Nuclear Thiol Peroxidase as a Functional Alkyl-hydroperoxide Reductase Necessary for Stationary Phase Growth of Saccharomyces cerevisiae

Abstract

Yeast nucleus-localized thiol peroxidase (nTPx) was characterized as a functional peroxidase. There are two cysteine residues in nTPx. Replacement of Cys-106 or Cys-111 with serine resulted in a complete loss of thioredoxin-linked peroxidase activity. However, when their activities were measured in terms of the ability to inhibit oxidation of glutamine synthetase, C111S showed the same antioxidant activity as the wild type protein. SDS-PAGE gel analysis revealed that only C111S existed as the dimer form. In addition to the identification of Cys-106 as the primary catalytic site, these data suggest the formation of the intradisulfide bond as a part of the catalytic cycle between nTPx and thioredoxin. nTPx preferentially reduced alkyl-hydroperoxides rather than H 2 O 2 . Furthermore, a nTPx mutant strain showed higher sensitivity toward alkyl-hydroperoxide than hydrogen peroxide. Also, reduction of the viability of nTPx mutant strain against various oxidants supports an in vivo antioxidant role for nTPx. nTPx transcriptional activity was not significantly detectable in log phase yeast, but the activity was exponentially increased after the diauxic shift. The transcriptional activity was highly induced even in the log phase yeast grown in nonfermentable carbon source. Deletion of Tor1p, Ras1p, and Ras2p resulted in considerable induction when compared with their parent strains, demonstrating the activation of the transcription of nTPx gene at the diauxic shift. Transcription of nTPx gene was induced in response to oxidative stress. Viability of a stationary phase nTPx mutant was considerably reduced compared with the isogenic strain. Collectively, these data demonstrate that nTPx is a thiol peroxidase family acting as alkyl-hydroperoxide reductase in the nucleus during post-diauxic growth.

The Response to Heat Shock and Oxidative Stress inSaccharomyces cerevisiae

“…Differences in the cytoplasmic Prx's are further highlighted by the finding that in contrast to Tsa1 and Tsa2, overoxidation of Ahp1 to cysteine-sulfinic acid is not reversed by Srx1 (Biteau et al 2003). Dot5 (nTPx) is a nuclear 2-Cys Prx, which is most active against alkyl hydroperoxides (Cha et al 2003). However, it has been proposed to play a minor role as an antioxidant, predominantly functioning in telomeric silencing (Izawa et al 2004).…”

Peroxiredoxin-null Yeast Cells Are Hypersensitive to Oxidative Stress and Are Genomically Unstable

“…Thus, the yeast serves as a good model for studying the biological functions of peroxiredoxins. Yeast peroxiredoxins are variously termed as Tsa1p/cTPxI/ YML028W (7,20), Tsa2p/cTPxII/YDR453C (11,20), Dot5p/ nTPx/BCP/YIL010W (23,24), Prx1p/mTPx/1CPrx/YBL064C (25), and Ahp1p/cTPxIII/PMP20/YLR109W (26). All five enzymes have similar antioxidant properties, but are differentially expressed and localized (20).…”

Vibrio cholerae Thiol Peroxidase-Glutaredoxin Fusion Is a 2-Cys TSA/AhpC Subfamily Acting as a Lipid Hydroperoxide Reductase

“…The 2-Cys-containing proteins exist as a homodimer via an intermolecular or intramolecular disulfide bond. Previously, we suggested that E. coli p20 (9, 21, 34) and yeast nuclear thiol peroxidase (30,31) are 2-Cys TPx and exist as a monomer in which the N-terminal Cys is bonded to the C-terminal Cys via intramolecular disulfide linkage. Pauwels et al (17) and Kim et al (18) demonstrated that HI TPx⅐Grx exists as the dimer linked via an intermolecular disulfide bond between 1-Cys of the TPx domains.…”