Search

Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis-Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase.

Alkylglycerol monooxygenase (AGMO; EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl ether bond of alkylglycerols in humans. It is an integral membrane protein with nine predicted transmembrane domains. We attempted to express and purify full-length and truncated forms of AGMO in Escherichia coli. Full-length AGMO could not be expressed in three different E. coli expression strains, three different expression vectors and several induction systems. We succeeded, however, in expression of three N-terminally strep-tagged truncated forms, named active sites 1, 2 and 3, with 205, 134 and 61 amino acids, respectively. Active site 1 fragment, containing two predicted transmembrane regions, a membrane associated region and all known amino acid residues important for catalytic activity, was not fully soluble even in 8 M urea. Active site 2 containing only one predicted membrane associated domain required 8 M urea for solubilisation and eluted in gel filtration in 1 M urea as a trimer. Active site 3 with no hydrophobic domain eluted in gel filtration in 1 M urea as monomer and dimer. These results show that even truncated forms of AGMO are barely soluble when expressed in E. coli and show a high tendency for aggregation.

Alkylglycerol monooxygenase is a tetrahydrobiopterin-dependent enzyme that cleaves the O-alkyl-bond of alkylglycerols. It is an exceptionally unstable, hydrophobic membrane protein which has never been purified in active form. Recently, we were able to identify the sequence of alkylglycerol monooxygenase. TMEM195, the gene coding for alkylglycerol monooxygenase, belongs to the fatty acid hydroxylases, a family of integral membrane enzymes which have an 8-histidine motif crucial for catalysis. Mutation of each of these residues resulted in a complete loss of activity. We now extended the mutational analysis to another 25 residues and identified three further residues conserved throughout all members of the fatty acid hydroxylases which are essential for alkylglycerol monooxygenase activity. Furthermore, mutation of a specific glutamate resulted in an 18-fold decreased affinity of the protein to tetrahydrobiopterin, strongly indicating a potential important role in cofactor interaction. A glutamate residue in a comparable amino acid surrounding had already been shown to be responsible for tetrahydrobiopterin binding in the aromatic amino acid hydroxylases. Ab initio modelling of the enzyme yielded a structural model for the central part of alkylglycerol monooxygenase where all essential residues identified by mutational analysis are in close spatial vicinity, thereby defining the potential catalytic site of this enzyme.

The tetrahydrobiopterin-dependent degradation of ether lipids by alkylglycerol monooxygenase (AGMO) produces fatty aldehydes, which are toxic to cells. Therefore, it is of great physiological importance that these harmful compounds are converted into their corresponding, less toxic fatty acids by fatty aldehyde dehydrogenase (FALDH). Dysfunction of this enzyme causes Sjögren-Larsson syndrome. This severe inherited disorder is accompanied by symptoms such as ichthyosis, mental retardation and spasticity. Surprisingly, fatty alcohols and not fatty aldehydes were found to accumulate in fibroblasts of Sjögren-Larsson syndrome patients, suggesting that there can be wide-ranging alterations in the lipid composition of patient cells. In particular, this has to be considered when searching for possible treatment options for patients suffering from Sjögren-Larsson syndrome. For example, inhibition of fatty aldehyde producing ether lipid degradation would have multiple implications on ether lipid- and fatty alcohol-mediated signalling pathways.

We investigated a possible involvement of nitric oxide formed by inducible nitric oxide synthase (iNOS) in the signaling cascade leading to growth inhibition and differentiation in the human neuroblastoma cell line SK-NSII. Treatment of SK-N-SH with interferon-γ (IFN-γ) plus interleukin-lß (IL-lß) led to induction of iNOS, growth inhibition and an altered cell shape. However two inhibitors of iNOS were not able to prevent cytokine induced changes. In addition, IFN-γ alone led to growth inhibition in absence of iNOS induction. Inhibition of the induced indoleamine 2,3-dioxygenase (IDO) activity also did not prevent growth inhibition. Our findings show that mechanisms other than NO and IDO can control interferon-y-induced growth inhibition of SK-N-SH cells.

Nitric oxide (NO) synthase has been found to be induced in Physarum polycephalum sporulation. P. polycephalum expresses a calcium-independent NO synthase like the inducible NO synthase (NOS) isoenzyme in mammals. It has been cloned and sequnced and is the first nonanimal NOS identified. Less than 39% of its amino acids are identical to those of known NOSs. The second messenger used for NO siglnaling in sporulation is cGMP. It links the signaling pathway to expression of lig1.

An analysis of ribosomal proteins MvaL1, MvaL10 and MvaL12, encoded by the MvaL1 operon of methanogenic archaebacterium Methanococcus vannielii, involved a study of the regulation of the preparation of these ribosomal proteins. M. vannielii possesses a unique translational feedback regulation. An mRNA sequence which lacks direct contact with the ribosome, contains the putative MvaL1 binding region.

Abstract Background Physarum polycephalum, an acellular plasmodial species belongs to the amoebozoa, a major branch in eukaryote evolution. Its complex life cycle and rich cell biology is reflected in more than 2500 publications on various aspects of its biochemistry, developmental biology, cytoskeleton, and cell motility. It now can be genetically manipulated, opening up the possibility of targeted functional analysis in this organism. Methods Here we describe a large fraction of the transcribed genes by sequencing a cDNA library from the plasmodial stage of the developmental cycle. Results In addition to the genes for the basic metabolism we found an unexpected large number of genes involved in sophisticated signaling networks and identified potential receptors for environmental signals such as light. In accordance with the various developmental options of the plasmodial cell we found that many P. polycephalum genes are alternatively spliced. Using 30 donor and 30 acceptor sites we determined the splicing signatures of this species. Comparisons to various other organisms including Dictyostelium, the closest relative, revealed that roughly half of the transcribed genes have no detectable counterpart, thus potentially defining species specific adaptations. On the other hand, we found highly conserved proteins, which are maintained in the metazoan lineage, but absent in D. discoideum or plants. These genes arose possibly in the last common ancestor of Amoebozoa and Metazoa but were lost in D. discoideum. Conclusion This work provides an analysis of up to half of the protein coding genes of Physarum polycephalum. The definition of splice motifs together with the description of alternatively spliced genes will provide a valuable resource for the ongoing genome project.

The transcription factor FOXO3 is associated with poor outcome in high-stage neuroblastoma (NB), as it facilitates chemoprotection and tumor angiogenesis. In other tumor entities, FOXO3 stimulates metastasis formation, one of the biggest challenges in the treatment of aggressive NB. However, the impact of FOXO3 on the metastatic potential of neuronal tumor cells remains largely unknown. In the present study, we uncover the small leucine-rich proteoglycan family member lumican (LUM) as a FOXO3-regulated gene that stimulates cellular migration in NB. By a drug-library screen we identified the small molecular weight compound repaglinide (RPG) as a putative FOXO3 inhibitor. Here, we verify that RPG binds to the FOXO3-DNA-binding-domain (DBD) and thereby silences the transcriptional activity of FOXO3. Consistent with the concept that the FOXO3/LUM axis enhances the migratory capacity of aggressive NB cells, we demonstrate that stable knockdown of LUM abrogates the FOXO3-mediated increase in cellular migration. Importantly, FOXO3 inhibition by RPG represses the binding of FOXO3 to the LUM promoter, inhibits FOXO3-mediated LUM RNA and protein expression, and efficiently abrogates FOXO3-triggered cellular “wound healing” as well as spheroid-based 3D-migration. Thus, silencing the FOXO3/LUM axis by the FDA-approved compound RPG represents a promising strategy for novel therapeutic interventions in NB and other FOXO3-dependent tumors. [ABSTRACT FROM AUTHOR]

The ribosomal protein MvaS7 in the archaeon Methanococcus vannielii appears to consist of 188 amino acids and its transcription begins at a TTG start codon. MvaS7 was thought to have one of three possible start codons. Each of the three possible forms of the protein was compared to the protein taken from the 30S ribosomal subunit. MvaS7 tuned out to be 42 amino acids longer than was previously thought and the TTG start codon functioned in Escherichia coli.

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases inAcanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer. [ABSTRACT FROM AUTHOR]

Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol monooxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a Cterminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidinemotif, onlymutation of Glu137 to alanine led to an 18-fold increase in the Michaelis-Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase. [ABSTRACT FROM AUTHOR]