Calcineurin activation

Tacrolimus (TRL), in the past also named FK506, belongs to the group of macrolides and is produced by a special actinomycete species. TRL binds to its cytosolic receptor FKBP-12, however, it also blocks calcineurin activity and subsequently cytokine synthesis. 

Addition of the L-732,531 FKBP binary complex to a calcineurin activity assay resulted in increasingly nonlinear progress curves with increasing binary complex concentration. The htting of the data to Equation (6.3) revealed an inhibitor concentration effect on v-, as well as on vs and obs, consistent with a two-step mechanism of inhibition as in scheme C of Figure 6.3. Salowe and Hermes analyzed the concentration-response effects of the binary complex on v, and determined an IC50 of 0.90 pM that, after correction for I.S I/A (assuming competitive inhibition), yielded a A) value for the inhibitor encounter complex of 625 nM. 

Newman, R. H. and Zhang, J. (2008). Visualization of phosphatase activity in living cells with a FRET-based calcineurin activity sensor. Mol. Biosyst. 4, 496-501. 

Luo, ]., ]. H. Yin, and Q. Wei. The effect of calcineurin activator, extracted from Chinese herbal medicine, on memory and immunity in mice. Pharmacol Biochem Behav... 

Fig. 4.1 Mechanism of action of cyclosporine. Cyclosporine readily diffuses into the cytoplasm of the target cells where it binds to cyclophilins. The cyclosporine-cyclophilin complex stably associates with calcineurin and inhibits calcineurin activity. Calcineurin is a Ca2+-dependent enzyme— serine/threonine phosphatase— which after activation by Ca2+, dephosphorylates a cytosolic component of NFAT (NFATc, cytosolic factor of activated T cells). After dephosphorylation, NFATc migrates from the cytoplasm to the nucleus where it associates with NFATn and induces transcription of several cytokine genes including IL-2. Cyclosporine inhibits calcineurin activity after associating with cyclophilins, resulting in the inhibition of IL-2 production and other cytokines (see Color Insert)...

Sirolimus binds to the cytosolic protein FK-binding protein R (FKBP-12) but does not block calcineurin activity. It does not bind to cyclophilins, which are cytosolic receptors for cyclosporine. Unlike cyclosporine and tacrolimus, sirolimus does not inhibit the activation of NFAT responsive genes. After binding to its cytosolic receptors, sirolimus inhibits a protein kinase, the mammalian target of rapamycin (mTOR) pathway, via suppression of PP2-A. When mTOR is inhibited, the cells will not proceed to the S phase, and the cell cycle will be blocked (Fig. 4.3). As a result, sirolimus blocks T-cell proliferation but its effects are downstream of the IL-2 receptors. IL-2 binding to its receptors activates intracellular protein kinases that in turn activate gene transcription and T-cell proliferation. 

Fig. 4.3 Mechanism of action of sirolimus. Sirolimus readily diffuses into the cytoplasm of the target cells where it binds to immunophilins (FK506-BP). The sirolimus-immunophilin complex does not inhibit calcineurin activity instead it binds to the mTOR. The sirolimus-immunophilin-mTOR complex stops the cell cycle progression from G1 to S phase. The targets of sirolimus include the eukaryotic initiation factor (eIF-4F), 70-kDa S6 protein kinase (p70S6 K) and several cyclin-dependent kinases (cdk). As a consequence, it blocks downstream signaling pathway initiated after activation of IL-2 receptors, resulting in blockage of T-cell proliferation (see Color Insert)...

Shi, J., Townsend, M., and Constantine-Paton, M. (2000). Activity-dependent induction of tonic calcineurin activity mediates a rapid developmental downregulation of NMDA receptor currents. 

With the identification of calcineurin-mediated NF-ATc translocation in T cells, a model of the cytosolic events involved in T-cell activation has emerged, as oudined in Fig. 4. Stimulation of the TCR results in a series of membrane-associated events culminating in a rise in intracellular Ca2+, which binds to and activates calmodulin/calcineurin. Activated calcineurin then causes the cytoplasmic-to-nuclear translocation of NF-ATc, an event potentially mediated via the dephosphorylation of critical serine and/or threonine residues on the NF-ATc protein (Beals et al., 1997a, 1997b). Once nuclear, NF-ATc binds DNA with its nuclear partner, NF-ATn to drive production of IL-2 and other early genes, leading to lymphocyte activation and proliferation. 

Wang, J. H., and Kelly, P. T. (1997). Postsynaptic calcineurin activity downregulates synaptic transmission by weakening intracellular Ca2+ signaling mechanisms in hippocampal CA1 neurons./. Neurosd. 17, 4600-4611. 

I. 2000. p-adrenergic pathway induces apoptosis through calcineurin activation in cardiac myocytes. J. Biol. Chem. 275 34528-34533. 

Calcineurin activity was measured at 30°C in a total volume of 150 fiL containing 50 mM Tris-HCl (pH 7.0), 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.01% Brij, 0.3 mg/mL bovine serum albumin 0.1 yiM calmodulin, 1 mM MnCl2, 1 mM CaCl2, and variable amounts of calcineurin (0.11-0.44 /u.g/150 /u.L). Aliquots (20 /u.L) were taken at intervals and the reaction was stopped with 70 fiL of 0.5% perchloric acid containing 0.1% Triton X-100 and the internal standard, probenecid (10 nmol/mL). After centrifugation, a 40 fiL aliquot of the supemate was directly applied on the HPLC column. The rate of enzyme activity was calculated from the linear appearance of product over the first 6 minutes. 

Sirohmus is a macrocychc lactone produced by the bacteria Streptomyces hygroscopious. Like the calcineurin inhibitors cyclosporine and tacrolimus its mechanism of action involves formation of a complex with an immunophiUn, in this case, FKBP-12. Unlike cyclosporine and tacrolimus, sirohmus does not affect calcineurin activity but binds to and inhibits the mammalian kinase, target of rapamycin (mTOR.). mTOR is a key enzyme in cell-cycle progression. When inhibited this kinase blocks cell cycle progression at the G1 to S phase transition (Dumont and Su, 1996 Sehgal, 2003). 

Figure 3. Model for the Activity of Coronin 1 in Macrophages. In resting macrophages, coronin 1 (coronin 1 A) is distributed between the cytoplasm as well as the cell cortex. Upon the entry of pathogenic mycobacteria, coronin 1 is recruited and actively retained at the phagosomal membrane, thereby ensuring the activation of calcineurin. Activation of calcineurin results in a block in the fusion of mycobacterial phagosomes with lysosomes. As a consequence, deletion of coronin 1 or inhibiting calcineurin activity results in the induction of phagosome lysosome fusion and mycobacterial killing.

Sirolimus is a macrocyclic lactone immunosuppressant that has anti-rejection activity through inhibition of T cell activation. In contrast to tacrohmus and ciclosporin, sirolimus has no effect on calcineurin activity. 

Tacrolimus capacity to blockade NO pathway is weU demonstrated. NO plays a major role in the pathogenesis of cerebral hypoxia-ischemia injury mediated by glutamate/N-methyl-D-aspartate (NDMA). This injury depends on intracellular calcium influx through NDMA receptor channels, which activate calcineurin with consequent dephosphorylation of constitutive NO synthase (cNOS). Tacrohmus addition to cultured neuronal cells reduced NDMA-mediated toxicity, through the inhibition of calcineurin activation, inhibition of cNOS dephosphorylation and consequent decrease in... 

Dendritic spine alteration is mediated by calcineurin activation, a calcium-dependent phosphatase involved in synapse signaling. Acute treatment of young and plaque-free transgenic mice with the calcineurin inhibitor FK506 leads to a complete rescue of longterm depression and postsynaptic density composition [550],... 

T-cell activation is caused by interactions between T-cell receptors, the MHC, cellular adhesion molecues, and costimulatory molecules. Among the series of events is calcineurin activation, which ultimately promotes interleukin 2 (IL-2) proliferation. After initial T-cell activation, the process of clonal expansion and immunologic progression is mediated by cytokines. IL-2 is released from T cells and activates T-lymphocytes locally and in other regions of the... 

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