NO-inducible nitrosothionein mediates NO removal in tandem with thioredoxin

Abstract

Nitric oxide (NO) is a toxic reactive nitrogen species that induces microbial adaption mechanisms. Screening a genomic DNA library identified a new gene, ntpA, that conferred growth tolerance upon Aspergillus nidulans against exogenous NO. The gene encoded a cysteine-rich 23-amino-acid peptide that reacted with NO and S-nitrosoglutathione to generate an S-nitrosated peptide. Disrupting ntpA increased amounts of cellular S-nitrosothiol and NO susceptibility. Thioredoxin and its reductase denitrosated the S-nitrosated peptide, decreased cellular S-nitrosothiol and conferred tolerance against NO, indicating peptide-mediated catalytic NO removal. The peptide binds copper(I) in vitro but is dispensable for metal tolerance in vivo. NO but not metal ions induced production of the peptide and ntpA transcripts. We discovered that the thionein family of peptides has NO-related functions and propose that the new peptide be named NO-inducible nitrosothionein (iNT). The ubiquitous distribution of iNT-like polypeptides constitutes a potent NO-detoxifying mechanism that is conserved among various organisms.

Stress Biology of Yeasts and Fungi

“…The chemistry of thiols (-SH) and NO is established, and their reactions under physiological conditions generates S -nitrosothiols (-SNO). This is also true for the iNT peptide, and thiols of the six cysteine residues are stoichemetrically converted to S -nitrosothiols in vitro (Zhou et al 2013 ). Studies using the gene disruptant and a strain that produces an excess of iNT found a negative correlation between cellular iNT levels and the amount of S -nitrosated proteins, indicating that iNT protects cellular protein thiols from S -nitrosation by scavenging NO under RNS stress.…”

“…The yeast GSH-GSNOR system and Fhb constitute a control mechanism of NO that eliminates stress imposed by RNS (Liu et al 2001 ). The recent fi ndings of Fhb in A. oryzae and A. nidulans (Zhou et al 2013 ) indicate that these fi lamentous fungi have NO-detoxifying functions. However, the functions of the GSH/GSNOR system and other proteins in the responses of fi lamentous fungi to NO remain unknown.…”

“…Another recently identifi ed NO-tolerating gene of A. nidulans encodes a 23-aminoacid peptide called inducible nitrosothionein (iNT) (Zhou et al 2013 ). This peptide is similar to the N-terminal half of metallothionein (MT) (Fig.…”

“…Studies using the gene disruptant and a strain that produces an excess of iNT found a negative correlation between cellular iNT levels and the amount of S -nitrosated proteins, indicating that iNT protects cellular protein thiols from S -nitrosation by scavenging NO under RNS stress. The NO scavenging role of iNT seems to be mediated by thioredoxin-dependent catalysis (Zhou et al 2013 ). Reconstitution studies in vitro have shown that thioredoxin (TrxA) rapidly reduces S -nitrosated iNT (iNT-SNO) to iNT, and the resultant oxidized thioredoxin is reduced by thioredoxin reductase (TrrA) and NADPH ( Fig.…”

Anti-Immune Strategies of Pathogenic Fungi

“…Furthermore, deletion of this gene results in hypersensitivity to NO and a moderate attenuation of virulence (Hromatka et al, 2005). Other proteins involved in the detoxification of NO are the porphobilinogen deaminase hemC , which promotes the activity of flavohemoglobin, the NO-inducible nitrosothionein ntpA , which scavenges NO through S-nitrosylation in A. nidulans (Zhou et al, 2012, 2013) and S-nitrosoglutathione (GSNO) reductase, which reduces GSNO to ammonia and glutathione disulfide and is important for the detoxification of RNS in C. neoformans (Fernández et al, 2003) and A. fumigatus (Lapp et al, 2014). …”