protein transduction domains such as polyarginine have been developed to deliver bioactive peptides and proteins into eukaryotic cells[9,10,11,12,13] and to successfully deliver covalently attached peptides and proteins in vivo into all mamalian tissues. To the VIVIT peptide, the RCAN-derived peptide does not inhibit the general calcineurin phosphatase activity, suggesting that it may have a specific immunosuppressive effect on the calcineurin-NFAT signaling pathway (Supplemental Fig. 1). We synthesized the RCAN peptide as an N-terminal fusion protein with 11-arginine (11R) in order to achieve the efficient in vivo delivery of the RCAN peptide into T cells
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