RESUMEN
Calcineurin is a critical cell-signaling protein that orchestrates growth, stress response, virulence, and antifungal drug resistance in several fungal pathogens. Blocking calcineurin signaling increases the efficacy of several currently available antifungals and suppresses drug resistance. We demonstrate the application of a novel scanning quadrupole DIA method for the analysis of changes in the proteins coimmunoprecipitated with calcineurin during therapeutic antifungal drug treatments of the deadly human fungal pathogen Aspergillus fumigatus. Our experimental design afforded an assessment of the precision of the method as demonstrated by peptide- and protein-centric analysis from eight replicates of the study pool QC samples. Two distinct classes of clinically relevant antifungal drugs that are guideline recommended for the treatment of invasive "aspergillosis" caused by Aspergillus fumigatus, the azoles (voriconazole) and the echinocandins (caspofungin and micafungin), which specifically target the fungal plasma membrane and the fungal cell wall, respectively, were chosen to distinguish variations occurring in the proteins coimmunoprecipitated with calcineurin. Novel potential interactors were identified in response to the different drug treatments that are indicative of the possible role for calcineurin in regulating these effectors. Notably, treatment with voriconazole showed increased immunoprecipitation of key proteins involved in membrane ergosterol biosynthesis with calcineurin. In contrast, echinocandin (caspofungin or micafungin) treatments caused increased immunoprecipitation of proteins involved in cell-wall biosynthesis and septation. Furthermore, abundant coimmunoprecipitation of ribosomal proteins with calcineurin occurred exclusively in echinocandins treatment, indicating reprogramming of cellular growth mechanisms during different antifungal drug treatments. While variations in the observed calcineurin immunoprecipitated proteins may also be due to changes in their expression levels under different drug treatments, this study suggests an important role for calcineurin-dependent cellular mechanisms in response to antifungal treatment of A. fumigatus that warrants future studies.
Asunto(s)
Aspergillus fumigatus/efectos de los fármacos , Calcineurina/aislamiento & purificación , Proteínas Fúngicas/aislamiento & purificación , Proteínas Ribosómicas/aislamiento & purificación , Voriconazol/farmacología , Antifúngicos/farmacología , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Caspofungina , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Pared Celular/química , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Cromatografía Liquida/métodos , Equinocandinas/farmacología , Ergosterol/biosíntesis , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Ontología de Genes , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunoprecipitación , Lipopéptidos/farmacología , Micafungina , Anotación de Secuencia Molecular , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
The effects of integrative medicine practices such as meditation and Ayurveda on human physiology are not fully understood. The aim of this study was to identify altered metabolomic profiles following an Ayurveda-based intervention. In the experimental group, 65 healthy male and female subjects participated in a 6-day Panchakarma-based Ayurvedic intervention which included herbs, vegetarian diet, meditation, yoga, and massage. A set of 12 plasma phosphatidylcholines decreased (adjusted p < 0.01) post-intervention in the experimental (n = 65) compared to control group (n = 54) after Bonferroni correction for multiple testing; within these compounds, the phosphatidylcholine with the greatest decrease in abundance was PC ae C36:4 (delta = -0.34). Application of a 10% FDR revealed an additional 57 metabolites that were differentially abundant between groups. Pathway analysis suggests that the intervention results in changes in metabolites across many pathways such as phospholipid biosynthesis, choline metabolism, and lipoprotein metabolism. The observed plasma metabolomic alterations may reflect a Panchakarma-induced modulation of metabotypes. Panchakarma promoted statistically significant changes in plasma levels of phosphatidylcholines, sphingomyelins and others in just 6 days. Forthcoming studies that integrate metabolomics with genomic, microbiome and physiological parameters may facilitate a broader systems-level understanding and mechanistic insights into these integrative practices that are employed to promote health and well-being.
Asunto(s)
Medicina Ayurvédica , Metabolómica , Yoga , Dieta Vegetariana , Femenino , Voluntarios Sanos , Humanos , Lipoproteínas/metabolismo , Masculino , Masaje , Meditación , Fosfatidilcolinas/metabolismo , Plantas Medicinales/metabolismo , Esfingomielinas/metabolismoRESUMEN
The echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition of Aspergillus fumigatus, a phenomenon known as the "paradoxical effect," which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies of A. fumigatus revealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca(2+) reporter aequorin expression system in A. fumigatus and showed that caspofungin elicits a transient increase in cytosolic free Ca(2+) ([Ca(2+)]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca(2+)]c was also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca(2+)]c increase was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca(2+)-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid], or the L-type Ca(2+) channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca(2+)]c levels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca(2+)]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.
Asunto(s)
Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/metabolismo , Calcineurina/metabolismo , Calcio/metabolismo , Equinocandinas/farmacología , Fosforilación/fisiología , Anidulafungina , Antifúngicos/farmacología , Caspofungina , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Proteínas Fúngicas/metabolismo , Lipopéptidos/farmacología , MicafunginaRESUMEN
While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes14-3-3ζ dissociation from caspase-2 in both egg extract and human cultured cells. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation.