Extensive in vivo metabolite-protein interactions revealed by large-scale systematic analyses

Natural small compounds comprise most cellular molecules and bind proteins as substrates, products, cofactors, and ligands. However, a large-scale investigation of in?vivo protein-small metabolite interactions has not been performed. We developed a mass spectrometry assay for the large-scale identification of in?vivo protein-hydrophobic small metabolite interactions in yeast and analyzed compounds that bind ergosterol biosynthetic proteins and protein kinases. Many of these proteins bind small metabolites; a few interactions were previously known, but the vast majority are new. Importantly, many key regulatory proteins such as protein kinases bind metabolites. Ergosterol was found to bind many proteins and may function as a general regulator. It is required for the activity of Ypk1, a mammalian AKT/SGK kinase homolog. Our study defines potential key regulatory steps in lipid biosynthetic pathways and suggests that small metabolites may play a more general role as regulators of protein activity and function than previously appreciated.     

Ecophysiology of Fe-cycling bacteria in acidic sediments

Using a combination of cultivation-dependent and -independent methods, this study aimed to elucidate the diversity of microorganisms involved in iron cycling and to resolve their in situ functional links in sediments of an acidic lignite mine lake. Using six different media with pH values ranging from 2.5 to 4.3, 117 isolates were obtained that grouped into 38 different strains, including 27 putative new species with respect to the closest characterized strains. Among the isolated strains, 22 strains were able to oxidize Fe(II), 34 were able to reduce Fe(III) in schwertmannite, the dominant iron oxide in this lake, and 21 could do both. All isolates falling into the Gammaproteobacteria (an unknown Dyella-like genus and Acidithiobacillus-related strains) were obtained from the top acidic sediment zones (pH 2.8). Firmicutes strains (related to Bacillus and Alicyclobacillus) were only isolated from deep, moderately acidic sediment zones (pH 4 to 5). Of the Alphaproteobacteria, Acidocella-related strains were only isolated from acidic zones, whereas Acidiphilium-related strains were isolated from all sediment depths. Bacterial clone libraries generally supported and complemented these patterns. Geobacter-related clone sequences were only obtained from deep sediment zones, and Geobacter-specific quantitative PCR yielded 8 × 10(5) gene copy numbers. Isolates related to the Acidobacterium, Acidocella, and Alicyclobacillus genera and to the unknown Dyella-like genus showed a broad pH tolerance, ranging from 2.5 to 5.0, and preferred schwertmannite to goethite for Fe(III) reduction. This study highlighted the variety of acidophilic microorganisms that are responsible for iron cycling in acidic environments, extending the results of recent laboratory-based studies that showed this trait to be widespread among acidophiles.     

The effects of dietary carotenoid intake on carotenoid accumulation in the retina of a wild bird, the house finch (Carpodacus mexicanus)

Carotenoid pigments accumulate in the retinas of many animals, including humans, where they play an important role in visual health and performance. Recently, birds have emerged as a model system for studying the mechanisms and functions of carotenoid accumulation in the retina. However, these studies have been limited to a small number of domesticated species, and the effects of dietary carotenoid access on retinal carotenoid accumulation have not been investigated in any wild animal species. The purpose of our studies was to examine how variation in dietary carotenoid types and levels affect retinal accumulation in house finches (Carpodacus mexicanus), a common and colorful North American songbird. We carried out three 8-week studies with wild-caught captive birds: (1) we tracked the rate of retinal carotenoid depletion, compared to other body tissues, on a very low-carotenoid diet, (2) we supplemented birds with two common dietary carotenoids (lutein + zeaxanthin) and measured the effect on retinal accumulation, and (3) we separately supplemented birds with high levels of zeaxanthin - an important dietary precursor for retinal carotenoids - or astaxanthin - a dominant retinal carotenoid not commonly found in the diet (i.e. a metabolic derivative). We found that carotenoids depleted slowly from the retina compared to other tissues, with a significant (∼50%) decline observed only after 8 weeks on a very low-carotenoid diet. Supplementation with lutein + zeaxanthin or zeaxanthin alone significantly increased only retinal galloxanthin and ε-carotene levels, while other carotenoid types in the retina remained unaffected. Concentrations of retinal astaxanthin were unaffected by direct dietary supplementation with astaxanthin. These results suggest highly specific mechanisms of retinal carotenoid metabolism and accumulation, as well as differential rates of turnover among retinal carotenoid types, all of which have important implications for visual health maintenance and interventions.     

Induced thyme product prevents VEGF-induced migration in human umbilical vein endothelial cells

Compounds with anti-angiogenic properties are useful in combating cancer by preventing new blood vessel formation to support the tumor. In this report we introduce a rapid method for screening potential anti-angiogenic compounds in a model system that stimulates the production of secondary defense chemicals in plants. This methodology identified an inducible vascular factor (IVF3), which was found to be inhibitory in all of the model systems tested. Thyme plants were exposed to highly vascular mint plants and the methanol extracts were analyzed by reverse phase HPLC. The thyme compounds induced by the invading mint tissue, and not present in the thyme plants grown alone, were tested in a vertical plate assay measuring root length as a quantitative assay for drug sensitivity. The HPLC-purified extract, referred to as IVF3, reduced the growth of root vascular tissue compared to the control and vehicle control, and 50% as well as known angiogenesis inhibitors, VEGF receptor tyrosine kinase inhibitor and amiloride hydrochloride. Extracted compounds that were effective inhibitors of plant roots were assayed in Madin Darby canine kidney epithelial cells (MDCK) for toxicity, and in human umbilical vein endothelial cells (HUVEC) for their effect on migration. IVF3 was effective at limiting HUVEC migration in VEGF-stimulated cultures. In vivo video capture of intersegmental vessel circulation between 48 and 72 h post fertilization in the developing vasculature of zebrafish embryos showed IVF3 also significantly reduced ISV functional circulation. This report demonstrates the anti-angiogenic effects of IVF3 extract in endothelial cells and in an intact vertebrate model for angiogenesis.     

Nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis

Nitronate monooxygenase (NMO), formerly referred to as 2-nitropropane dioxygenase, is an FMN-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from Neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. Over the past 5 years, a resurgence of interest on the enzymology of NMO has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. This review article summarizes the knowledge gained from these studies on NMO, which has been emerging as a model system for the investigation of anionic flavosemiquinone intermediates in the oxidative catalysis of organic molecules, and for the effect that branching of reaction intermediates has on both the kinetic parameters and isotope effects associated with enzymatic reactions. A comparison of the catalytic mechanism of NMO with other flavin-dependent enzymes that oxidize nitroalkane and nitronates is also presented.