RESUMEN
Group III hybrid histidine kinases are fungal-specific proteins and part of the multistep phosphorelay, representing the initial part of the high osmolarity glycerol (HOG) pathway. TcsC, the corresponding kinase in Aspergillus fumigatus, was expected to be a cytosolic protein but is targeted to the nucleus. Activation of TcsC by the antifungal fludioxonil has lethal consequences for the fungus. The agent triggers a fast and TcsC-dependent activation of SakA and later on a redistribution of TcsC to the cytoplasm. High osmolarity also activates TcsC, which then exits the nucleus or concentrates in spot-like, intra-nuclear structures. The sequence corresponding to the N-terminal 208 amino acids of TcsC lacks detectable domains. Its loss renders TcsC cytosolic and non-responsive to hyperosmotic stress, but it has no impact on the antifungal activity of fludioxonil. A point mutation in one of the three putative nuclear localization sequences, which are present in the N-terminus, prevents the nuclear localization of TcsC, but not its ability to respond to hyperosmotic stress. Hence, this striking intracellular localization is no prerequisite for the role of TcsC in the adaptive response to hyperosmotic stress, instead, TcsC proteins that are present in the nuclei seem to modulate the cell wall composition of hyphae, which takes place in the absence of stress. The results of the present study underline that the spatiotemporal dynamics of the individual components of the multistep phosphorelay is a crucial feature of this unique signaling hub. IMPORTANCE: Signaling pathways enable pathogens, such as Aspergillus fumigatus, to respond to a changing environment. The TcsC protein is the major sensor of the high osmolarity glycerol (HOG) pathway of A. fumigatus and it is also the target of certain antifungals. Insights in its function are therefore relevant for the pathogenicity and new therapeutic treatment options. TcsC was expected to be cytoplasmic, but we detected it in the nucleus and showed that it translocates to the cytoplasm upon activation. We have identified the motif that guides TcsC to the nucleus. An exchange of a single amino acid in this motif prevents a nuclear localization, but this nuclear targeting is no prerequisite for the TcsC-mediated stress response. Loss of the N-terminal 208 amino acids prevents the nuclear localization and renders TcsC unable to respond to hyperosmotic stress demonstrating that this part of the protein is of crucial importance.
Asunto(s)
Aspergillus fumigatus , Núcleo Celular , Dioxoles , Proteínas Fúngicas , Histidina Quinasa , Pirroles , Aspergillus fumigatus/genética , Aspergillus fumigatus/enzimología , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/efectos de los fármacos , Histidina Quinasa/metabolismo , Histidina Quinasa/genética , Histidina Quinasa/química , Núcleo Celular/metabolismo , Pirroles/farmacología , Pirroles/metabolismo , Dioxoles/farmacología , Dioxoles/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Antifúngicos/farmacología , Antifúngicos/metabolismo , Presión Osmótica , Citoplasma/metabolismo , Transporte de Proteínas , Regulación Fúngica de la Expresión Génica , Concentración Osmolar , Transducción de SeñalRESUMEN
Rupture or dissection of thoracic aortic aneurysms is still the leading cause of death for patients diagnosed with Marfan syndrome. Inflammation and matrix digestion regulated by matrix metalloproteases (MMPs) play a major role in the pathological remodeling of the aortic media. Regnase-1 is an endoribonuclease shown to cleave the mRNA of proinflammatory cytokines, such as interleukin-6. Considering the major anti-inflammatory effects of regnase-1, here, we aimed to determine whether adeno-associated virus (AAV)-mediated vascular overexpression of the protein could provide protection from the development and progression of aortic aneurysms in Marfan syndrome. The overexpression of regnase-1 resulted in a marked decrease in inflammatory parameters and elastin degradation in aortic smooth muscle cells in vitro. Intravenous injection of a vascular-targeted AAV vector resulted in the efficient transduction of the aortic wall and overexpression of regnase-1 in a murine model of Marfan syndrome, associated with lower circulating levels of proinflammatory cytokines and decreased MMP expression and activity. Regnase-1 overexpression strongly improved elastin architecture in the media and reduced aortic diameter at distinct locations. Therefore, AAV-mediated regnase-1 overexpression may represent a novel gene therapy approach for inhibiting aortic aneurysms in Marfan syndrome.