Yeast glutamine-fructose-6-phosphate aminotransferase (Gfa1) requires methionine aminopeptidase activity for proper function.
ABSTRACT Methionine aminopeptidase (MetAP) catalyzes the co-translational processing of initiator methionine from nascent proteins. A cellular requirement for MetAP activity is likely due to dysfunction of MetAP substrates that require methionine removal for proper protein function. Glutamine-fructose-6-phosphate aminotransferase (Gfa1) is an essential enzyme in yeast that catalyzes the first and rate-limiting step in hexosamine biosynthesis. The alpha-amino group of Gfa1 Cys-1 has been proposed to act as a nucleophile in the catalytic mechanism. We used two mutational strategies to evaluate whether removal of initiator methionine, catalyzed by MetAP, is required for Gfa1 function. Our results demonstrate that exposure of the alpha-amino group of Cys-1 is required for normal Gfa1 function as failure to do so results in decreased enzyme activity and slow growth. Further, either isoform of MetAP in yeast is sufficient for Gfa1 processing in vivo. These results are the first demonstration of an endogenous yeast protein that requires the exposure of the alpha-amino group by MetAP action for normal function. Additionally, Gfa1 will be a relevant target in therapeutic or physiological applications in which MetAP activity is inhibited.
- SourceAvailable from: Shridhar Bhat[Show abstract] [Hide abstract]
ABSTRACT: [*F.Z. and S.B. are joint first authors] Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (HsMetAP1)-selective inhibitors (1-4), but all of them failed to inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate HsMetAP1, we found that 1-4 could only effectively inhibit purified HsMetAP1 in the presence of physiologically unachievable concentrations of Co(II). In an effort to seek Co(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that 11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an HsMetAP1-selective inhibitor is effective against its target in cells.Journal of Medicinal Chemistry 05/2013; 56(10):3996-4016. · 5.48 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Paracoccidioides is the causative agent of paracoccidioidomycosis, a systemic mycosis endemic to Latin America. Infection is initiated by inhalation of conidia (C) or mycelial (M) fragments, which subsequently differentiate into yeast (Y). Epidemiological studies show a striking predominance of paracoccidioidomycosis in adult men compared to premenopausal women. In vitro and in vivo studies suggest that the female hormone (17β-estradiol, E(2)) regulates or inhibits M-or-C-to-Y transition. In this study we have profiled transcript expression to understand the molecular mechanism of how E(2) inhibits M-to-Y transition. We assessed temporal gene expression in strain Pb01 in the presence or absence of E(2) at various time points through 9 days of the M-to-Y transition using an 11,000 element random-shear genomic DNA microarray and verified the results using quantitative real time-PCR. E(2)-regulated clones were sequenced to identify genes and biological function. E(2)-treatment affected gene expression of 550 array elements, with 331 showing up-regulation and 219 showing down-regulation at one or more time points (p≤0.001). Genes with low expression after 4 or 12 h exposure to E(2) belonged to pathways involved in heat shock response (hsp90 and hsp70), energy metabolism, and several retrotransposable elements. Y-related genes, α-1,3-glucan synthase, mannosyltransferase and Y20, demonstrated low or delayed expression in E(2)-treated cultures. Genes potentially involved in signaling, such as palmitoyltransferase (erf2), small GTPase RhoA, phosphatidylinositol-4-kinase, and protein kinase (serine/threonine) showed low expression in the presence of E(2), whereas a gene encoding for an arrestin domain-containing protein showed high expression. Genes related to ubiquitin-mediated protein degradation, and oxidative stress response genes were up-regulated by E(2). This study characterizes the effect of E(2) at the molecular level on the inhibition of the M-to-Y transition and is indicative that the inhibitory actions of E(2) may be working through signaling genes that regulate dimorphism.PLoS ONE 12/2011; 6(12):e28402. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Fusarium graminearum sensu stricto (s.s.) is an ubiquitous pathogen of cereals. The economic impact of Fusarium head blight (FHB) is characterized by crop losses and mycotoxin contamination. Our objective was to associate SNP diversity within candidate genes with phenotypic traits. A total of 77 F. graminearum s.s. isolates was tested for severity of fungal infection (= aggressiveness) and deoxynivalenol (DON) production in an inoculated field experiment at two locations in each of two years. For seven genes known to control fungal growth (MetAP1, Erf2) or DON production (TRI1, TRI5, TRI6 TRI10 and TRI14) single nucleotides polymorphic sites (SNPs) were determined and evaluated for the extent of linkage disequilibrium (LD). Associations of SNPs with both phenotypic traits were tested using linear mixed models. Decay of LD was in most instances fast. Two neighboring SNPs in MetAP1 and one SNP in Erf2 were significantly (P < 0.05) associated with aggressiveness explaining proportions of genotypic variance (pG) of 25.6%, 0.5%, and 13.1%, respectively. One SNP in TRI1 was significantly associated with DON production (pG = 4.4). We argue that using the published sequence information of Fusarium graminearum as a template to amplify comparative sequence parts of candidate genes is an effective method to detect quantitative trait loci. Our findings underline the potential of candidate gene association mapping approaches to identify functional SNPs underlying aggressiveness and DON production for F. graminearum s.s populations.BMC Genetics 03/2012; 13:14. · 2.36 Impact Factor