RESUMO
Gß-like/RACK1 functions as a key mediator of various pathways and contributes to numerous cellular functions in eukaryotic organisms. In the pathogenic fungus Cryptococcus neoformans, noncanonical Gß Gib2 promotes cAMP signaling in cells lacking normal Gpa1 function while displaying versatility in interactions with Gα Gpa1, protein kinase Pkc1, and endocytic intersectin Cin1. To elucidate the Gib2 functional mechanism(s), we demonstrate that Gib2 is required for normal growth and virulence. We show that Gib2 directly binds to Gpa1 and Gγ Gpg1/Gpg2 and that it interacts with phosphodiesterase Pde2 and monomeric GTPase Ras1. Pde2 remains functionally dispensable, but Ras1 is found to associate with adenylyl cyclase Cac1 through the conserved Ras association domain. In addition, the ras1 mutant exhibits normal capsule formation, whereas the ras1 gpa1 mutant displays enhanced capsule formation, and the ras1 gpa1 cac1 mutant is acapsular. Collectively, these findings suggest that Gib2 promotes cAMP levels by relieving an inhibitory function of Ras1 on Cac1 in the absence of Gpa1. In addition, using GST affinity purification combined with mass spectrometry, we identified 47 additional proteins that interact with Gib2. These proteins have putative functions ranging from signal transduction, energy generation, metabolism, and stress response to ribosomal function. After establishing and validating a protein-protein interactive network, we believe Gib2 to be a key adaptor/scaffolding protein that drives the formation of various protein complexes required for growth and virulence. Our study reveals Gib2 as an essential component in deciphering the complexity of regulatory networks that control growth and virulence in C. neoformans.
Assuntos
Adenilil Ciclases/metabolismo , Cryptococcus neoformans/metabolismo , AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Adenilil Ciclases/genética , Sequência de Aminoácidos , Western Blotting , Cryptococcus neoformans/genética , Cryptococcus neoformans/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/classificação , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Filogenia , Ligação Proteica , Mapas de Interação de Proteínas , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas ras/genéticaRESUMO
Water samples from two of 17 field sites in Arizona (USA) inhibited growth of the amphibian pathogen, Batrachochytrium dendrobatidis. Chemical analyses of samples revealed statistically significant facilitating or inhibitory activity of certain elements. Although low levels of copper were found in environmental samples demonstrating facilitated growth, growth was inhibited at concentrations of copper sulfate (CuSO(4)) at or greater than 100 ppm.
