Georg Golderer

Tetrahydrobiopterin Biosynthesis, Utilization and Pharmacological Effects

Tetrahydrobiopterin (H4-biopterin) is an essential cofactor of a set of enzymes that are of central metabolic importance, i.e. the hydroxylases of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan, of ether lipid oxidase, and of the three nitric oxide synthase (NOS) isoenzymes. As a consequence, H4-biopterin plays a key role in a vast number of biological processes and pathological states associated with neurotransmitter formation, vasorelaxation, and immune response. In mammals, its biosynthesis is controlled by hormones, cytokines and certain immune stimuli. This review aims to summarize recent developments concerning regulation of H4-biopterin biosynthetic and regulatory enzymes and pharmacological effects of H4-biopterin in various conditions, e.g. endothelial dysfunction or apoptosis of neuronal cells. Also, approaches towards gene therapy of diseases like the different forms of phenylketonuria or of Parkinsons disease are reviewed. Additional emphasis is given to H4-biopterin biosynthesis and function in non-mammalian species such as fruit fly, zebra fish, fungi, slime molds, the bacterium Nocardia as well as to the parasitic protozoan genus of Leishmania that is not capable of pteridine biosynthesis but has evolved a sophisticated salvage network for scavenging various pteridine compounds, notably folate and biopterin.

A first glimpse at the transcriptome of Physarum polycephalum

BackgroundPhysarum 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.MethodsHere we describe a large fraction of the transcribed genes by sequencing a cDNA library from the plasmodial stage of the developmental cycle.ResultsIn 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.ConclusionThis 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.

Enzymes involved in the dynamic equilibrium of core histone acetylation of Physarum polycephalum

DEAE‐Sepharose chromatography of extracts from plasmodia of the myxomycete Physarum polycephalum revealed the presence of multiple histone acetyltransferases and histone deacetylases. A cytoplasmic histone acetyltransferase B, specific for histone H4, and two nuclear acetyltransferases A1 and A2 were identified; A1 acetylates all core histones with a preference for H3 and H2A, whereas A2 is specific for H3 and also slightly for H2B. Two histone deacetylases, HD1 and HD2, could be discriminated. They differ with respect to substrate specificity and pH dependence. For the first time the substrate specificity of histone deacetylases was determined using HPLC‐purified individual core histone species. The order of acetylated substrate preference is H2A>H3>‐H4>H2B for HD1 and H3>H2A>H4 for HD2, respectively; HD2 is inactive with H2B as substrate. Moreover histone deacetylases are very sensitive to butyrate, since 2 mM butyrate leads to more than 50% inhibition of enzyme activity.

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling.

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 in Acanthamoeba 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.

Analysis of von Willebrand Factor Multimers by Simultaneous High- and Low-Resolution Vertical SDS-Agarose Gel Electrophoresis and Cy5-Labeled Antibody High-Sensitivity Fluorescence Detection

Analysis of von Willebrand factor (vWF) multimers allows classification of the subtypes of von Willebrand disease (vWD) in human serum and platelet lysates. A novel method for multimer analysis of vWF by 2-chamber, vertical (sodium dodecyl sulfate), agarose gel electrophoresis, designed for comparing discontinuous high- and low-resolving gels for plasma and platelets, followed by Western blotting and high-sensitivity fluorescence detection (HSFD) of cyanine (Cy)5-labeled vWF multimers is presented. HSFD shows that this method has high discriminatory power for visualization and densitometric analysis of platelets and plasma vWF multimers in various types of vWD and allows rapid classification of vWD types, to separate types 2A and 2B. The described procedures of vWF multimer analysis with high-sensitivity Cy5 fluorescence detection and direct comparison of high- and low-resolving gels for screening and detection of the complete range of high- and low-molecular vWF multimers is efficient and useful for screening, detecting, and classifying vWD subtypes and makes this method diagnostically and clinically relevant.

Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome

Significance We have recently identified the sequence of the ether lipid-cleaving enzyme alkylglycerol monooxygenase. Like nitric oxide synthases and aromatic amino acid hydroxylases, alkylglycerol monooxygenase needs tetrahydrobiopterin as cofactor. Whereas the former enzymes have well-established roles in the cell, the physiology of alkylglycerol monooxygenase is still not clear. Here we show its regulation in murine macrophage differentiation and its dependence on the cofactor tetrahydrobiopterin in live murine macrophage-like RAW264.7 cells. Upon modulation of the activity of alkylglycerol monooxygenase and the key enzyme in tetrahydrobiopterin biosynthesis, we observe extensive changes in various lipid classes ranging from ether lipids to far more complex lipids. These findings point to an important role of tetrahydrobiopterin in cellular lipid homeostasis. Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and “alternatively activated” M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide–induced expression of inducible nitric oxide synthase, interleukin-1β, and interleukin 1 receptor antagonist but not transforming growth factor β1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.

A gatekeeper helix determines the substrate specificity of Sjögren–Larsson Syndrome enzyme fatty aldehyde dehydrogenase

Mutations in the gene coding for membrane-bound fatty aldehyde dehydrogenase (FALDH) lead to toxic accumulation of lipid species and development of the Sjögren–Larsson Syndrome (SLS), a rare disorder characterized by skin defects and mental retardation. Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase. The dimeric FALDH displays a previously unrecognized element in its C-terminal region, a ‘gatekeeper’ helix, which extends over the adjacent subunit, controlling the access to the substrate cavity and helping orientate both substrate cavities towards the membrane surface for efficient substrate transit between membranes and catalytic site. Activity assays demonstrate that the gatekeeper helix is important for directing the substrate specificity of FALDH towards long-chain fatty aldehydes. The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases. Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations.

The amounts of alpha 1 antitrypsin protein are reduced in the vascular wall of the acutely dissected human ascending aorta

BACKGROUND Aneurysm and dissection of the ascending aorta carry the risk of life-threatening complications. The anti-protease alpha 1 antitrypsin plays an important role in the tissue protease - anti-protease equilibrium. We aim to investigate the molecular pathology of these diseases by differential proteomics and mass-spectrometric analysis. METHODS From ascending aortic wall specimens of aneurysms, acute dissections and controls, protein amounts were analysed by the differential in-gel electrophoresis (DIGE). Significantly, different spots underwent qualitative analysis by nanospray mass spectrometry. RESULTS Among the most significant differentially expressed protein spots in the DIGE analysis, the most notable protein identified by nanospray mass spectrometry was alpha 1 antitrypsin. This was significantly reduced in aneurysms and aortic dissections compared with controls (p<0.05). Western blot analysis confirmed the reduced amounts of alpha 1 antitrypsin in aortic dissections (p=0.008 vs controls) but not for aneurysms (p=0.258). By quantitative reverse transcription polymerase chain reaction (RT-PCR), mRNA level of alpha 1 antitrypsin was found to be increased in aortic dissections (p=0.035 vs controls), whereas in aneurysms a non-significant reduction of alpha 1 antitrypsin mRNA was present (p=0.123 vs controls). CONCLUSION In the vascular wall of ascending aortic dissections, alpha 1 antitrypsin protein amounts are reduced compared with healthy aortas. Local alpha 1 antitrypsin deficiency in the human ascending aorta might lead to proteolytic damage easing aortic dissection.

Proteomic profiling of acute cardiac allograft rejection.

Background. Proteome analysis has emerged as a valuable tool for the study of large-scale protein expression profiles. Here, we applied this novel technology to identify specific biomarkers for acute cardiac allograft rejection. Methods. Hearts of C57BL/10 mice were placed in fully major histocompatibility complex-mismatched C3H/He recipients. Syngeneic transplants served as controls. Intragraft protein expression analysis was performed using fluorescence two-dimensional difference gel electrophoresis on day 6 posttransplant. Spots of interest were subsequently subjected to nanospray ionization tandem mass spectrometry (MS/MS) for protein identification. In addition, expression of selected proteins was confirmed by Western blot analysis and by immunohistochemistry. Results. Two-dimensional difference gel electrophoresis enabled detection of 1541 protein spots. For 95 protein spots, the expression level during acute rejection differed by more than 1.5-fold from that observed in syngeneic grafts. Spots with significant differential regulation identified by tandem mass spectrometry were derived from peroxiredoxin 6, pyruvate kinase isozyme M2, coronin 1A, protein disulfide isomerase A3 precursor, and aconitate hydratase. Conclusion. These identified proteins may constitute novel biomarkers of acute cardiac allograft rejection and might hold great potential as surrogate markers for monitoring in vivo alloimmune response.

Widespread occurrence of glyceryl ether monooxygenase activity in rat tissues detected by a novel assay

An assay was set up for glyceryl ether monooxygenase activity in tissue samples using the novel substrate 1-O-pyrenedecyl-sn-glycerol and high-performance liquid chromatographic analysis of reaction mixtures with fluorescence detection, allowing robust detection of enzymatic activity in microgram amounts of tissue homogenates. The activity partially purified from rat liver strictly depended on the presence of a tetrahydropteridine. Tetrahydrobiopterin-dependent glyceryl ether monooxygenase activity was observed in all rat tissues tested except female heart, with highest activities in liver, intestine, and cerebellum. Activity was not uniformly distributed in brain: it was higher in cerebellum than in striatum or cortex. These data demonstrate that tetrahydrobiopterin-dependent glyceryl ether monooxygenase is found not only in liver and the gastrointestinal tract but also in brain and other organs of the rat and provide an additional goal for tetrahydrobiopterin biosynthesis in these organs.