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)

Calcineurin (CN), a Ca(2+)/calmodulin (CaM)-dependent serine/threonine protein phosphatase, is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). The activity of CNA is under the control of two functionally distinct, but structurally similar Ca(2+)-regulated proteins, CaM and CNB. The crystal structure of the holoenzyme reveals that the N-terminus and C-terminus of CNB and the N-terminus of CNA each have a long arm not involved in the active site. We constructed a fusion of the genes of CaM, CNB and CNA in that order using linker primers containing six and ten codons of glycine. A single-chain CaM-CNB-CNA (CBA) complex was expressed and purified to near homogeneity. The single-chain complex was fully soluble, and had biochemical properties and kinetic parameters similar to single-chain CNB-CNA (BA) activated by CaM. It was not regulated by CaM and CNB, but was strongly stimulated by Mn2+, Ni2+ and Mg2+. Intrinsic fluorescence spectroscopy of the complex showed a change in the environment of tryptophan in the presence of Ca2+ and circular dichroism (CD) spectropolarimetry revealed an increase in alpha-helical content. Our findings suggest that fusion of CaM, CNB and CNA does not prevent the structural changes required for their functioning; in particular, CaM within the complex could still interact correctly with CN in the presence of Ca2+.
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PMID:Preparation and characterization of a single-chain calcineurin-calmodulin complex. 1569 51

Rat calcineurin (CaN) A alpha isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EGFP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared. The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results verified that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A alpha (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.
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PMID:Construction of rat calcineurin A alpha cDNA recombinant adenovirus vector and its identification. 1671 Sep 96

Calmodulin is an essential protein in the model organism Dictyostelium discoideum. As in other organisms, this small, calcium-regulated protein mediates a diversity of cellular events including chemotaxis, spore germination, and fertilization. Calmodulin works in a calcium-dependent or -independent manner by binding to and regulating the activity of target proteins called calmodulin-binding proteins. Profiling suggests that Dictyostelium has 60 or more calmodulin-binding proteins with specific subcellular localizations. In spite of the central importance of calmodulin, the study of these target proteins is still in its infancy. Here we critically review the history and state of the art of research into all of the identified and presumptive calmodulin-binding proteins of Dictyostelium detailing what is known about each one with suggestions for future research. Two individual calmodulin-binding proteins, the classic enzyme calcineurin A (CNA; protein phosphatase 2B) and the nuclear protein nucleomorphin (NumA), which is a regulator of nuclear number, have been particularly well studied. Research on the role of calmodulin in the function and regulation of the various myosins of Dictyostelium, especially during motility and chemotaxis, suggests that this is an area in which future active study would be particularly valuable. A general, hypothetical model for the role of calmodulin in myosin regulation is proposed.
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PMID:Calmodulin-binding proteins in the model organism Dictyostelium: a complete & critical review. 1789 9

The protein serine/threonine phosphatase calcineurin (CN) is activated by calmodulin (CaM) in response to intracellular calcium mobilization. A widely accepted model for CN activation involves displacement of the CN autoinhibitory peptide (CN(467-486)) from the active site upon binding of CaM. However, CN activation requires calcium binding both to the low affinity sites of CNB and to CaM, and previous studies did not dissect the individual contributions of CNB and CaM to displacement of the autoinhibitory peptide from the active site. In this work we have produced separate CN fragments corresponding to the CNA regulatory region (CNRR(381-521), residues 381-521), the CNA catalytic domain truncated at residue 341, and the CNA-CNB heterodimer with CNA truncated at residue 380 immediately after the CNB binding helix. We show that the separately expressed regulatory region retains its ability to inhibit CN phosphatase activity of the truncated CN341 and CN380 and that the inhibition can be reversed by calcium/CaM binding. Tryptophan fluorescence quenching measurements further indicate that the isolated regulatory region inhibits CN activity by occluding the catalytic site and that CaM binding exposes the catalytic site. The results provide new support for a model in which calcium binding to CNB enables CaM binding to the CNA regulatory region, and CaM binding then instructs an activating conformational change of the regulatory region that does not depend further on CNB. Moreover, the secondary structural content of the CNRR(381-521) was tentatively addressed by Fourier transform infrared spectroscopy. The results indicate that the secondary structure of CNRR(381-521) fragment is predominantly random coil, but with significant amount of beta-strand and alpha-helix structures.
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PMID:The secondary structure of calcineurin regulatory region and conformational change induced by calcium/calmodulin binding. 1829 42

Calcineurin (CN), the Ca(2+)/calmodulin (CaM)-dependant protein phosphatase, is the target for immunosuppressive drugs cyclosporine A (CsA) and FK506. These immunosuppressants can inhibit CN activity after binding with respective immunophilins. Based on the model of screening by using p-nitrophenyl phosphate as a substrate for preliminary screening and (32)P-labeled 19-residue phosphopeptide as a specific substrate for final determination, we found Kaempferol, a natural flavonol, could inhibit CN activity in purified enzyme and Jurkat T-cells. Unlike CsA and FK506, CN inhibition by kaempferol is independent of matchmaker protein and the inhibitory manner is noncompetitive. Through investigation of inhibitions for CNA and a series of its truncated mutants, we suggested that Kaempferol could directly act on the catalytic domain. Data also indicated that the CN inhibition by kaempferol could be enhanced when the enzyme was activated in the presence of CaM and CNB. CNB is necessary for mediating inhibition of enzyme by kaempferol. The result of RT-PCR also indicated that kaempferol had an inhibitory activity against IL-2 gene expression in activated Jurkat cells. All data suggested that kaempferol could be a new immunosuppressant of CN.
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PMID:Kaempferol: a new immunosuppressant of calcineurin. 1850 53

The protein phosphatase 1 catalytic subunit (PP1c) and the protein phosphatase 2B (PP2B or calcineurin) catalytic subunit (CNA) contain nonconserved N-terminal regions followed by conserved phosphatase cores. To examine the role of the N-termini of these two phosphatases, we substituted the residues 1-8 of PP1c with residues 1-42 of CNA, which is designated CNA(1-42)-PP1(9-330). The activities of CNA(1-42)-PP1(9-330) were similar to those of PP2B and different from those of PP1. The chimera was at least fourfold less sensitive to inhibition by okadaic acid, but was stimulated by nickel ions and chlorogenic acid, characteristics of PP2B not of PP1. These observations suggest that the N-terminus of CNA shifts the properties of PP1 toward those of PP2B. Our findings provide evidence that the nonconserved N-terminus of PP2B not only functions as important regulatory domain but also confers itself particular characteristics. This region may be targeted for regulation of PP2B activities in vivo.
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PMID:The nonconserved N-terminus of protein phosphatase 2B confers its properties to protein phosphatase 1. 1892 49

Many types of serine/threonine protein phosphatase have been cloned and characterized in plants, such as Type-1 serine/threonine protein phosphatase (PP1), Type-2A serine/threonine protein phosphatase (PP2A), Type-2C serine/threonine protein phosphatase (PP2C). However no Type-2B serine/threonine protein phosphatase (PP2B, calcineurin), or calcineurin A subunit-like protein (CaNAL), has been identified. We detected protein phosphatase activity in mixtures of CaM-binding proteins from three plants (Nicotiana tabacum, Brassica oleracea and Arabidopsis thaliana). Two-dimensional electrophoresis (2-D) and Western blot analysis with an anti-rat CNA antibody revealed a small protein of 60 kDa that we believe is a CaNAL. The isoelectric point (pI) of this protein in N. tabacum was approximately 5.69. The protein phosphatase activity in the mixture of CaM-binding proteins from N. tabacum was regulated by Ca(2+) and Calmodulin (CaM) with either RII peptides or pNPP as substrate. The immunosuppressive drugs, CsA and FK506, also inhibited the protein phosphatase activity significantly.
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PMID:The primary identification of a calcineurin A subunit-like protein in plants. 1914 13

The protein phosphatase 1 has conserved cores in PPP gene family flanked by non-conserved N-terminal domains. PP1 with residues 1-8 deleted or substituted by residues 1-42 of calcineurin catalytic subunit were designated PP1-(9-330) and CNA(1-42)-PP1(9-330), respectively. When compared with PP1, PP1-(9-330) had higher and CNA(1-42)-PP1(9-330) had lower activity with three kinds of substrates; PP1-(9-330) has higher and CNA(1-42)-PP1(9-330) has lower sensitivity to okadaic acid. These results imply that the N-terminal residues influence the activity and sensitivity to inhibitors of PP1. PP1-(9-330), PP1, and CNA(1-42)-PP1(9-330) displayed increasing K (m) and decreasing V (max) with three kinds of substrates, which suggest that the N-terminal residues are connected with the substrates affinity and catalytic efficiency of PP1. PP1-(9-330) has higher and CNA(1-42)-PP1(9-330) has lower fluorescence intensity than PP1, and the emission wavelength maximum was blue-shifted from PP1 to PP1-(9-330) and red-shifted from PP1 to CNA(1-42)-PP1(9-330). Our findings provide evidence that the N-terminal domain is an important region influencing the structure and properties of PP1.
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PMID:The N-terminal domain influences the structure and property of protein phosphatase 1. 1924 55

Calcineurin subunit B (CNB), a regulatory subunit of calcineurin, is a member of EF-hand calcium (Ca(2+))-binding protein superfamily. In this study, we performed phosphatase activity, pull-down, and circular dichroism analyses, which shows Ca(2+)-free CNB (apo-CNB) is able to bind CNA (Calcineurin subunit A) and regulate its phosphatase activity, albeit to a lesser extent than Ca(2+)-saturated CNB (holo-CNB). This is supported by subsequent molecular dynamics (MD) simulations which aimed to examine calcium-induced conformational changes of CNB, using both holo-CNB and apo-CNB. Based on the trajectories of MD, we found that there were no drastic changes between holo-CNB and apo-CNB. The modest differences seen are mainly reflected in the interhelical angles of the four EF-hand motifs and solvent accessible surface area of the hydrophobic groove which is responsible for CNA binding. Interestingly, this hydrophobic groove was largely retained in both holo-CNB and apo-CNB. Comparative analyses show that the calcium-induced conformational change in CNB is remarkably different from that of the two prototypical EF-hand calcium-binding proteins, namely, calmodulin and recoverin. This is the first time that CNB is shown to activate CNA in the absence of Ca(2+).
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PMID:Calcineurin regulatory subunit B is a unique calcium sensor that regulates calcineurin in both calcium-dependent and calcium-independent manner. 1953 97

A Setosphaeria turcica gene encoding the catalytic subunit of calcineurin was cloned using degenerated primers corresponding to conserved domains of Ser/Thr protein phosphatases and its complete cDNA (GenBank accession No. EF 407562) was obtained with RACE method. It's validated single copied model by southern hybridization. Furthermore, the CNA inhibitor Cyclosporin A (CsA) exhibited potent antifungal activity against conidial germination and appressorium formation of S. turcica. The inhibition ratio was positively correlated to CsA concentration. However, appressorium formation was more sensitive than conidium germination to the inhibitor at the same concentration. It was suggested that CNA might play an important role in the pathogenicity of S. turcica.
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PMID:[Molecular cloning and characterization of calcineurin A in Setosphaeria turcica]. 1984 Sep 29


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