Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA

The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the NiRD, NES exposure, and relocation of the inactive NirA to the cytosol

Search & retrieval in CAD databases: A user-centric state-of-the-art overview

This article presents a state-of-the-art overview on shape, information and design retrieval systems in the context of CAD engineering. In contrast to existing surveys, we classify the different approaches from a CAD application user point of view. As a consequence, we focus on features of surveyed techniques such as: supported shape data types, handling of geometric invariances, support of metadata, supported query types, quality of retrieval results, and the availability of implementations

Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA

The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed,Peer reviewe

N-Glycan Modification in Aspergillus Species▿

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional α-1,2-mannosidase producing increased amounts of N-glycans of the Man5GlcNAc2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan5GlcNac2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man3GlcNAc2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi

Overview of correlations between NES sequences, oxidation status of M169 (red/ox), NirA-GFP subcellular localization and NirA transcriptional activity in different genetic backgrounds of strains grown under non-inducing or inducing conditions.

<p>(n.a., not applicable, n.d., not determined)</p><p>Overview of correlations between NES sequences, oxidation status of M169 (red/ox), NirA-GFP subcellular localization and NirA transcriptional activity in different genetic backgrounds of strains grown under non-inducing or inducing conditions.</p

N-octylamine treatment does not influence NirA subcellular localization in KapK/CRM1-inactivated cells.

<p><b>(A)</b> The <i>KapK</i>1 strain was incubated with 10 ng/ml Leptomycin B (+LMB) for 30 minutes and subsequently treated with 10 mM n-octylamine (+LMBo). <b>(B)</b> The control strain expressing histone H1 tagged with RFP (<i>hhoA</i>-<i>mrfp</i>) was used to verify that n-octylamine has no damaging effect on the subcellular localization of nuclear proteins.-O, in the absence of n-octylamine; +O, in the presence of 10 mM n-octylamine. Cells were grown 16 h on GMM with arginine 3 mM as nitrogen source. <b>(C)</b> Comparison of NirA sub-cellular localization between the control strain and the <i>ERE</i>_<i>p</i><i>-nirA</i>^<i>M169I</i><i>-gfp</i> strain under non inducing (NI, arginine 3 mM), inducing (IND, nitrate 10 mM, 2 minutes), inducing plus n-octylamine (INDo, nitrate 10 mM, 10 mM n-octylamine, added after 2 minutes of nitrate induction) conditions. Scale bars refer to 5 μm. <b>(D)</b> Growth test of <i>nirA637</i>, <i>ERE</i>_<i>p</i><i>-nirA</i>-<i>gfp</i> and <i>ERE</i>_<i>p</i><i>-nirA</i>^<i>M169I</i>-<i>gfp</i> strains in the presence of DES (diethylsilbestrol), inducer of <i>ERE</i> promoter on nitrate. Western panels (a, b, c) show the amount of NirA-GFP fusion protein (~130 kDa) in each strain tested for growth.</p

<i>In vivo</i> and <i>in vitro</i> characteristics of FmoB.

<p><b>(A)</b> Sub-cellular localization of FmoB as a GFP fusion protein. FmoB::GFP localizes both in the cytoplasm and in the nuclei. Nuclei were visualized by DAPI staining. Cells were grown for 16 h in AMM with 3 mM arginine as a sole nitrogen source. Scale bar refers to 5 μm. <b>(B)</b> Preparative size exclusion chromatography elution profile of purified recombinant FmoB. An UV-visible absorbance spectra of FAD saturated FmoB is shown as inset. <b>(C)</b> SDS-PAGE analysis of FmoB purification steps ([<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005297#pgen.1005297.ref001" target="_blank">1</a>] HIS-Select load; [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005297#pgen.1005297.ref002" target="_blank">2</a>] HIS-Select flow through; [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005297#pgen.1005297.ref003" target="_blank">3</a>] HIS-Select pool; [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005297#pgen.1005297.ref004" target="_blank">4</a>] size exclusion chromatography pool). The FmoB band corresponds to the molecular weight of ~ 60 kDa. <b>(D)</b> HPLC monitoring of L-methionine sulfoxide formation by FmoB. FmoB was incubated with indicated L-methionine concentrations for 60, 150 and 300 seconds and the formation of L-methionine sulfoxide was quantitatively analyzed by HPLC. Detected amounts (nmol) of L-methionine sulfoxide are displayed over the time and data points were fitted by the hyperbolic function y = ax/(b+x) using SigmaPlot 10. On the left panel, the concentrations of free methionine and the corresponding colours are indicated. <b>(E)</b> FmoB activity assay with methimazole. FmoB was incubated with indicated concentrations of methimazole as described in Supplementary Materials and Methods. Absolute values of change in NADPH absorption at 340 nm were plotted versus time. NADPH oxidase activity of the enzyme in the absence of substrate and heat inactivated FmoB in the reaction are shown as controls. There is only a slight increase in NADPH consumption when methimazole concentrations raise from 0,25 mM to 0.5 mM. On the left side of the graph, methimazole concentrations and the corresponding colours are indicated. <b>(F)</b> FmoB activity assays with different substrates. NADPH levels are expressed as absorbance (OD_340nm) and are plotted against time. All samples received 2 μM FmoB and 200 μM NADPH. After 120 seconds pre-incubation time, samples received additionally 2 mM of putative substrates and kinetics of OD_340nm reduction indicates enzyme activities on the tested substrates. Cysteine, DTT and β-mercaptoethanol are apparently preferred substrates for FmoB under these conditions. <b>(G)</b> FmoB NADPH-oxidase activity is enhanced by n-octylamine. NADPH levels are expressed as absorbance (OD_340nm) and are plotted against time. All samples received 0.5 μM FmoB and 200 μM NADPH and in addition indicated concentrations of n-octylamine. A specific substrate was omitted from this assay. Stimulation of FmoB activity apparently is optimal at concentrations between 1 mM and 2 mM, higher concentrations apparently inhibit the enzyme activity in these conditions.</p

The C-terminus and a central portion of NirA participate in determining NES accessibility.

<p><b>(A)</b> Lacking of the NirA activation domain (panel <i>kapK</i>1 NirA^-AD) leads to permanent, nitrate-independent nuclear localization of a NirA-GFP fusion protein. Strain <i>kapK</i>1 served as wild type control. Fluorescence microscopy shows cells grown in GMM with 3 mM arginine as a sole nitrogen source (NI) and cells induced for 5 minutes by 10 mM nitrate (IND). This strain shows in the conditions tested a stronger GFP signal if compared to the other analysed strains. <b>(B)</b> The NirA-form lacking the central region cannot accumulate in the nucleus and is only moderately active. The expressed construct fused 364 amino acids of the N-terminal part (containing the nuclear localization sequence, DNA-binding domain and the NES) with the last 154 amino acids of the C-terminal AD region. The figure captures fluorescence microscopy pictures of the strain expressing NirA^NiRDΔ-GFP under non-induced (NI, 3 mM arginine) or induced (IND, 10 mM nitrate) growth conditions. Scale bars refer to 5 μm. <b>(C)</b> Comparison between the growth on ammonium (NH₄), nitrate (NO₃^-) and nitrite (NO₂^-) of <i>gpdA</i>_p-<i>nirA</i>^NiRDΔ-<i>gfp</i> strain, the wild-type strain (WT) and two different strains harbouring respectively the <i>nirA</i> deletion (<i>nirA</i>^Δ) and the functional <i>nirA-gfp</i> expression driven by <i>gpdA</i> promoter (<i>gpdA</i>_<i>p</i><i>-nirA-gfp</i>). The strain lacking NiRD domain of <i>nirA</i> shows growth on nitrate and nitrite comparable to that one on ammonium. The approximate number of inoculated spores is indicated.</p

Amino acid changes and phenotypes of NirA mutants.

<p>Amino acid changes and phenotypes of NirA mutants.</p