Binding, Immunophilins leads to Inhibition, Calcineurin Activity

The phosphatase activity of calcineurin (CN) is well known to be inhibited by CN inhibitors such as FK506 and cyclosporine A through complex formation with immunophilins.

Immunophilins are a general class of proteins that exhibit peptidyl-propyl isomerase (PPIase) activity, such as FKBP (FK506-binding protein) or cyclophilin (Barik. 2006). FKBP and cyclophilin bind with calcineurin (CN)-inhibitors FK506 and cyclosporin A to form complexes, which inhibit CN activity (Barik. 2006).

While FKBP12, FKBP12.6, FKBP13, and FKBP52 are all part of the FK506-binding FKBP family, FKBP12 has a significant involvement in the mechanism of action for FK506-induced immunosuppression (Siekierka et al. 1989, Kang et al. 2008).

FKBP12 is a 12-kDa protein localized in cytoplasm and has been isolated from Jurkat T-cells as a receptor that binds with the CN inhibitor FK506 (Bram et al. 1993). FKBP12 has an FK506-binding domain (FKBD) that comprises 108 amino acids, and is expressed in T‑cells, B‑cells, Langerhans cells, and mast cells (Siekierka et al. 1990, Panhans-Gross et al. 2001, Hultsch et al. 1991).

Cyclophilin and FKBP both exhibit PPIase activity, but no structural similarities have been found between them. Additionally, while immunophilin complexes formed with either substance do inhibit CN phosphatase activity, the PPlase activity and the inhibition of activity that they indicate are unrelated to CN regulation.

CN is a heterodimer that comprises a catalytic subunit (CnA) and a Ca-binding regulatory subunit (CnB). CnA handles phosphatase activity as well as calmodulin binding, and CnB regulates intracellular calcium and CnA (Klee et al. 1988, Zhang et al. 1996). CnA is a 59kDa protein with a serine-threonine phosphatase domain. A FK506-FKBP complex binds directly to CnA in the cell, causing steric hindrance of substrate binding to CN, which in turn inhibits phosphatase activity of CN (Schreiber and Crabtree 1992, Liu et al. 1993, Bierer et al. 1993, Bram et al. 1993, Rao et al. 1997, Liu et al. 1991). Cyclophilin-Cyclosprine A (CsA) complexes also function in the same manner, binding directly to CnA in the cell, which in turn inhibits CN phosphatase activity.

The nuclear factor of activated T cells (NFAT) is a substrate of calcineurin (Rao et al. 1997). When CN activates through stimulus from outside of the cell, it binds directly to the N‑terminal of NFAT in cytoplasm, after which dephosphorylation of SP motifs exposes nuclear localization signal (NLS) and covers nuclear export signal (NES), thereby promoting nuclear localization of NFAT (Matsuda and Koyasu 2000, Zhu and McKeon 1999). When T-cell activation takes place, T-cell receptor (TCR)-mediated stimulus increases the intracellular concentration of calcium and activates CnB, which subsequently induces CnA phosphatase activation, leading to dephosphorylation of NFAT followed by nuclear localization.

When CN activity is inhibited by the binding of immunophilin complexes, dephosphorylation does not occur in NFAT, thereby interfering with nuclear localization.

MIE:

In the phosphatase assay for CN, addition of 1 molar equivalent of FKBP-FK506 complex inhibited over 80% of the CN protein phosphatase activity (mixed with 300 nM calmodulin-CN and 30 µM FK506) (Liu et al. 1992). Dose-response analysis of the effects of FK506 on CN-mediated phosphatase activity in KiSVMC4W cells showed that increased expression of FKBP12 resulted in a greater than ten-fold increase in the sensitivity of the KiSV-MC4W cells containing human FKBP12 cDNA to FK506-mediated inhibition of CN phosphatase activity, as indicted by an IC50 value of ~3 nM (Fruman et al.1995). The phosphatase assay showed that FK506 inhibition of CN activity was concentration-dependent and that IC50 values for CN inhibition were approximately 0.5 nM for FK 506 (Fruman et al.1992).

Interference with translocation of NFAT to the nucleus is detected using a gel mobility shift assay to test nuclear extracts and cytoplasmic extracts (Flanagan et al. 1991). NFAT translocation has been found to be regulated by the concentration of CN inhibitor additives.

KE1:

Dose-dependent interference with nuclear translocation of NFAT1 was observed with increasing calcineurin inhibitor concentrations up to 1 μM (1000 nM). Higher concentrations induced cellular toxicity and resulted in cell death. Dose-dependent interference of nuclear NFAT1 translocation per calcineurin inhibition was also observed in CD4+ T cells from healthy donors, again at maximal concentrations of 1 μM. (Maguire et al. 2013).

FKBP is found in a wide variety of organisms, from prokaryotes to multicellular organisms (Siekierka et al. 1989). Multiple subfamilies of FKBP have been reported, with at least eight types having been found in mammals. FKBP12 is reported to be expressed in B-cells, Langerhans cells, and mast cells as well as in T-cells of humans, mice, and other mammalian species.

Cyclophilins have been found in mammals, plants, insects, fungi, and bacteria. They are structurally conserved throughout evolution and all have PPIase activity (Wang P et al. 2005).

CN is broadly distributed throughout the body, including T- and B-cells, and the structure of CnA and CnB is highly conserved from yeasts to humans. Also highly conserved are the amino acid sequences of the catalytic and regulatory domains of CnA isoforms from different organisms (Kincaid. 1993).

NFAT expresses in B cells, mast cells, neutrophil granulocytes, dendritic cells, macrophages, and natural killer cells as well as T cells from humans, rodents, and other mammalian species (Rao et al. 1997).