Role of Glycoside Phosphorylases in Mannose Foraging by Human Gut Bacteria

To metabolize both dietary fiber constituent carbohydrates and host glycans lining the intestinal epithelium, gut bacteria produce a wide range of carbohydrate-active enzymes, of which glycoside hydrolases are the main components. In this study, we describe the ability of phosphorylases to participate in the breakdown of human N-glycans, from an analysis of the substrate specificity of UhgbMP, a mannoside phosphorylase of the GH130 protein family discovered by functional metagenomics. UhgbMP is found to phosphorolyze β-d-Manp-1,4-β-d-GlcpNAc-1,4-d-GlcpNAc and is also a highly efficient enzyme to catalyze the synthesis of this precious N-glycan core oligosaccharide by reverse phosphorolysis. Analysis of sequence conservation within family GH130, mapped on a three-dimensional model of UhgbMP and supported by site-directed mutagenesis results, revealed two GH130 subfamilies and allowed the identification of key residues responsible for catalysis and substrate specificity. The analysis of the genomic context of 65 known GH130 sequences belonging to human gut bacteria indicates that the enzymes of the GH130_1 subfamily would be involved in mannan catabolism, whereas the enzymes belonging to the GH130_2 subfamily would rather work in synergy with glycoside hydrolases of the GH92 and GH18 families in the breakdown of N-glycans. The use of GH130 inhibitors as therapeutic agents or functional foods could thus be considered as an innovative strategy to inhibit N-glycan degradation, with the ultimate goal of protecting, or restoring, the epithelial barrier.     

Novel Key Metabolites Reveal Further Branching of the Roquefortine/Meleagrin Biosynthetic Pathway

Metabolic profiling and structural elucidation of novel secondary metabolites obtained from derived deletion strains of the filamentous fungus Penicillium chrysogenum were used to reassign various previously ascribed synthetase genes of the roquefortine/meleagrin pathway to their corresponding products. Next to the structural characterization of roquefortine F and neoxaline, which are for the first time reported for P. chrysogenum, we identified the novel metabolite roquefortine L, including its degradation products, harboring remarkable chemical structures. Their biosynthesis is discussed, questioning the exclusive role of glandicoline A as key intermediate in the pathway. The results reveal that further enzymes of this pathway are rather unspecific and catalyze more than one reaction, leading to excessive branching in the pathway with meleagrin and neoxaline as end products of two branches.     

Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4^−/−Abca4^−/−Rdh8^−/− mice displayed milder retinal degenerative phenotypes than Abca4^−/−Rdh8^−/− mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.     

New Trends in Cytochrome P450 Research at the Half-Century Mark

Cytochrome P450 enzymes have major roles in the metabolism of steroids, drugs, carcinogens, eicosanoids, and numerous other chemicals. The P450s are collectively considered the most diverse catalysts known in biochemistry, although they operate from a basic structural fold and catalytic mechanism. The four minireviews in this thematic series deal with the unusual aspects of catalytic reactions and electron transfer pathway organization, the structural diversity of P450s, and the expanding roles of P450s in disease and medicine.     

hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria

Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency.     

A Metabolomics-driven Elucidation of the Anti-obesity Mechanisms of Xanthohumol

Mild, mitochondrial uncoupling increases energy expenditure and can reduce the generation of reactive oxygen species (ROS). Activation of cellular, adaptive stress response pathways can result in an enhanced capacity to reduce oxidative damage. Together, these strategies target energy imbalance and oxidative stress, both underlying factors of obesity and related conditions such as type 2 diabetes. Here we describe a metabolomics-driven effort to uncover the anti-obesity mechanism(s) of xanthohumol (XN), a prenylated flavonoid from hops. Metabolomics analysis of fasting plasma from obese, Zucker rats treated with XN revealed decreases in products of dysfunctional fatty acid oxidation and ROS, prompting us to explore the effects of XN on muscle cell bioenergetics. At low micromolar concentrations, XN acutely increased uncoupled respiration in several different cell types, including myocytes. Tetrahydroxanthohumol also increased respiration, suggesting electrophilicity did not play a role. At higher concentrations, XN inhibited respiration in a ROS-dependent manner. In myocytes, time course metabolomics revealed acute activation of glutathione recycling and long term induction of glutathione synthesis as well as several other changes indicative of short term elevated cellular stress and a concerted adaptive response. Based on these findings, we hypothesize that XN may ameliorate metabolic syndrome, at least in part, through mitochondrial uncoupling and stress response induction. In addition, time course metabolomics appears to be an effective strategy for uncovering metabolic events that occur during a stress response.     

High Dietary Fat Selectively Increases Catalase Expression within Cardiac Mitochondria

Obesity is a predictor of diabetes and cardiovascular disease. One consequence of obesity is dyslipidemia characterized by high blood triglycerides. It has been proposed that oxidative stress, driven by utilization of lipids for energy, contributes to these diseases. The effects of oxidative stress are mitigated by an endogenous antioxidant enzyme network, but little is known about its response to high fat utilization. Our experiments used a multiplexed quantitative proteomics method to measure antioxidant enzyme expression in heart tissue in a mouse model of diet-induced obesity. This experiment showed a rapid and specific up-regulation of catalase protein, with subsequent assays showing increases in activity and mRNA. Catalase, traditionally considered a peroxisomal protein, was found to be present in cardiac mitochondria and significantly increased in content and activity during high fat feeding. These data, coupled with the fact that fatty acid oxidation enhances mitochondrial H₂O₂ production, suggest that a localized catalase increase is needed to consume excessive mitochondrial H₂O₂ produced by increased fat metabolism. To determine whether the catalase-specific response is a common feature of physiological conditions that increase blood triglycerides and fatty acid oxidation, we measured changes in antioxidant expression in fasted versus fed mice. Indeed, a similar specific catalase increase was observed in mice fasted for 24 h. Our findings suggest a fundamental metabolic process in which catalase expression is regulated to prevent damage while preserving an H₂O₂-mediated sensing of diet composition that appropriately adjusts insulin sensitivity in the short term as needed to prioritize lipid metabolism for complete utilization.