An Ustilago maydis gene involved in H2O2 detoxification is required for virulence

The fungus Ustilago maydis is a biotrophic pathogen of maize (Zea mays). In its genome we have identified an ortholog of YAP1 (for Yeast AP-1-like) from Saccharomyces cerevisae that regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H(2)O(2) than wild-type cells, and their virulence was significantly reduced. U. maydis yap1 could partially complement the H(2)O(2) sensitivity of a yap1 deletion mutant of S. cerevisiae, and a Yap1-green fluorescent protein fusion protein showed nuclear localization after H(2)O(2) treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two Cys residues prevented accumulation in the nucleus, and the respective mutant strains showed the same virulence phenotype as Deltayap1 mutants. Diamino benzidine staining revealed an accumulation of H(2)O(2) around yap1 mutant hyphae, which was absent in the wild type. Inhibition of the plant NADPH oxidase prevented this accumulation and restored virulence. During the infection, Yap1 showed nuclear localization after penetration up to 2 to 3 d after infection. Through array analysis, a large set of Yap1-regulated genes were identified and these included two peroxidase genes. Deletion mutants of these genes were attenuated in virulence. These results suggest that U. maydis is using its Yap1-controlled H(2)O(2) detoxification system for coping with early plant defense responses.     

Proteome-wide profiling of carbonylated proteins and carbonylation sites in HeLa cells under mild oxidative stress conditions

A number of oxidative protein modifications have been well characterized during the past decade. Presumably, reversible oxidative posttranslational modifications (PTMs) play a significant role in redox signaling pathways, whereas irreversible modifications including reactive protein carbonyl groups are harmful, as their levels are typically increased during aging and in certain diseases. Despite compelling evidence linking protein carbonylation to numerous disorders, the underlying molecular mechanisms at the proteome remain to be identified. Recent advancements in analysis of PTMs by mass spectrometry provided new insights into the mechanisms of protein carbonylation, such as protein susceptibility and exact modification sites, but only for a limited number of proteins. Here we report the first proteome-wide study of carbonylated proteins including modification sites in HeLa cells for mild oxidative stress conditions. The analysis relied on our recent strategy utilizing mass spectrometry-based enrichment of carbonylated peptides after DNPH derivatization. Thus a total of 210 carbonylated proteins containing 643 carbonylation sites were consistently identified in three replicates. Most carbonylation sites (284, 44.2%) resulted from oxidation of lysine residues (aminoadipic semialdehyde). Additionally, 121 arginine (18.8%), 121 threonine (18.8%), and 117 proline residues (18.2%) were oxidized to reactive carbonyls. The sequence motifs were significantly enriched for lysine and arginine residues near carbonylation sites (±10 residues). Gene Ontology analysis revealed that 80% of the carbonylated proteins originated from organelles, 50% enrichment of which was demonstrated for the nucleus. Moreover, functional interactions between carbonylated proteins of kinetochore/spindle machinery and centrosome organization were significantly enriched. One-third of the 210 carbonylated proteins identified here are regulated during apoptosis.