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Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|All life stages||Moderate|
Key Event Description
Calcineurin is a heterodimer that comprises a catalytic subunit (CnA), which handles phosphatase activity as well as calmodulin binding, and a Ca-binding regulatory subunit (CnB), which regulates intracellular calcium as well as CnA (Klee et al. 1988, Zhang et al. 1996). CnA, a 59kDa protein, has a serine-threonine phosphatase domain.
Immunophilins are a general class of proteins that exhibit peptidyl-propyl isomerase (PPIase) activity (Barik. 2006) and an immunophilin-Cn inhibitor complex such as FKBP12-FK506 and cyclophilin-CsA binds directly to CnA in the cell, causing steric hindrance of substrate binding to calcineurin, which inhibits the phosphatase activity of calcineurin without any contribution of PPIase activity (Schreiber and Crabtree 1992, Liu et al. 1993, Bierer et al. 1993, Bram et al. 1993, Rao et al. 1997, Liu et al. 1991).
How It Is Measured or Detected
Phosphatase activity can be measured using a phosphatase assay. Calcineurin, calmodulin, FK506, and FKBP are incubated together, and the phosphatase activity is measured at various concentrations of FKBP. Kinetic analysis of FKBP12 concentration-dependent phosphatase activity and calculation of Ki inhibition of calcineurin by the FKBP12-FK506 complex are conducted. (Bram et al. 1993). Phosphatase activity of calcineurin in the presence of CsA and cyclophilin can also be determined in the manner described above.
Immunophilin-calcineurin inhibitor complexes directly inhibit phosphatase activity of calcineurin, therefore, as a surrogate measurement of the calcineurin activity, the binding of cyclosporin A with cyclophilin can be detected using an ELISA kit.Microtiter plates precoated with BSA and conjugated to cyclosporin are incubated with cyclophilin. Bound cyclophilin is then revealed by incubation with anti-cyclophilin rabbit antiserum followed by incubation with anti-rabbit globulin goat IgG coupled to alkaline phosphatase (Quesniaux et al. 1987).
Domain of Applicability
Calcineurin is broadly distributed in T-cells, B‑cells, and throughout the body. 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 calcineurin A isoforms from different organisms (Kincaid. 1996).
As for immunophilins, of which complexes inhibit the calcineurin activity, FKBP is found in a wide variety of organisms, from prokaryotes to multicellular organisms. (Siekierka et al. 1989a) 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).
Therefore, inhibition of calcineurin phosphatase activity through immunophilin-calnineurin inhibitor complex deems to be in common among mammalian species.
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