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)

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), inhibiting the function of the eIF2 complex and continued initiation of translation. When bound to an activating RNA and ATP, PKR undergoes autophosphorylation reactions at multiple serine and threonine residues. This autophosphorylation reaction stimulates the eIF2alpha kinase activity of PKR. The binding of certain viral RNAs inhibits the activation of PKR. Wild-type PKR is obtained as a highly phosphorylated protein when overexpressed in Escherichia coli. We report here that treatment of the isolated phosphoprotein with the catalytic subunit of protein phosphatase 1 dephosphorylates the enzyme. The in vitro autophosphorylation and eIF2alpha kinase activities of the dephosphorylated enzyme are stimulated by addition of RNA. Thus, inactivation by phosphatase treatment of autophosphorylated PKR obtained from overexpression in bacteria generates PKR in a form suitable for in vitro analysis of the RNA-induced activation mechanism. Furthermore, we used gel mobility shift assays, methidiumpropyl-EDTA.Fe footprinting and affinity chromatography to demonstrate differences in the RNA-binding properties of phospho- and dephosphoPKR. We found that dephosphorylation of PKR increases binding affinity of the enzyme for both kinase activating and inhibiting RNAs. These results are consistent with an activation mechanism that includes release of the activating RNA upon autophosphorylation of PKR prior to phosphorylation of eIF2alpha.
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PMID:Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity. 1145 27

Herpes simplex viruses (HSV) are resistant to the antiviral action of interferon. However, the underlying mechanisms are not well understood. In this report, we show that unlike that of wild-type HSV-1, replication of the gamma 1 34.5 null mutants was significantly inhibited by exogenous interferon-alpha in cells devoid of interferon-alpha/beta genes. Using a series of gamma 1 34.5 deletion mutants, the domain required for interferon resistance was mapped to the region containing amino acids 146 to 263 in the gamma 1 34.5 protein. Interestingly, Val193 Glu and Phe195 Leu substitutions in the protein phosphatase 1 interacting motif of the gamma 1 34.5 protein rendered HSV-1 sensitive to interferon-alpha. Furthermore, gamma 1 34.5 null mutants were sensitive to interferon-alpha/beta in PKR+/+ but not in PKR-/- mouse embryo fibroblasts. These findings provide evidence that the gamma 1 34.5 protein contributes to HSV-1 resistance to interferon-alpha/beta by inhibiting PKR function.
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PMID:Val193 and Phe195 of the gamma 1 34.5 protein of herpes simplex virus 1 are required for viral resistance to interferon-alpha/beta. 1188 96

By virtue of its binding to cyclophilin, the cellular receptor for cyclosporine (CsA), we could identify a new compound D-43787 [N-[(1-tert-butyloxycarbonyl)-indolin-2-(S)-carbonyl]-indolin-2-(S)-carbonacid-[N-epsilon-benzyloxycarbonyl)-2-(S)-lysin methylester]-amide] exhibiting immunomodulating properties. It inhibited cell proliferation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA)/ionomycin and anti-CD3/CD28 with an IC(50) of 0.3 microM. The protein phosphatase calcineurin, which is the target of the CsA-cyclophilin complex, is not inhibited by D-43787. It inhibited T helper cell (Th) 2 cytokines interleukin (IL)-4, -5, and -13 more effectively than the Th1 cytokine interferon (IFN)-gamma in human primary T cells. The IC(50) for IL-5 and IL-13 in TPA/ionomycin-stimulated peripheral blood mononuclear cells (PBMC) is 0.7 +/- 0.1 and 0.5 +/- 0.1 microM, respectively, whereas the IC(50) for IFN-gamma is 2.0 +/- 0.4 microM. When PBMC were stimulated with anti-CD3/CD28, the IC(50) for IL-4, -5, and -13 were 1.5 +/- 0.2, 1.8 +/- 0.2, and 1.9 +/- 0.4 microM, respectively. IFN-gamma was only partially inhibited under these conditions. This effect was even more pronounced in pure CD4(+) T cells. Pretreatment of human monocytes with D-43787 inhibited lipopolysaccharide-induced proinflammatory cytokines IL-6 and TNFalpha with an IC(50) of 1.2 +/- 0.1 and 4.7 +/- 0.9 microM, respectively. In vivo, D-43787 potently inhibited late-phase eosinophilia in actively sensitized and challenged guinea pigs (10 mg/kg, i.p.: 51%) and Brown-Norway rats (1 mg/kg, intrapulmonary: 66% 30 mg/kg, i.p.: 50%). In adjuvant-induced arthritis, D-43787 (10-40 mg/kg, b.i.d., i.p.) dose dependently reduced edema development on both hind paws. The potency of D-43787 was comparable with that of indomethacin and dexamethasone. In conclusion, we characterized a novel Th2 selective immunosuppressive drug with possible anti-asthmatic/anti-inflammatory effects. Its mode of action is distinct from that of CsA.
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PMID:Anti-inflammatory effects of a cyclosporine receptor-binding compound, D-43787. 1196 Oct 80

The PKR protein kinase is among the best-studied effectors of the host interferon (IFN)-induced antiviral and antiproliferative response system. In response to stress signals, including virus infection, the normally latent PKR becomes activated through autophosphorylation and dimerization and phosphorylates the eIF2alpha translation initiation factor subunit, leading to an inhibition of mRNA translation initiation. While numerous virally encoded or modulated proteins that bind and inhibit PKR during virus infection have been studied, little is known about the cellular proteins that counteract PKR activity in uninfected cells. Overexpression of PKR in yeast also leads to an inhibition of eIF2alpha-dependent protein synthesis, resulting in severe growth suppression. Screening of a human cDNA library for clones capable of counteracting the PKR-mediated growth defect in yeast led to the identification of the catalytic subunit (PP1(C)) of protein phosphatase 1alpha. PP1(C) reduced double-stranded RNA-mediated auto-activation of PKR and inhibited PKR transphosphorylation activities. A specific and direct interaction between PP1(C) and PKR was detected, with PP1(C) binding to the N-terminal regulatory region regardless of the double-stranded RNA-binding activity of PKR. Importantly, a consensus motif shared by many PP1(C)-interacting proteins was necessary for PKR binding to PP1(C). The PKR-interactive site was mapped to a C-terminal non-catalytic region that is conserved in the PP1(C)2 isoform. Indeed, co-expression of PP1(C) or PP1(C)2 inhibited PKR dimer formation in Escherichia coli. Interestingly, co-expression of a PP1(C) mutant lacking the catalytic domain, despite retaining its ability to bind PKR, did not prevent PKR dimerization. Our findings suggest that PP1(C) modulates PKR activity via protein dephosphorylation and subsequent disruption of PKR dimers.
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PMID:The direct binding of the catalytic subunit of protein phosphatase 1 to the PKR protein kinase is necessary but not sufficient for inactivation and disruption of enzyme dimer formation. 1213 6

The gamma(1)34.5 protein of herpes simplex virus 1 (HSV-1) is a virus-encoded protein phosphatase 1 (PP1) regulatory protein that contributes to viral resistance to interferon. It functions to block the shutoff of protein synthesis mediated by the double-stranded RNA-dependent protein kinase. This requires the carboxyl terminus of the gamma(1)34.5 protein to recruit PP1, forming a high-molecular-weight complex that dephosphorylates the alpha subunit of translation initiation factor eIF-2 (eIF-2alpha). In the present study, we introduced a series of point mutations into a region in the effector domain of the gamma(1)34.5 protein, which is adjacent to the PP1-binding domain. Analysis of these mutants in virus-infected cells shows that Ser209Ala, Ser209Asp, Ser218Ala, or Trp219Tyr substitution does not affect viral response to interferon-alpha. In contrast, Arg215Leu or Ser218Asp substitution rendered the virus hypersensitive to interferon-alpha, which correlates with the inability of these gamma(1)34.5 mutants to mediate dephosphorylation of eIF-2alpha. However, Arg215Leu or Ser218Asp substitution does not disrupt the formation of a high-molecular-weight complex required for eIF-2alpha dephosphorylation or binding of the gamma(1)34.5 protein to PP1. These results suggest that concerted action of the PP1-binding domain and the effector domain of the gamma(1)34.5 protein is required to confer HSV-1 interferon resistance.
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PMID:Amino acid substitutions in the effector domain of the gamma(1)34.5 protein of herpes simplex virus 1 have differential effects on viral response to interferon-alpha. 1266 99

There is always a second infection with HCV during the post-transplant period. Recurrence of the disease is one of the main present therapeutic challenges because there are still many questions to answer. Some transplant groups suggest that recurrence is earlier in certain circumstances, especially in face of repetitive episodes of acute rejection in the early post-transplant period. Different transplant centers have various approaches to minimize the risk of recurrence secondary to excessive immunosuppression. They go from absolute avoidance of steroids to single therapy with inhibitors of calcineurin. Some investigators advocate for a fast reduction in the dose of corticosteroids (in less than 3 months), elimination of steroids from the therapy or administration of a single steroid dose in the immediate post-transplant period. Some are trying to establish a regime based on a single inhibitor of calcineurin and supported with mofetil micofenolate if it's effective. There are other inhibitors being studied, like anti-CD25 antibodies. The goals of the treatment for HCV recurrence must be aimed at obtaining an effective therapy that may be able to prevent the damage and to inhibit the viral replication and subsequent inflammation on long term basis. Recently there have been reports about different schemes with IFN and ribavirin, either as single therapy or in combination, that showed modest advances in early viral elimination and delaying the recurrence of the disease. One of the risks of therapy with interferon that has immunomodulation properties, is organ rejection. Ribavirin administration as single therapy is contraindicated. There are still some unanswered questions, since nobody knows for sure how long therapy for HCV should be continued.
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PMID:[Post-transplant hepatitis C. Immunosuppression and antiviral treatment]. 1271 58

Interferon-alpha (IFN-alpha)-2b is known to have antiproliferative effects on hematological malignant cells, especially chronic myelogenous leukaemia (CML). However, it can induce cytogenetical remissions in a very small percentage of the patients. Also during interferon therapy, resistance can emerge in the CML clones. K562 is an in vitro model cell line transformed from a Ph positive CML patient. It can be induced to differentiate to granulocytic and/or monocytic lineages with certain molecules. IFN-alpha-2b generally exerts its effects on CML cells by Janus family kinases (Jak/Stat) pathway, mostly through tyrosine kinase system. However, there is almost no data on the relevance of serine/threonine (Ser/Thr) protein phosphatase (PP) system in the interferon induced signal transduction pathways. In this study, we investigated serine/threonine protein phosphatases in the IFN-alpha-2b induced K562 cytotoxicity. Trypan blue dye exclusion test and MTT assay were utilised for determining cytotoxicity. IC(50) of IFN-alpha-2b on K562 cells was found to be 600IU/ml. However, no differentiation was determined by analysis of cell surface antigen expressions. Serine/threonine protein phosphatase inhibitors calyculin A (Cal A) and okadaic acid (OKA) augmented the IFN-alpha-2b induced cytotoxicity. Apoptosis assay by the mono-oligonucleosome detection and acridine orange/propidium iodide dye revealed marked apoptosis underlying cytotoxicity. Phosphatase enzyme assay revealed a gradual increase in protein phosphatase 2A (PP2A) activity during interferon induced cytotoxicity. Conversely, immunoblots showed no change in the expression of PP2A catalytic and regulatory subunits. In conclusion, PP2A plays a role in IFN-alpha-2b induced apoptosis of K562 cells and should be investigated as a new window furthermore.
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PMID:Involvement of protein phosphatase 2A in interferon-alpha-2b-induced apoptosis in K562 human chronic myelogenous leukaemia cells. 1280 29

Graft rejections as well as tolerance are true representation of the specificity, sophistication and redundancy of an elegantly and meticulously designed immune system. Tolerance is in a way similar to the process of self-recognition where lymphoid clones, during development, baring self-reactive receptor are eliminated or rendered in active by "clonal deletion" leading to a state of accommodation and acceptance (anergic). On the other hand, both acute and chronic rejections are manifestation of the purpose of existence of the immune system, which is to defend the host against foreign invaders. Thus, in order to treat (control) graft rejection it is necessary to determine and understand the steps leading to recognition, stimulation, activation, and amplification of the immune system. The first step leading to the initiation of the immune system cascade is recognition. Which can either be direct where donor antigens of the major histocompatibility complex (MHC) expressed on the donor cells (passenger leukocytes) or tissues are recognised by the host immune system. The direct recognition pathway initiates acute graft rejection. Alternatively processed donor MHC peptides presented by the recipient antigen presenting cells (APC) initiate the indirect pathway of immune response, which is as important as the direct recognition especially in chronic rejection. Recognition is followed by the ligation of a series of adhesion molecules starting with an antigen to its specific T-cell receptor (TCR)/cluster of differentiation (CD) complex, expressed on the surface of the T cell. In order for the activation to precede additional costimulatory signals, such as ligation of the CD28/B7, CD4/HLA class II and CD/HLA class I antigens are required. The activation process is accompanied by an increase of cytokines production such as interleukin (IL)-2, IL-12, interferon (INF) and tumour necrosis factor (TNF) by the primed T cell. The complexity and the polymorphic nature of the immune system have necessitated designing agents that inhibit the immune system at different levels. Cyclosporine and Tacrolimus, collectively known as calcineurin inhibitors, seems to act on the IL-2 by inhibiting its production thus leading to a decrease in the proliferation of the activated lymphocyte. Rapamycin, which is similar to Tacrolimus, inhibits graft rejection by blocking IL-2 activation and phosphorylation of 70 S6 kinase thus inhibiting the progression of T-cell from G to S phase. While Cellcept (MMF) reduce the proliferation of T cell by inhibiting purine synthesis and by its action on ionosine monophosphate dehydrogenase. Anti-lymphocyte antibodies (ATG) deplete circulating lymphocytes while selective monoclonal antibodies are directed against IL-2 receptor thus reducing the rate of proliferation of activated T cells. Recently, antibodies to the CD40/CD40 ligand have been shown to induce long-term graft survival with the inhibition of the Th1 cytokines (INF), IL-2 and IL-12 and upregulating the Th2 cytokines IL-4 and IL-10. Lastly graft rejection can be reduced by blockade of the B7/CD28 costimulation pathway with the fusion protein CTLA-4Ig. With the availability of such potent and diverse agents it is now possible to develop multi drug regiments that can depress the immune system at the different steps of the activation cascade, with minimal side effects, thus improving graft and patient survival rates.
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PMID:The mosaic of immunosuppressive drugs. 1283 79

The gamma(1)34.5 protein of herpes simplex virus type 1 (HSV-1) functions to block the shutoff of protein synthesis involving double-stranded RNA-dependent protein kinase (PKR). In this process, the gamma(1)34.5 protein recruits cellular protein phosphatase 1 (PP1) to form a high-molecular-weight complex that dephosphorylates eIF-2alpha. Here we show that the gamma(1)34.5 protein is capable of mediating eIF-2alpha dephosphorylation without any other viral proteins. While deletion of amino acids 1 to 52 from the gamma(1)34.5 protein has no effect on eIF-2alpha dephosphorylation, further truncations up to amino acid 146 dramatically reduce the activity of the gamma(1)34.5 protein. An additional truncation up to amino acid 188 is deleterious, indicating that the carboxyl-terminal domain alone is not functional. Like wild-type HSV-1, the gamma(1)34.5 mutant with a truncation of amino acids 1 to 52 is resistant to interferon, and resistance to interferon is coupled to eIF-2alpha dephosphorylation. Intriguingly, this mutant exhibits a similar growth defect seen for the gamma(1)34.5 null mutant in infected cells. Restoration of the wild-type gamma(1)34.5 gene in the recombinant completely reverses the phenotype. These results indicate that eIF-2alpha dephosphorylation mediated by the gamma(1)34.5 protein is required for HSV response to interferon but is not sufficient for viral replication. Additional functions or activities of the gamma(1)34.5 protein contribute to efficient viral infection.
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PMID:Dephosphorylation of eIF-2alpha mediated by the gamma(1)34.5 protein of herpes simplex virus type 1 is required for viral response to interferon but is not sufficient for efficient viral replication. 1294 28

Calmodulin (CaM) antagonists have been shown to inhibit tumor cell invasion and metastasis and to induce apoptosis in various tumor models, but the molecular mechanism of CaM antagonist-mediated apoptosis is poorly understood. Here, we demonstrate that interferon (IFN)-gamma induces susceptibility to CaM antagonist-mediated apoptosis in human cholangiocarcinoma cells weakly expressing Fas (Fas-low cells). During CaM antagonist-mediated apoptosis in IFN-gamma-pretreated Fas-low cells, cleavage of caspases-8, -9, and -3 and Bid, release of cytochrome c from the mitochondria and an increase in the free cytosolic calcium concentration were observed. CaM antagonists also caused depolarization of the mitochondrial membrane independent of caspase activation. Although a broad-range caspase inhibitor partially blocked CaM antagonist-mediated apoptosis, the neutralizing Fas antibody had no effect, suggesting that CaM antagonist-mediated apoptosis does not require interaction between CaM antagonists and surface Fas. CaM antagonists induce apoptosis via mechanisms other than inhibition of CaM-dependent protein kinase II and calcineurin, as their inhibitors, KN93 and cyclosporine A, had no effect on apoptosis. Taken together, these results indicate that CaM antagonists induce apoptosis in both caspase-dependent and -independent manners, and that susceptibility to CaM antagonists is modulated by IFN-gamma. The combination of IFN-gamma and CaM antagonists, including tamoxifen, may be a potential therapeutic modality for cholangiocarcinoma and possibly other malignancies.
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PMID:The combination of calmodulin antagonists and interferon-gamma induces apoptosis through caspase-dependent and -independent pathways in cholangiocarcinoma cells. 1457 4


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