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)

Here we report the recombinant expression of the catalytically active phosphatase domain of the Saccharomyces cerevisiae protein phosphatase 1 (Ppt1) in E. coli. Ppt1 consists of two domains: a 20 kDa TPR (tetratricopeptide repeat) domain, which mediates protein-protein interactions and directs Ppt1 to potential substrate proteins, e.g. the molecular chaperone Hsp90. The second, a 40 kDa phosphatase domain, exhibits catalytic activity and dephosphorylates phosphorylated serine/threonine residues of respective substrate proteins. The Ppt1 phosphatase domain was cloned and expressed in E. coli in unsoluble inclusion bodies. After isolating these, the aggregates were denatured with guanidinium hydrochloride and soluble protein was purified using affinity chromatography. Optimal renaturation conditions led to large amounts of the refolded phosphatase domain in high purity. Interestingly, further enzymatic studies revealed that the domain is not only correctly folded, but also shows higher catalytic activity compared to the full length protein.
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PMID:Expression, purification and refolding of the phosphatase domain of protein phosphatase 1 (Ppt1) from Saccharomyces cerevisiae. 1644 12

CHIP proteins are E3 ubiquitin ligases that promote degradation of Hsp70 and Hsp90 substrate proteins through the 26S proteasome in animal systems. A CHIP-like protein in Arabidopsis, AtCHIP, also has E3 ubiquitin ligase activity and has important roles to play under conditions of abiotic stress. In an effort to study the mode of action of AtCHIP in plant cells, proteins that physically interact with it were identified. Like its animal orthologs, AtCHIP interacts with a unique class of ubiquitin-conjugating enzymes (UBC or E2) that belongs to the stress-inducible UBC4/5 class in yeast. AtCHIP also interacts with other proteins, including an A subunit of protein phosphatase 2A (PP2A). This PP2A subunit appears to be a substrate of AtCHIP, because it can be ubiquitylated by AtCHIP in vitro and because the activity of PP2A is increased in AtCHIP-overexpressing plants in the dark or under low-temperature conditions. Unlike the rcn1 mutant, that has reduced PP2A activity due to a mutation in one of the A subunit genes of PP2A, AtCHIP-overexpressing plants are more sensitive to ABA treatment. Since PP2A was previously shown to be involved in low-temperature responses in plants, the low-temperature-sensitive phenotype observed in AtCHIP-overexpressing plants might be partly due to the change in PP2A activity. These data suggest that the E3 ubiquitin ligase AtCHIP may function upstream of PP2A in stress-responsive signal transduction pathways under conditions of low temperature or in the dark.
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PMID:AtCHIP functions as an E3 ubiquitin ligase of protein phosphatase 2A subunits and alters plant response to abscisic acid treatment. 1664 Jun 1

Hsp90 potentiates the evolution of azole resistance in the model yeast Saccharomyces cerevisiae and the opportunistic pathogen Candida albicans via calcineurin. Here, we explored effectors downstream of calcineurin regulating this Hsp90-dependent trait. Using S. cerevisiae erg3 mutants as a model, we determined that both Crz1 and Hph1 modulate azole resistance.
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PMID:Genetic architecture of Hsp90-dependent drug resistance. 1705 42

Compelled activation of Ca(2+) signaling by exposure of zds1Delta strain Saccharomyces cerevisiae cells to external CaCl(2) leads to characteristic physiological consequences such as growth inhibition in the G(2) phase and polarized bud growth. Screening of microbial metabolites for activity alleviating the deleterious physiological effects of external CaCl(2) identified the Hsp90 inhibitor radicicol as an inhibitor of Ca(2+)-signal-dependent cell-cycle regulation in yeast. Radicicol alleviated analogous physiological effects due to the expression of a constitutively active form of calcineurin or overexpression of Swe1, the negative regulatory kinase of the Cdc28-Clb complex. Western blot analysis indicated that radicicol inhibited Ca(2+)-induced accumulation of Swe1 and Cln2.
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PMID:Inhibition of Ca2+-signal-dependent growth regulation by radicicol in budding yeast. 1817 93

Microbes evolved to produce natural products that inhibit growth of competing soil microorganisms. In many cases these compounds act on fungi, which are eukaryotes with conserved gene sequences closely related to metazoans, including humans. The calcineurin inhibitors cyclosporin A and FK-506, the Tor inhibitor rapamycin, and the Hsp90 inhibitor geldanamycin, all act via targets conserved from yeast to humans. This allows the use of genetically tractable fungi as models to elucidate how these drugs and their targets function in yeast and human cells. These inhibitors also enable studies aimed at harnessing their intrinsic antimicrobial activities to develop novel antifungal therapies. Extensive studies have revealed a globally conserved role for the Tor protein in regulating growth and proliferation in response to nutrients, and targeting its essential functions results in robust antifungal action. Similarly, a conserved and essential role for calcineurin in fungal virulence has been established and could be targeted by inhibitors for therapeutic uses in a variety of clinical settings. Finally, the discovery that inhibitors of calcineurin or Hsp90 result in dramatic synergism with either azoles or glucan synthase inhibitors (candins) provides another therapeutic vantage point. Taken together, these fungal targets and their inhibitors provide a robust platform from which to develop novel antimicrobial therapies.
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PMID:Signaling cascades as drug targets in model and pathogenic fungi. 1866 33

Endoplasmic reticulum (ER) stress can trigger apoptosis and necrosis in many types of mammalian cells. Previous studies in yeast found little or no cell death in response to the ER stressor tunicamycin, but a recent study suggested widespread apoptosis-like death. Here we show that wild-type laboratory Saccharomyces cerevisiae cells responding to tunicamycin die by nonapoptotic mechanisms in low-osmolyte culture media and survive for long periods of time in standard synthetic media. Survival requires calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase, but none of its known targets. The Ca(2+)/calmodulin-dependent protein kinase Cmk2 was identified as an indirect target of calcineurin that suppresses death of calcineurin-deficient cells. Death of Cmk2- and/or calcineurin-deficient S. cerevisiae cells was preceded by accumulation of reactive oxygen species but was not associated with hallmarks of apoptosis and was not dependent on Mca1, Aif1, Nuc1, or other factors implicated in apoptosis-like death. Cmk2 and calcineurin also independently suppressed the death of S. cerevisiae cells responding to dithiothreitol or miconazole, a common azole-class antifungal drug. Though inhibitors of Hsp90 have been shown to diminish calcineurin signaling in S. cerevisiae and to synergistically inhibit growth in combination with azoles, they did not stimulate death of S. cerevisiae cells in combination with miconazole or tunicamycin, and instead they prevented the death of calcineurin- and Cmk2-deficient cells. These findings reveal a novel prodeath role for Hsp90 and antideath roles for calcineurin and Cmk2 that extend the life span of S. cerevisiae cells responding to both natural and clinical antifungal compounds.
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PMID:Nonapoptotic death of Saccharomyces cerevisiae cells that is stimulated by Hsp90 and inhibited by calcineurin and Cmk2 in response to endoplasmic reticulum stresses. 1880 10

Fungi have evolved an elegant repertoire of mechanisms to survive the cellular stress exerted by antifungal drugs such as azoles, which inhibit ergosterol biosynthesis inducing cell membrane stress. The evolution and maintenance of diverse resistance phenotypes is contingent upon cellular circuitry regulated by the molecular chaperone Hsp90 and its client protein calcineurin. Here, we establish a novel role for nutrients and nutrient signaling in azole resistance. The vulnerability of Saccharomyces cerevisiae azole resistance phenotypes to perturbation was contingent upon specific auxotrophies. Using strains that acquired azole resistance by Erg3 loss of function as a model for resistance that depends on cellular stress responses, we delineated genetic and environmental factors that mitigate the translation of genotype into resistance phenotype. Compromising a global regulator that couples growth and metabolism to environmental cues, Tor kinase, provides a powerful strategy to abrogate drug resistance of S. cerevisiae and Candida albicans with broad therapeutic potential.
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PMID:Metabolic control of antifungal drug resistance. 1959 84

Candida albicans is the leading fungal pathogen of humans, causing life-threatening disease in immunocompromised individuals. Treatment of candidiasis is hampered by the limited number of antifungal drugs whose efficacy is compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. We previously established that the molecular chaperone Hsp90, which regulates the form and function of diverse client proteins, potentiates resistance to the azoles in C. albicans and in the model yeast Saccharomyces cerevisiae. Genetic studies in S. cerevisiae revealed that Hsp90's role in azole resistance is to enable crucial cellular responses to the membrane stress exerted by azoles via the client protein calcineurin. Here, we demonstrate that Hsp90 governs cellular circuitry required for resistance to the only new class of antifungals to reach the clinic in decades, the echinocandins, which inhibit biosynthesis of a critical component of the fungal cell wall. Pharmacological or genetic impairment of Hsp90 function reduced tolerance of C. albicans laboratory strains and resistance of clinical isolates to the echinocandins and created a fungicidal combination. Compromising calcineurin function phenocopied compromising Hsp90 function. We established that calcineurin is an Hsp90 client protein in C. albicans: reciprocal co-immunoprecipitation validated physical interaction; Hsp90 inhibition blocked calcineurin activation; and calcineurin levels were depleted upon genetic reduction of Hsp90. The downstream effector of calcineurin, Crz1, played a partial role in mediating calcineurin-dependent stress responses activated by echinocandins. Hsp90's role in echinocandin resistance has therapeutic potential given that genetic compromise of C. albicans HSP90 expression enhanced the efficacy of an echinocandin in a murine model of disseminated candidiasis. Our results identify the first Hsp90 client protein in C. albicans, establish an entirely new role for Hsp90 in mediating resistance to echinocandins, and demonstrate that targeting Hsp90 provides a promising therapeutic strategy for the treatment of life-threatening fungal disease.
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PMID:Hsp90 governs echinocandin resistance in the pathogenic yeast Candida albicans via calcineurin. 1964 12

Optimal therapy for invasive aspergillosis is unknown, and many clinicians have attempted to utilize a combination antifungal approach to improve outcomes. However, while numerous in vitro studies, animal models, and clinical reports suggest the possibility that combination antifungal therapy might offer improved results, there is no definitive accepted strategy. The currently available antifungals used in various combination approaches have not demonstrated clear improvement over monotherapy. The current classes of drugs targeting the cell wall and cell membrane may need adjunctive agents focused on separate cellular pathways, such as cell stress response or cellular signaling, to maximize efficacy. The calcineurin and the Hsp90 pathways are two such untouched arenas in which targeted manipulation may lead to great advances against aspergillosis.
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PMID:Newer combination antifungal therapies for invasive aspergillosis. 2060 84

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.
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PMID:PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90. 2086 72


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