A novel peptide exerts potent immunosuppression by blocking the two-site interaction of NFAT with calcineurin

Lu Wang,Na Cheng,Ping Wang,Jing Li,Anna Jia,Wenying Li,Nan Zhang,Yanxia Yin,Li Tong,Qun Wei,Guangwei Liu,Zhimei Li,Jing Luo

doi:10.1074/jbc.ra119.010254

Abstract

The calcineurin/nuclear factor of activated T cell (CN/NFAT) signaling pathway plays a critical role in the immune response. Therefore, inhibition of the CN/NFAT pathway is an important target for inflammatory disease. The conserved PXIXIT and LXVP motifs of CN substrates and targeting proteins have been recognized. Based on the affinity ability and inhibitory effect of these docking sequences on CN, we designed a bioactive peptide (named pep3) against the CN/NFAT interaction, which has two binding sites derived from the RCAN1-PXIXIT motif and the NFATc1-LXVP motif. The shortest linker between the two binding sites in pep3 is derived from A238L, a physiological binding partner of CN. Microscale thermophoresis revealed that pep3 has two docking sites on CN. Pep3 also has the most potent inhibitory effect on CN. It is suggested that pep3 contains an NFATc1-LXVP-substrate recognition motif and RCAN1-PXIXIT-mediated anchoring to CN. Expression of this peptide significantly suppresses CN/NFAT signaling. Cell-permeable 11-arginine-modified pep3 (11R-pep3) blocks the NFAT downstream signaling pathway. Intranasal administration of the 11R-pep3 peptide inhibits airway inflammation in an ovalbumin-induced asthma model. Our results suggest that pep3 is promising as an immunosuppressive agent and can be used in topical remedies.

Highlights

The calcineurin/nuclear factor of activated T cell (CN/nuclear factor of activated T cells (NFAT)) signaling pathway plays a critical role in the immune response
To carry out its biological functions, it interacts with multiple substrates and targeting proteins, such as nuclear factor of activated T cells (NFAT) [2], endogenous regulator of calcineurin 1 (RCAN1) [3], transcription factor EB (TFEB) [4], and dynamin-related protein 1 (Drp1) [5]
We previously reported that two LXVP-type peptides derived from RCAN1 and TFEB could bind to CN and inhibit its phosphatase activity [13,14,15]

Summary

Introduction

The calcineurin/nuclear factor of activated T cell (CN/NFAT) signaling pathway plays a critical role in the immune response. Based on the affinity ability and inhibitory effect of these docking sequences on CN, we designed a bioactive peptide (named pep3) against the CN/NFAT interaction, which has two binding sites derived from the RCAN1-PXIXIT motif and the NFATc1-LXVP motif. They developed a high-affinity PVIVIT peptide (MAGPHPVIVITGPHEE, PVIVIT peptide) based on the PXIXIT docking motif [7] This peptide potently inhibited NFAT activation (NFATc1-c3) and NFAT-dependent gene expression in T cells without affecting CN activity. The DQYLAVPQHPYQWAK peptide derived from NFATc1-LXVP inhibits CN activity directly and NFAT-dependent transcription regulation. It is well-known that the T cell receptor (TcR)/Ca2ϩ/CN signaling pathway plays a critical role in T cell activation. Our previous study demonstrated that the binding between CN and the RCAN1-

Methods

Results

Conclusion