Odd André Karlsen

Human Naa50p (Nat5/San) Displays Both Protein Nα- and Nϵ-Acetyltransferase Activity

Protein acetylation is a widespread modification that is mediated by site-selective acetyltransferases. KATs (lysine Nϵ-acetyltransferases), modify the side chain of specific lysines on histones and other proteins, a central process in regulating gene expression. Nα-terminal acetylation occurs on the ribosome where the α amino group of nascent polypeptides is acetylated by NATs (N-terminal acetyltransferase). In yeast, three different NAT complexes were identified NatA, NatB, and NatC. NatA is composed of two main subunits, the catalytic subunit Naa10p (Ard1p) and Naa15p (Nat1p). Naa50p (Nat5) is physically associated with NatA. In man, hNaa50p was shown to have acetyltransferase activity and to be important for chromosome segregation. In this study, we used purified recombinant hNaa50p and multiple oligopeptide substrates to identify and characterize an Nα-acetyltransferase activity of hNaa50p. As the preferred substrate this activity acetylates oligopeptides with N termini Met-Leu-Xxx-Pro. Furthermore, hNaa50p autoacetylates lysines 34, 37, and 140 in vitro, modulating hNaa50p substrate specificity. In addition, histone 4 was detected as a hNaa50p KAT substrate in vitro. Our findings thus provide the first experimental evidence of an enzyme having both KAT and NAT activities.

Analysing the outer membrane subproteome of Methylococcus capsulatus (Bath) using proteomics and novel biocomputing tools

High-resolution two-dimensional gel electrophoresis and mass spectrometry has been used to identify the outer membrane (OM) subproteome of the Gram-negative bacterium Methylococcus capsulatus (Bath). Twenty-eight unique polypeptide sequences were identified from protein samples enriched in OMs. Only six of these polypeptides had previously been identified. The predictions from novel bioinformatic methods predicting β-barrel outer membrane proteins (OMPs) and OM lipoproteins were compared to proteins identified experimentally. BOMP (http://www.bioinfo.no/tools/bomp) predicted 43 β-barrel OMPs (1.45%) from the 2,959 annotated open reading frames. This was a lower percentage than predicted from other Gram-negative proteomes (1.8–3%). More than half of the predicted BOMPs in M. capsulatus were annotated as (conserved) hypothetical proteins with significant similarity to very few sequences in Swiss-Prot or TrEMBL. The experimental data and the computer predictions indicated that the protein composition of the M. capsulatus OM subproteome was different from that of other Gram-negative bacteria studied in a similar manner. A new program, Lipo, was developed that can analyse entire predicted proteomes and give a list of recognised lipoproteins categorised according to their lipo-box similarity to known Gram-negative lipoproteins (http://www.bioinfo.no/tools/lipo). This report is the first using a proteomics and bioinformatics approach to identify the OM subproteome of an obligate methanotroph.

Characterization of a prokaryotic haemerythrin from the methanotrophic bacterium Methylococcus capsulatus (Bath)

For a long time, the haemerythrin family of proteins was considered to be restricted to only a few phyla of marine invertebrates. When analysing differential protein expression in the methane‐oxidizing bacterium, Methylococcus capsulatus (Bath), grown at a high and low copper‐to‐biomass ratio, respectively, we identified a putative prokaryotic haemerythrin expressed in high‐copper cultures. Haemerythrins are recognized by a conserved sequence motif that provides five histidines and two carboxylate ligands which coordinate two iron atoms. The diiron site is located in a hydrophobic pocket and is capable of binding O2. We cloned the M. capsulatus haemerythrin gene and expressed it in Escherichia coli as a fusion protein with NusA. The haemerythrin protein was purified to homogeneity cleaved from its fusion partner. Recombinant M. capsulatus haemerythrin (McHr) was found to fold into a stable protein. Sequence similarity analysis identified all the candidate residues involved in the binding of diiron (His22, His58, Glu62, His77, His81, His117, Asp122) and the amino acids forming the hydrophobic pocket in which O2 may bind (Ile25, Phe59, Trp113, Leu114, Ile118). We were also able to model a three‐dimensional structure of McHr maintaining the correct positioning of these residues. Furthermore, UV/vis spectrophotometric analysis demonstrated the presence of conjugated diiron atoms in McHr. A comprehensive genomic database search revealed 21 different prokaryotes containing the haemerythrin signature (PROSITE 00550), indicating that these putative haemerythrins may be a conserved prokaryotic subfamily.

Global transcriptome analysis of Atlantic cod (Gadus morhua) liver after in vivo methylmercury exposure suggests effects on energy metabolism pathways.

Methylmercury (MeHg) is a widely distributed contaminant polluting many aquatic environments, with health risks to humans exposed mainly through consumption of seafood. The mechanisms of toxicity of MeHg are not completely understood. In order to map the range of molecular targets and gain better insights into the mechanisms of toxicity, we prepared Atlantic cod (Gadus morhua) 135k oligonucleotide arrays and performed global analysis of transcriptional changes in the liver of fish treated with MeHg (0.5 and 2 mg/kg of body weight) for 14 days. Inferring from the observed transcriptional changes, the main pathways significantly affected by the treatment were energy metabolism, oxidative stress response, immune response and cytoskeleton remodeling. Consistent with known effects of MeHg, many transcripts for genes in oxidative stress pathways such as glutathione metabolism and Nrf2 regulation of oxidative stress response were differentially regulated. Among the differentially regulated genes, there were disproportionate numbers of genes coding for enzymes involved in metabolism of amino acids, fatty acids and glucose. In particular, many genes coding for enzymes of fatty acid beta-oxidation were up-regulated. The coordinated effects observed on many transcripts coding for enzymes of energy pathways may suggest disruption of nutrient metabolism by MeHg. Many transcripts for genes coding for enzymes in the synthetic pathways of sulphur containing amino acids were also up-regulated, suggesting adaptive responses to MeHg toxicity. By this toxicogenomics approach, we were also able to identify many potential biomarker candidate genes for monitoring environmental MeHg pollution. These results based on changes on transcript levels, however, need to be confirmed by other methods such as proteomics.

An Oxidized Tryptophan Facilitates Copper Binding in Methylococcus capsulatus-secreted Protein MopE

Proteins can coordinate metal ions with endogenous nitrogen and oxygen ligands through backbone amino and carbonyl groups, but the amino acid side chains coordinating metals do not include tryptophan. Here we show for the first time the involvement of the tryptophan metabolite kynurenine in a protein metal-binding site. The crystal structure to 1.35Å of MopE* from the methane-oxidizing Methylococcus capsulatus (Bath) provided detailed information about its structure and mononuclear copper-binding site. MopE* contains a novel protein fold of which only one-third of the structure displays similarities to other known folds. The geometry around the copper ion is distorted tetrahedral with one oxygen ligand from a water molecule, two histidine imidazoles (His-132 and His-203), and at the fourth distorted tetrahedral position, the N1 atom of the kynurenine, an oxidation product of Trp-130. Trp-130 was not oxidized to kynurenine in MopE* heterologously expressed in Escherichia coli, nor did this protein bind copper. Our findings indicate that the modification of tryptophan to kynurenine and its involvement in copper binding is an innate property of M. capsulatus MopE*.

The Surface-Associated and Secreted MopE Protein of Methylococcus capsulatus (Bath) Responds to Changes in the Concentration of Copper in the Growth Medium

ABSTRACT Expression of surface-associated and secreted protein MopE of the methanotrophic bacterium Methylococcus capsulatus (Bath) in response to the concentration of copper ions in the growth medium was investigated. The level of protein associated with the cells and secreted to the medium changed when the copper concentration in the medium varied and was highest in cells exposed to copper stress.

Precision-cut liver slices of Atlantic cod (Gadus morhua): An in vitro system for studying the effects of environmental contaminants

The Atlantic cod (Gadus morhua) is an economically important species commonly consumed by humans. The widespread distribution of cod in the North Atlantic Ocean makes it vulnerable to effluents from human activities, such as coastal industries and offshore petroleum exploration. It has been demonstrated that many effluents have adverse effects on cod reproduction and health, e.g. by disrupting endocrine signaling pathways. The liver, expressing important components of the biotransformation and the endocrine system, is one of the main target organs. Thus, reliable and reproducible in vitro systems of the liver are important for studying effects of environmental contaminants. The aim of this study was to investigate precision-cut liver slices (PCLS) as an alternative in vitro system for toxicological studies of the Atlantic cod liver. Slices of 8 mm in diameter and 250 μm thickness were prepared and cultivated from immature cod. Several analyses to measure the liver slice viability were performed: enzyme assays, histology, and morphometric analysis, all confirming cell viability for up to 72 h in culture. The liver slices were also exposed to two well-known model environmental contaminants, β-naphthoflavone (BNF) and 17α-ethynylestradiol (EE2), representing established agonists for the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER), respectively. The results showed increased transcription of the target genes cytochrome P450 1A (CYP1A) and vitellogenin (VTG), both well-established biomarkers for exposure of fish to the selected compounds. In conclusion, PCLS is a promising in vitro system for toxicological studies of cod liver cells. The liver slices are viable in culture for several days and respond to environmental contaminants in a dose- and time-specific manner.

The presence of multiple c-type cytochromes at the surface of the methanotrophic bacterium Methylococcus capsulatus (Bath) is regulated by copper

Identification of surface proteins is essential to understand bacterial communication with its environment. Analysis of the surface‐associated proteins of Methylococcus capsulatus (Bath) revealed a highly dynamic structure responding closely to the availability of copper in the medium in the range from ∼0 to 10 μM. Several c‐type cytochromes, including three novel multihaem proteins, are present at the cellular surface, a feature that is otherwise a peculiarity of dissimilatory metal‐reducing bacteria. At low copper concentrations, the cytochrome c553o and the cytochrome c553o family protein, encoded by the MCA0421 and MCA0423 genes, respectively, are major constituents of the surfaceome and show a fine‐tuned copper‐dependent regulation of expression. Two novel members of the cytochrome c553o family were identified: MCA0338 was abundant between 5 and 10 μM copper, while MCA2259 was detected only in the surface fraction obtained from ∼0 μM copper cultures. The presence at the bacterial surface of several c‐type cytochromes, generally involved in energy transduction, indicates strongly that redox processes take place at the bacterial surface. Due to the unique role of copper in the biology of M. capsulatus (Bath), it appears that c‐type cytochromes have essential functions in copper homeostasis allowing the cells to adapt to varying copper exposure.

Identification of a copper-repressible C-type heme protein of Methylococcus capsulatus (Bath). A member of a novel group of the bacterial di-heme cytochrome c peroxidase family of proteins.

Genomic sequencing of the methanotrophic bacterium, Methylococcus capsulatus (Bath), revealed an open reading frame (MCA2590) immediately upstream of the previously described mopE gene (MCA2589). Sequence analyses of the deduced amino acid sequence demonstrated that the MCA2590‐encoded protein shared significant, but restricted, sequence similarity to the bacterial di‐heme cytochrome c peroxidase (BCCP) family of proteins. Two putative C‐type heme‐binding motifs were predicted, and confirmed by positive heme staining. Immunospecific recognition and biotinylation of whole cells combined with MS analyses confirmed expression of MCA2590 in M. capsulatus as a protein noncovalently associated with the cellular surface of the bacterium exposed to the cell exterior. Similar to MopE, expression of MCA2590 is regulated by the bioavailability of copper and is most abundant in M. capsulatus cultures grown under low copper conditions, thus indicating an important physiological role under these growth conditions. MCA2590 is distinguished from previously characterized members of the BCCP family by containing a much longer primary sequence that generates an increased distance between the two heme‐binding motifs in its primary sequence. Furthermore, the surface localization of MCA2590 is in contrast to the periplasmic location of the reported BCCP members. Based on our experimental and bioinformatical analyses, we suggest that MCA2590 is a member of a novel group of bacterial di‐heme cytochrome c peroxidases not previously characterized.

Conservation and divergence of chemical defense system in the tunicate Oikopleura dioica revealed by genome wide response to two xenobiotics.

BackgroundAnimals have developed extensive mechanisms of response to xenobiotic chemical attacks. Although recent genome surveys have suggested a broad conservation of the chemical defensome across metazoans, global gene expression responses to xenobiotics have not been well investigated in most invertebrates. Here, we performed genome survey for key defensome genes in Oikopleura dioica genome, and explored genome-wide gene expression using high density tiling arrays with over 2 million probes, in response to two model xenobiotic chemicals - the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) the pharmaceutical compound Clofibrate (Clo).ResultsOikopleura genome surveys for key genes of the chemical defensome suggested a reduced repertoire. Not more than 23 cytochrome P450 (CYP) genes could be identified, and neither CYP1 family genes nor their transcriptional activator AhR was detected. These two genes were present in deuterostome ancestors. As in vertebrates, the genotoxic compound BaP induced xenobiotic biotransformation and oxidative stress responsive genes. Notable exceptions were genes of the aryl hydrocarbon receptor (AhR) signaling pathway. Clo also affected the expression of many biotransformation genes and markedly repressed genes involved in energy metabolism and muscle contraction pathways.ConclusionsOikopleura has the smallest number of CYP genes among sequenced animal genomes and lacks the AhR signaling pathway. However it appears to have basic xenobiotic inducible biotransformation genes such as a conserved genotoxic stress response gene set. Our genome survey and expression study does not support a role of AhR signaling pathway in the chemical defense of metazoans prior to the emergence of vertebrates.