Good fungi gone bad: the corruption of calcineurin

Calcineurin is a Ca(2+)/calmodulin-activated protein phosphatase that is conserved in eukaryotes, from yeast to humans, and is the conserved target of the immunosuppressive drugs cyclosporin A (CsA) and FK506. Genetic studies in yeast and fungi established the molecular basis of calcineurin inhibition by the cyclophilin A-CsA and FKBP12-FK506 complexes. Calcineurin also functions in fungi to control a myriad of physiological processes including cell cycle progression, cation homeostasis, and morphogenesis. Recent investigations into the molecular mechanisms of pathogenesis in Candida albicans and Cryptococcus neoformans, two fungi that cause life-threatening infections in humans, have revealed an essential role for calcineurin in morphogenesis, virulence, and antifungal drug action. Novel non-immunosuppressive analogs of the calcineurin inhibitors CsA and FK506 that retain antifungal activity have been identified and hold promise as candidate antifungal drugs. In addition, comparisons of calcineurin function in both fungi and humans may identify fungal-specific components of calcineurin-signaling pathways that could be targeted for therapy, as well as conserved elements of calcium signaling events.     

Calcineurin and intracellular Ca2+-release channels: regulation or association?

The Ca(2+)- and calmodulin-dependent phosphatase calcineurin was reported to interact with the inositol 1,4,5-trisphosphate receptor (IP(3)R) and the ryanodine receptor (RyR) and to modulate their phosphorylation status and activity. However, controversial data on the molecular mechanisms involved and on the functional relevance of calcineurin for these channel-complexes have been described. Hence, we will focus on the functional importance of calcineurin for IP(3)R and RyR function and on the different mechanisms by which Ca(2+)-dependent dephosphorylation can affect the gating of those intracellular Ca(2+)-release channels. Since many studies made use of immunosuppressive drugs that are inhibiting calcineurin activity, we will also have to take the different side effects of these drugs into account for the proper interpretation of the effects of calcineurin on intracellular Ca(2+)-release channels. In addition, it became recently known that various other phosphatases and kinases can associate with these channels, thereby forming macromolecular complexes. The relevance of these enzymes for IP(3)R and RyR functioning will be reviewed since in some cases they could interfere with the effects ascribed to calcineurin. Finally, we will discuss the downstream effects of calcineurin on the regulation of the expression levels of intracellular Ca(2+)-release channels as well as the relation between IP(3)R- and RyR-mediated Ca(2+) release and calcineurin-dependent gene expression.     

Effect of calcineurin inhibitors on myotoxic activity of crotoxin and Bothrops asper phospholipase A2 myotoxins in vivo and in vitro

Previous studies have shown that calcineurin activity plays a critical role in the myotoxic activity induced by crotoxin (CTX), a group II phospholipase A(2) (PLA(2)) with neurotoxic and myotoxic actions. In order to address whether calcineurin is also important for the activity of non-neurotoxic group II PLA(2) myotoxins we have compared the effects of calcineurin inhibition on the myotoxic capacity of CTX and the non-neurotoxic PLA(2)s, myotoxin II (Mt II) and myotoxin III (Mt III) from Bothrops asper venom. Rats were treated with cyclosporin A (CsA) or FK506, calcineurin inhibitors, and received an intramuscular injection of either CTX, Mt II or Mt III into the tibialis anterior. Animals were killed 24 h after injection of toxins. Tibialis anterior was removed and stored in liquid nitrogen. Myofibers in culture were also treated with CsA or FK506 and exposed to CTX, Mt II and Mt III. It was observed that, in contrast to CTX, CsA and FK506 do not attenuate myotoxic effects induced by both Mt II and Mt III in vivo and in vitro. The results of the present study suggest that calcineurin is not essential for the myotoxic activity of Mt II and Mt III, indicating that distinct intracellular pathways might be involved in myonecrosis induced by neurotoxic CTX and non-neurotoxic Bothrops sp. PLA(2) myotoxins. Alternatively, calcineurin dependent fast fiber type shift might render the muscle resistant to the action of CTX, without affecting its susceptibility to Bothrops sp. myotoxins.     

Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised patients. Its growth is sensitive to the immunosuppressants FK506 and cyclosporin, which inhibit the Ca2+- calmodulin-activated protein phosphatase calcineurin. Calcineurin is required for growth at 37 degrees C and virulence of C.neoformans. We found that calcineurin is also required for mating. FK506 blocks mating of C.neoformans via FKBP12-dependent inhibition of calcineurin, and mutants lacking calcineurin are bilaterally sterile. Calcineurin is not essential for the initial fusion event, but is required for hyphal elongation and survival of the heterokaryon produced by cell fusion. It is also required for hyphal elongation in diploid strains and during asexual haploid fruiting of MATalpha cells in response to nitrogen limitation. Because mating and haploid fruiting produce infectious basidiospores, our studies suggest a second link between calcineurin and virulence of C.neoformans. Calcine urin regulates filamentation and 37 degrees C growth via distinct pathways. Together with studies revealing that calcineurin mediates neurite extension and neutrophil migration in mammals, our findings indicate that calcineurin plays a conserved role in the control of cell morphology.     

Use of fluorinated tyrosine phosphates to probe the substrate specificity of the low molecular weight phosphatase activity of calcineurin

Calcineurin, a calmodulin-activated protein phosphatase, is known to dephosphorylate certain low molecular weight phosphate esters. The low molecular weight phosphatase activity of calcineurin has been studied by utilizing tyrosine phosphate derivatives. Kinetic studies suggest that the substrate specificity is dependent upon the electronic nature of the substrate in contrast to results obtained with alkaline phosphatase from Escherichia coli. Comparison of calcineurin and acid-catalyzed hydrolyses indicates a 1:1 correlation between the rate constants for the two processes. This correlation and other model studies have been utilized to provide insight into the chemical mechanism of calcineurin. Possible chemical mechanisms for calcineurin are discussed.     

Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans

Calcineurin is a Ca2+-calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37 degrees C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12-FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12-FK506-calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.     

Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae.

The PMC1 gene in Saccharomyces cerevisiae encodes a vacuolar Ca2+ ATPase required for growth in high-Ca2+ conditions. Previous work showed that Ca2+ tolerance can be restored to pmc1 mutants by inactivation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase sensitive to the immunosuppressive drug FK506. We now report that calcineurin decreases Ca2+ tolerance of pmc1 mutants by inhibiting the function of VCX1, which encodes a vacuolar H+/Ca2+ exchanger related to vertebrate Na+/Ca2+ exchangers. The contribution of VCX1 in Ca2+ tolerance is low in strains with a functional calcineurin and is high in strains which lack calcineurin activity. In contrast, the contribution of PMC1 to Ca2+ tolerance is augmented by calcineurin activation. Consistent with these positive and negative roles of calcineurin, expression of a vcx1::lacZ reporter was slightly diminished and a pmc1::lacZ reporter was induced up to 500-fold by processes dependent on calcineurin, calmodulin, and Ca2+. It is likely that calcineurin inhibits VCX1 function mainly by posttranslational mechanisms. Activities of VCX1 and PMC1 help to control cytosolic free Ca2+ concentrations because their function can decrease pmc1::lacZ induction by calcineurin. Additional studies with reporter genes and mutants indicate that PMR1 and PMR2A, encoding P-type ion pumps required for Mn2+ and Na+ tolerance, may also be induced physiologically in response to high-Mn2+ and -Na+ conditions through calcineurin-dependent mechanisms. In these situations, inhibition of VCX1 function may be important for the production of Ca2+ signals. We propose that elevated cytosolic free Ca2+ concentrations, calmodulin, and calcineurin regulate at least four ion transporters in S. cerevisiae in response to several environmental conditions.     

Coping with stress: calmodulin and calcineurin in model and pathogenic fungi

Calcium signaling via calmodulin and calcineurin is critical for the regulation of stress responses in fungi. The functions of calmodulin and calcineurin are largely conserved among pathogenic fungi and model fungi, however, the mechanisms of action have diverged. Saccharomyces cerevisiae is an excellent model for understanding the framework of calcium-mediated signal transduction pathways, and considerable progress has been made in understanding the details of how Ca(2+)-calmodulin and calcineurin control adaptation to environmental stress. Studies using the divergent human pathogenic fungi Candida albicans and Cryptococcus neoformans reveal that calcineurin is critical for virulence, yet it acts via distinct mechanisms in each fungus. These differences in function may reflect the requirements of each pathogen to survive inside the host, and illustrate that studies must be conducted in each organism in order to elucidate the details of the molecular mechanisms of calmodulin and calcineurin-mediated signaling pathways.     

Regulation of organelle transport in melanophores by calcineurin

Previous studies have shown that pigment granule dispersion and aggregation in melanophores of the African cichlid, Tilapia mossambica, are regulated by protein phosphorylation and dephosphorylation, respectively (Rozdzial, M. M., and L. T. Haimo. 1986. Cell. 47:1061- 1070). The present studies suggest that calcineurin, a Ca2+/calmodulin- stimulated phosphatase, is the endogenous phosphatase that mediates pigment aggregation in melanophores. Aggregation, but not dispersion, is inhibited by okadaic acid at concentrations consistent with an inhibition of calcineurin activity. Inhibition of aggregation in melanophores that have been BAPTA loaded or treated with calmodulin antagonists implicate Ca2+ and calmodulin, respectively, in this process. Moreover, addition of calcineurin rescues aggregation in lysed melanophores which are otherwise incapable of aggregating pigment. Immunoblotting with an anticalcineurin IgG reveals that calcineurin is a component of the dermis, which contains the melanophores, and indirect immunofluorescence localizes calcineurin specifically to the melanophores. Finally, this antibody, which inhibits calcineurin's phosphatase activity (Tash, J. S., M. Krinks, J. Patel, R. L. Means, C. B. Klee, and A. R. Means. 1988. J. Cell Biol. 106:1625-1633), inhibits aggregation but has no effect on pigment granule dispersion. Together these studies indicate that retrograde transport of pigment granules to the melanophore cell center depends upon the participation of calcineurin.     

Effects of sulfhydryl agents,trifluoperazine, phosphatase inhibitors and tryptic proteolysis on calcineurin isolated from bovine cerebral cortex

Summary Calcineurin was dicovered as an inhibitor of calmodulin stimulated cyclic AMP phosphodiesterase and its ability to act as a calmodulin binding protein largely explains its inhibitory action on calmodulin regulated enzymes. Recent studies establish calcineurin as the enzyme protein phosphatase whose activity is regulated by calmodulin and a variety of divalent metals. In this work, we have investigated the effects of several agents including sulfhydryl agents, trifluoperazine (a calmodulin antagonist), PP_i, NaF and orthovanadate and of tryptic proteolysis on the calcineurin inhibition of cyclic AMP phosphodiesterase (called inhibitory activity) and on protein phosphatase activity. Inhibitors for sulfhydryl groups (pHMB, NEM) inhibited phosphatase activity without any effect on the inhibitory activity. Dithioerythritol completely reversed the inhibition by pHMB. Limited proteolysis of calcineurin caused an activation of basal phosphatase activity with a complete loss of inhibitory activity. Phosphatase activity of the proteolyzed calcineurin was not stimulated by calmodulin. The presence of calmodulin along with calcineurin during tryptic digestion appeared to preserve the stimulation of phosphatase by Ca²⁺-calmodulin. [³H]-Trifluoperazine (TFP) was found to be incorporated irreversibly into calcineurin in the presence of ultraviolet light. This incorporation was evident into the A and B subunits of calcineurin. TFP-caused a decrease in the phosphatase activity and an increase in its inhibitory activity. [³H]-TFP incorporation into the A subunit was drastically decreased in the proteolyzed calcineurin. This was also true when the [³H]-TFP incorporated calcineurin was subjected to tryptic proteolysis. The incorporation into the B unit was essentially unaffected in the trypsinized calcineurin. Phosphatase activity was inhibited by orthovanadate, NaF, PP_i, and EDTA. Inhibitions by these compounds were more pronounced when the phosphatase was determined in the presence of Ca²⁺-cahnodulin than in their absence.     

A significant increase in both basal and maximal calcineurin activity in the rat pilocarpine model of status epilepticus

This study focused on the effects of status epilepticus on the activity of calcineurin, a neuronally enriched, calcium-dependent phosphatase. Calcineurin is an important modulator of many neuronal processes, including learning and memory, induction of apoptosis, receptor function and neuronal excitability. Therefore, a status epilepticus-induced alteration of the activity of this important phosphatase would have significant physiological implications. Status epilepticus was induced by pilocarpine injection and allowed to continue for 60 min. Brain region homogenates were then assayed for calcineurin activity by dephosphorylation of p-nitrophenol phosphate. A significant status epilepticus-dependent increase in both basal and Mn(2+)-dependent calcineurin activity was observed in homogenates isolated from the cortex and hippocampus, but not the cerebellum. This increase was resistant to 150 nM okadaic acid, but sensitive to 50 microM okadaic acid. The increase in basal activity was also resistant to 100 microM sodium orthovanadate. Both maximal dephosphorylation rate and substrate affinity were increased following status epilepticus. However, the increase in calcineurin activity was not found to be due to an increase in calcineurin enzyme levels. Finally, increase in calcineurin activity was found to be NMDA-receptor activation dependent. The data demonstrate that status epilepticus resulted in a significant increase in both basal and maximal calcineurin activity.     

A safety assessment of topical calcineurin inhibitors in the treatment of atopic dermatitis

Atopic dermatitis (AD) is a common childhood disease that can disrupt the lives of patients and their families and, in turn, affect their quality of life. The goal of treatment is the long-term control of AD by minimizing the frequency and severity of flares. Topical corticosteroids of various potencies have been the mainstay of pharmacologic treatment of AD flares. In the past few years, the introduction of topical calcineurin inhibitors (TCIs) has provided physicians with an effective, well-tolerated alternative to topical corticosteroids. In January 2006, a boxed warning and a patient medication guide were added to TCI product labels in the United States after the US Food and Drug Administration raised concerns about their safety. These concerns were based on rare cases of skin malignancy and lymphoma, and a theoretical risk stemming from the systemic use of calcineurin inhibitors in animal studies and transplant patients. However, the boxed warning states that no causal link has been established between TCI use and malignancy. Pharmacokinetic studies have also shown that treatment with TCIs leads to only minimal systemic absorption. In addition, controlled, blinded studies have found no evidence of systemic immunosuppression and no causal relationship between the use of TCIs and the occurrence of lymphoma or other malignancies. Overall, TCIs have been shown to be an effective and valuable treatment option for AD.