Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin.
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PMID:Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition. 133 79

FK-506 inhibits Ca(2+)-dependent transcription of lymphokine genes in T cells, and thereby acts as a powerful immunosuppressant. However, its potential therapeutic applications may be seriously limited by several side effects, including nephrotoxicity and neurotoxicity. At present, it is unclear whether these immunosuppressive and toxic effects result from interference with related biochemical processes. FK-506 is known to interact with FK-binding protein-12 (FKBP-12), an abundant cytosolic protein with cis-trans peptidyl-prolyl isomerase activity (PPIase) activity. Because rapamycin (RAP) similarly binds to FKBP-12, although it acts in a manner different from FK-506, by inhibiting T cell responses to lymphokines, such an interaction with FKBP-12 is not sufficient to mediate immunosuppression. Recently, it was found that the complex of FKBP-12 with FK-506, but not with RAP, inhibits the phosphatase activity of calcineurin. Here, we used L-685,818, the C18-hydroxy, C21-ethyl derivative of FK-506, to explore further the role of FKBP-12 in the immunosuppressive and toxic actions of FK-506. Although L-685,818 bound with high affinity to FKBP-12 and inhibited its PPIase activity, it did not suppress T cell activation, and, when complexed with FKBP-12, did not affect calcineurin phosphatase activity. However, L-685,818 was a potent antagonist of the immunosuppressive activity of both FK-506 and RAP. Moreover, L-685,818 did not induce any toxicity in dogs and rats or in a mouse model of acute FK-506 nephrotoxicity, but it blocked the effect of FK-506 in this model. Therefore, FK-506 toxicity involves the disruption of biochemical mechanisms related to those implicated in T cell activation. Like immunosuppression, this toxicity is not due to the inhibition of the PPIase activity of FKBP-12, but may be linked to the inhibition of the phosphatase activity of calcineurin by the drug FKBP-12 complex.
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PMID:The immunosuppressive and toxic effects of FK-506 are mechanistically related: pharmacology of a novel antagonist of FK-506 and rapamycin. 138 Sep 76

The peptidyl-prolyl isomerases FKBP12 and cyclophilin A (immunophilins) form complexes with the immunosuppressants FK506 and cyclosporin A that inhibit the phosphatase calcineurin. With the yeast two hybrid system, we detect complexes between FKBP12 and the calcineurin A catalytic subunit in both the presence and absence of FK506. Mutations in FKBP12 surface residues or the absence of the calcineurin B regulatory subunit perturb the FK506-dependent, but not the ligand-independent, FKBP12-calcineurin complex. By affinity chromatography, both FKBP12 and cyclophilin A bind calcineurin A in the absence of ligand, and FK506 and cyclosporin A respectively potentiate these interactions. Both in vivo and in vitro, the peptidyl-prolyl isomerase active sites are dispensable for ligand-independent immunophilin-calcineurin complexes. Lastly, by genetic analyses we demonstrate that FKBP12 modulates calcineurin functions in vivo. These findings reveal that immunophilins interact with calcineurin in the absence of exogenous ligands and suggest that immunosuppressants may take advantage of the inherent ability of immunophilins to interact with calcineurin.
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PMID:Immunophilins interact with calcineurin in the absence of exogenous immunosuppressive ligands. 752 75

The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action.
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PMID:FKBP51, a novel T-cell-specific immunophilin capable of calcineurin inhibition. 754 43

The 12- and 13-kDa FK506 binding proteins (FKBP12 and FKBP13) are cis-trans peptidyl-prolyl isomerases that bind the macrolides FK506 (Tacrolimus) and rapamycin (Sirolimus). The FKBP12.FK506 complex is immunosuppressive, acting as an inhibitor of the protein phosphatase calcineurin. We have examined the role of the key surface residues of FKBP12 and FKBP13 in calcineurin interactions by generating substitutions at these residues by site-directed mutagenesis. All mutants are active catalysts of the prolyl isomerase reaction, and bind FK506 or rapamycin with high affinity. Mutations at FKBP12 residues Asp-37, Arg-42, His-87, and Ile-90 decrease calcineurin affinity of the mutant FKBP12.FK506 complex by as much as 2600-fold in the case of I90K. Replacement of three FKBP13 surface residues (Gln-50, Ala-95, and Lys-98) with the corresponding homologous FKBP12 residues (Arg-42, His-87, and Ile-90) generates an FKBP13 variant that is equivalent to FKBP12 in its affinity for FK506, rapamycin, and calcineurin. These results confirm the role of two loop regions of FKBP12 (residues 40-44 and 84-91) as part of the effector face that interacts with calcineurin.
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PMID:FK506 binding protein mutational analysis. Defining the surface residue contributions to stability of the calcineurin co-complex. 764 51

In complex with the peptidyl-prolyl isomerase cyclophilin A, the immunosuppressive antifungal drug cyclosporin A (CsA) inhibits a Ca2+/calmodulin-dependent protein phosphatase, calcineurin, which regulates signal transduction. We isolated and characterized cyclophilin A mutations that confer CsA resistance in a Saccharomyces cerevisiae strain whose growth is CsA-sensitive. Three mutations (G70S, H90Y, and G102A) alter single amino acids conserved between yeast and human cyclophilin A, which structural analyses implicate in CsA binding to human cyclophilin A. By Western analysis, all three mutant proteins are expressed in yeast. In vitro, two purified mutant cyclophilins (G70S, G102A) retain prolyl isomerase activity and have moderately reduced affinity for CsA and calcineurin but, when bound to CsA, do bind and inhibit calcineurin phosphatase activity. In contrast, the purified H90Y mutant cyclophilin is dramatically decreased in prolyl isomerase activity, CsA affinity, and calcineurin binding and inhibition. These studies identify conserved cyclophilin A residues that participate in CsA binding and catalysis.
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PMID:Mutations that perturb cyclophilin A ligand binding pocket confer cyclosporin A resistance in Saccharomyces cerevisiae. 767 24

Mutation of three cationic surface residues of human cyclophilin A (hCyPA), R69, K125, and R148, to both anionic and neutral residues left its intrinsic peptidyl-prolyl isomerase (PPIase) activity and cyclosporin A (CsA) binding unaffected, but altered its ability to inhibit the serine phosphatase activity of calcineurin (CN). R69E was 13-fold less effective (Ki = 3400 nM) than wild-type hCyPA (Ki = 270 nM) in presenting CsA for calcineurin phosphatase inhibition, while R148E was 17-fold more effective (Ki < or = 16 nM), and human CyPB was 13-fold better (Ki < or = 21 nM), establishing that a composite drug/protein surface is being recognized. The phosphoserine phosphatase reaction catalyzed by CN using unlabeled phosphoserine RII19 peptide was coupled to a continuous spectrophotometric assay to measure inorganic phosphate production using the enzyme purine ribonucleoside phosphorylase and the substrate N7-methyl-2-thioguanosine [Webb, M. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4884-4887]. With this assay, we have determined that human cyclophilin A complexed with the immunosuppressive drug cyclosporin A is a noncompetitive inhibitor of calcineurin phosphatase activity. This mutational analysis identified hCyPA residues that interact with CN, and comparison to similar data on FKBP allowed us to begin to map out the CN recognition surface. The p-nitrophenylphosphatase activity of CN was stimulated ca. 3-fold by CyP.CsA, presumably reflecting altered active site geometry and selective access of this small substrate.
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PMID:Cyclophilin residues that affect noncompetitive inhibition of the protein serine phosphatase activity of calcineurin by the cyclophilin.cyclosporin A complex. 811 97

The solution structure of the periplasmic cyclophilin type cis-trans peptidyl-prolyl isomerase from Escherichia coli (167 residues, MW > 18.200) has been determined using multidimensional heteronuclear NMR spectroscopy and distance geometry calculations. The structure determination is based on a total of 1720 NMR-derived restraints (1566 distance and 101 phi and 53 chi 1 torsion angle restraints). Twelve distance geometry structures were calculated, and the average root-mean-square (rms) deviation about the mean backbone coordinate positions is 0.84 +/- 0.18 A for the backbone atoms of residues 5-165 of the ensemble. The three-dimensional structure of E. coli cyclophilin consists of an eight-stranded antiparallel beta-sheet barrel capped by alpha-helices. The average coordinates of the backbone atoms of the core residues of E. coli cyclophilin have an rms deviation of 1.44 A, with conserved regions in the crystal structure of unligated human T cell cyclophilin [Ke, H. (1992) J. Mol. Biol. 228, 539-550]. Four regions proximal to the active site differ substantially and may determine protein substrate specificity, sensitivity to cyclosporin A, and the composite drug:protein surface required to inhibit calcineurin. A residue essential for isomerase activity in human T cell cyclophilin (His126) is replaced by Tyr122 in E. coli cyclophilin without affecting enzymatic activity.
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PMID:Three-dimensional solution structure of Escherichia coli periplasmic cyclophilin. 813 Jan 88

The periplasmic Escherichia coli cyclophilin is distantly related to human cyclophilin (34% sequence identity). Peptidyl-prolyl isomerase activity, cyclosporin A binding, and inhibition of the calcium-dependent phosphatase calcineurin are compared for human and E. coli wild-type and mutant proteins. Like human cyclophilin, the E. coli protein is a cis-trans peptidyl-prolyl isomerase. However, while the human protein binds cyclosporin A tightly (Kd = 17 nM), the E. coli protein does not (Kd = 3.4 microM). The mutant F112W E. coli cyclophilin has enhanced cyclosporin binding (Kd = 170 nM). As for the human protein, the complex of the E. coli mutant with cyclosporin A inhibits calcineurin. Here we describe the structure at pH 6.2 of cyclosporin A bound to the mutant E. coli cyclophilin as solved with solution NMR methods. Despite the low overall sequence identity, the structure of the bound cyclosporin A is virtually identical in both proteins. To assess differences of the cyclosporin binding site, the solution structure of wild-type E. coli cyclophilin was compared with structures of uncomplexed human cyclophilin A and with cyclosporin bound. Despite the structural similarity of bound cyclosporin A, the architecture of the binding site in the E. coli protein is substantially different at the site most distant to tryptophan 121 (human sequence). This site is constructed by a five-residue insertion in a loop of the E. coli protein, replacing another loop in the human protein.
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PMID:The mutant Escherichia coli F112W cyclophilin binds cyclosporin A in nearly identical conformation as human cyclophilin. 818 Jan 97

Human cyclophilin A (CypA), a ubiquitous intracellular protein of 165 amino acids, is the major receptor for the cyclic undecapeptide immunosuppressant drug cyclosporin A (CsA), which prevents allograft rejection after transplant surgery and is efficacious in the field of autoimmune diseases. CsA prevents T-cell proliferation by blocking the calcium-activated pathway leading to interleukin-2 transcription. Besides their ability to bind CsA, the cyclophilin isoforms also have peptidyl-prolyl isomerase activity and enhance the rate of protein folding. The macrolide FK506 acts similarly to CsA and its cognate receptor FKBP also has peptidyl-prolyl isomerase activity. Inhibition of this enzymatic activity alone is not sufficient to achieve immunosuppression. A direct molecular interaction between the drug-immunophilin complex (CsA-CypA, or FK506-FKBP) and the phosphatase calcineurin, is responsible for modulating the T-cell receptor signal transduction pathway. Here we describe the crystal structure of a decameric CypA-CsA complex. The crystallographic asymmetric unit is composed of a pentamer of 1:1 cyclophilin-cyclosporin complexes of rather exact non-crystallographic fivefold symmetry. The 2.8 A electron density map is of high quality. The five independent cyclosporin molecules are clearly identifiable, providing an unambiguous picture of the detailed interactions between a peptide drug and its receptor. It broadly confirms the results of previous NMR, X-ray and modelling studies, but provides further important structural details which will be of use in the design of drugs that are analogues of CsA.
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PMID:X-ray structure of a decameric cyclophilin-cyclosporin crystal complex. 842 1


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