Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12–FK506 in <i>Cryptococcus neoformans</i>

Fox, Donald T.; Cruz, Maricel Dela; Sia, Rey A. L.; Ke, Hengming; Cox, Gary M.; Cárdenas, María E.; Heitman, Joseph

doi:10.1046/j.1365-2958.2001.02295.x

Cited by 177 publications

(180 citation statements)

References 49 publications

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“…This can be explained by the contacts of both residues with CsA. The CNB mutant of Cryptococcus neoformans with an insertion of two amino acids between Asn-121 and Gln-127 has reduced binding affinity for FKBP-FK506 both in vivo and in vitro (27). The crystal structures show that the loop 121-127 forms three hydrogen bonds and numerous van der Waals contacts with FKBP and FK506.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of calcineurin–cyclophilin–cyclosporin shows common but distinct recognition of immunophilin–drug complexes

Huai

Kim

Liu

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Calcineurin, a Ca 2؉ ͞calmodulin-dependent protein phosphatase, is the common target for two immunophilin-immunosuppressant complexes, cyclophilin A-cyclosporin A (CyPA-CsA) and FKBP-FK506. How the two structurally distinct immunophilin-drug complexes bind the same target has remained unknown. We report the crystal structure of calcineurin (CN) in complex with CyPA-CsA at 2.8-Å resolution. The CyPA-CsA complex binds to a composite surface formed by the catalytic and regulatory subunits of CN, where the complex of FK506 and its binding protein FKBP also binds. While the majority of the CN residues involved in the binding are common for both immunophilin-immunosuppressant complexes, a significant number of the residues are distinct. Unlike immunosuppressants ͉ FK506 ͉ FK506-binding protein ͉ protein phosphatase ͉ T cell C alcineurin (CN) is a unique serine͞threonine protein phosphatase (PP3 or PP2B) in its regulation by the second messenger Ca 2ϩ and calmodulin. CN is involved in many biological processes, including immune responses (1-5), the second messenger cAMP pathway (3, 6), Na ϩ ͞K ϩ ion transportation in the nephron (7), cell cycle progression in lower eukaryotes (8), cardiac hypertrophy (9), and memory formation (10, 11). The most extensively studied function of CN is its role in the signal transduction pathway during T cell activation. CN has been shown to be a common receptor for two immunophilinimmunosuppressant complexes, cyclophilin A-cyclosporin A (CyPA-CsA) and FK506-binding protein (FKBP)-FK506 (12, 13). The binding of CyPA-CsA or FKBP-FK506 inhibits the calcium-dependent dephosphorylation of the transcription factor nuclear factor of activated T cell (NFAT) by CN, thus blocking T cell receptor-mediated cytokine transcription and T-cell activation (14,15).Since the discovery of CN as a common target for both CyPA-CsA and FKBP-FK506 complexes, how two structurally distinct immunophilin-immunosuppressant complexes recognize a common target has remained a mystery. While the crystal structures of CN and its complex with FKBP-FK506 have shed light on the molecular recognition of CN by 17), the CyPA-CsA-CN structure has remained elusive. We report here the crystal structure of CyPA-CsA-CN at 2.8-Å resolution. The structure revealed that CyPA-CsA and FKBP-FK506 are bound to the same surface of CN, thus providing a molecular basis for understanding the regulation of CN activity by the immunophilin-immunosuppressant. MethodsProtein Purification. The catalytic subunit (CNA, 61 kDa) and regulatory subunit (CNB, 19 kDa) of the ␣ isoform of human CN were coexpressed in Escherichia coli and purified by using a previously described procedure with slight modifications (18). The recombinant human CyPA was expressed as a glutathione S-transferase (GST) fusion protein in E. coli BL21 and purified by glutathione-agarose beads (Sigma). The CyPACsA-CN complex was prepared by a four-step procedure: (i) purification of recombinant human CN by Ni NTA (Qiagen, Valencia, CA) and Q-Sepharose (Pharmacia) columns, (ii) purific...

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Section: Resultsmentioning

confidence: 99%

Crystal structure of calcineurin–cyclophilin–cyclosporin shows common but distinct recognition of immunophilin–drug complexes

Huai

Kim

Liu

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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252

191

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“…All these components showed a high degree of sequence identity (40-90%) among the fungal species studied. In the human pathogens C. albicans, C. neoformans and A. fumigatus, calcineurin was required for survival in serum and for virulence (Bader et al, 2003;Blankenship et al, 2003;Da Silva Ferreira et al, 2007;Fox et al, 2001;Odom et al, 1997;Steinbach et al, 2006Steinbach et al, , 2007. In the plant pathogen Sclerotinia sclerotiorum, the calcineurin orthologue controls sclerotial development and infection (Harel et al, 2006).…”

Section: The Calcium/calcineurin Signalling Pathwaymentioning

confidence: 99%

Comparative genomics of MAP kinase and calcium–calcineurin signalling components in plant and human pathogenic fungi

Rispail

Soanes

Ant

et al. 2009

Fungal Genetics and Biology

276

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a b s t r a c tMitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.

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“…Due to such reasons, signaling cascades governing thermotolerance in fungi have been intensively investigated toward their exploitation as potential antifungal drug targets (Bahn et al 2007;Bahn and Jung 2013). In C. neoformans, temperature-sensing signaling cascades include the calmodulin/calcineurin (Fox et al 2001;Kraus et al 2005), Ras (Alspaugh et al 2000;Nichols et al 2007), HOG (Bahn et al , 2006, Msi1-like protein (Yang et al 2012), protein kinase C 1 (Pkc1)/ Mpk1 MAPK (Kraus et al 2003;Gerik et al 2005), and unfolded protein response (UPR) (Cheon et al 2011) pathways. Unlike other temperature-sensing signaling components described above, a protein kinase, Sch9, suppresses C. neoformans thermotolerance (Wang et al 2004).…”

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confidence: 99%

Rewiring of Signaling Networks Modulating Thermotolerance in the Human Pathogen Cryptococcus neoformans

et al. 2017

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Thermotolerance is a crucial virulence attribute for human pathogens, including the fungus Cryptococcus neoformans that causes fatal meningitis in humans. Loss of the protein kinase Sch9 increases C. neoformans thermotolerance, but its regulatory mechanism has remained unknown. Here, we studied the Sch9-dependent and Sch9-independent signaling networks modulating C. neoformans thermotolerance by using genome-wide transcriptome analysis and reverse genetic approaches. During temperature upshift, genes encoding for molecular chaperones and heat shock proteins were upregulated, whereas those for translation, transcription, and sterol biosynthesis were highly suppressed. In this process, Sch9 regulated basal expression levels or induced/repressed expression levels of some temperature-responsive genes, including heat shock transcription factor (HSF1) and heat shock proteins (HSP104 and SSA1). Notably, we found that the HSF1 transcript abundance decreased but the Hsf1 protein became transiently phosphorylated during temperature upshift. Nevertheless, Hsf1 is essential for growth and its overexpression promoted C. neoformans thermotolerance. Transcriptome analysis using an HSF1 overexpressing strain revealed a dual role of Hsf1 in the oxidative stress response and thermotolerance. Chromatin immunoprecipitation demonstrated that Hsf1 binds to the step-type like heat shock element (HSE) of its target genes more efficiently than to the perfect-or gap-type HSE. This study provides insight into the thermotolerance of C. neoformans by elucidating the regulatory mechanisms of Sch9 and Hsf1 through the genome-scale identification of temperature-dependent genes. KEYWORDS Hsf1; Sch9; transcriptome analysis; high temperature; Cryptococcus neoformans R ESPONSE and adaptation to their host's physiological temperature, 37°, are key virulence attributes for most human fungal pathogens that infect from natural environments at ambient temperature. Cryptococcus neoformans is an example of such a pathogen. This basidiomycetous fungus exists in diverse environment niches and generates infectious yeast cells or spores through bisexual and unisexual differentiation, which are inhaled through the respiratory tract in the host and initially colonize the lungs (Idnurm et al. 2005;Lin and Heitman 2006). C. neoformans is subsequently disseminated into the bloodstream, breaches the central nervous system, and eventually elicits fatal meningoencephalitis, responsible for an estimated hundreds of thousands of deaths annually (Park et al. 2009). During this infectious process, the ability to sense and adapt to temperature upshift are crucial factors for the pathogen to establish the initial colonization of the lungs. Therefore, most mutants that are severely defective in thermotolerance tend to be highly attenuated in virulence. Due to such reasons, signaling cascades governing thermotolerance in fungi have been intensively investigated toward their exploitation as potential antifungal drug targets (Bahn et al. 2007;Bahn and Jung 2013). In...

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

Cited by 177 publications

References 49 publications

Crystal structure of calcineurin–cyclophilin–cyclosporin shows common but distinct recognition of immunophilin–drug complexes

Crystal structure of calcineurin–cyclophilin–cyclosporin shows common but distinct recognition of immunophilin–drug complexes

Comparative genomics of MAP kinase and calcium–calcineurin signalling components in plant and human pathogenic fungi

Rewiring of Signaling Networks Modulating Thermotolerance in the Human Pathogen Cryptococcus neoformans

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