Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme

Heterodisulfide reductase (Hdr) from methanogenic Archaea catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Upon reaction of the oxidized enzyme with CoM-SH a unique paramagnetic species is formed, which has been shown to be due to a novel type of [4Fe-4S](3+) cluster. In this work, it was addressed whether CoM-SH is directly attached to this [4Fe-4S] cluster using CoM-(33)SH as substrate and purified Hdr from Methanothermobacter marburgensis and Methanosarcina barkeri. With both enzymes treatment with CoM-(33)SH in the presence of duroquinone as an oxidant resulted in a significant broadening of the electron paramagnetic resonance spectrum as compared to CoM-SH as substrate. The signal broadening resulted from an unresolved anisotropic hyperfine coupling between the (33)S nucleus and the paramagnetic center. The results provide compelling evidence for a direct binding of CoM-SH to the [4Fe-4S] cluster in the active site of the enzyme.     

Compositional biases and polyalanine runs in humans

Human proteins containing polyalanine tracts tend to have runs of other amino acids and their open reading frames (ORFs) display a biased codon usage. Their alanine, glycine, proline, and histidine content strongly correlates with the GC content of the third codon base, suggesting that the compositional specificity of these proteins is dictated to a great extent by the evolution of their ORFs.     

Function and subnuclear distribution of Rpp21, a protein subunit of the human ribonucleoprotein ribonuclease P

Rpp21, a protein subunit of human nuclear ribonuclease P (RNase P) was cloned by virtue of its homology with Rpr2p, an essential subunit of Saccharomyces cerevisiae nuclear RNase P. Rpp21 is encoded by a gene that resides in the class I gene cluster of the major histocompatibility complex, is associated with highly purified RNase P, and binds precursor tRNA. Rpp21 is predominantly localized in the nucleoplasm but is also observed in nucleoli and Cajal bodies when expressed at high levels. Intron retention and splice-site selection in Rpp21 precursor mRNA regulate the intranuclear distribution of the protein products and their association with the RNase P holoenzyme. Our study reveals that dynamic nuclear structures that include nucleoli, the perinucleolar compartment and Cajal bodies are all involved in the production and assembly of human RNase P.     

Plasma cortisol in men--relationship with atherosclerosis of retinal arteries

The production of cortisol increases in acute stress but the effects of chronic stress on plasma cortisol are still controversial. Stress on the other hand plays a role in coronary artery disease (CAD) and carotid atherosclerosis. Since there is no data about plasma cortisol and atherosclerosis of the retinal arteries, the purpose of this study was to explore the relationship between plasma cortisol in 101 adult males with the degree of their retinal vessels atherosclerosis. The results were compared with those in 47 matched apparently healthy men with no retinal vessels changes. The atherosclerotic changes of retinal vessels were determined by direct ophthalmoscopy and graded (1-4) according to Scheie. Morning plasma cortisol levels were determined by radioimmunoassay using commercial kits. The results were compared by using chi-square test. No association between morning plasma cortisol concentrations and retinal vessels atherosclerosis could be found. The results of this study do not support a role for physiological levels of plasma cortisol in the development of atherosclerosis, at least of the retinal arteries, in men.     

Leishmania EF-1alpha activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation

The human leishmaniasis are persistent infections of macrophages caused by protozoa of the genus Leishmania. The chronic nature of these infections is in part related to induction of macrophage deactivation, linked to activation of the Src homology 2 domain containing tyrosine phosphatase-1 (SHP-1) in infected cells. To investigate the mechanism of SHP-1 activation, lysates of Leishmania donovani promastigotes were subjected to SHP-1 affinity chromatography and proteins bound to the matrix were sequenced by mass spectrometry. This resulted in the identification of Leishmania elongation factor-1alpha (EF-1alpha) as a SHP-1-binding protein. Purified Leishmania EF-1alpha, but not host cell EF-1alpha, bound directly to SHP-1 in vitro leading to its activation. Three independent lines of evidence indicated that Leishmania EF-1alpha may be exported from the phagosome thereby enabling targeting of host SHP-1. First, cytosolic fractions prepared from macrophages infected with [(35)S]methionine-labeled organisms contained Leishmania EF-1alpha. Second, confocal, fluorescence microscopy using Leishmania-specific antisera detected Leishmania EF-1alpha in the cytosol of infected cells. Third, co-immunoprecipitation showed that Leishmania EF-1alpha was associated with SHP-1 in vivo in infected cells. Finally, introduction of purified Leishmania EF-1alpha, but not the corresponding host protein into macrophages activated SHP-1 and blocked the induction of inducible nitric-oxide synthase expression in response to interferon-gamma. Thus, Leishmania EF-1alpha is identified as a novel SHP-1-binding and activating protein that recapitulates the deactivated phenotype of infected macrophages.     

Effects of T4 lysozyme release from transgenic potato roots on bacterial rhizosphere communities are negligible relative to natural factors

Rhizosphere bacterial communities of two transgenic potato lines which produce T4 lysozyme for protection against bacterial infections were analyzed in comparison to communities of wild-type plants and transgenic controls not harboring the lysozyme gene. Rhizosphere samples were taken from young, flowering, and senescent plants at two field sites in three consecutive years. The communities were characterized in a polyphasic approach. Cultivation-dependent methods included heterotrophic plate counts, determination of species composition and diversity based on fatty acid analysis of isolates, and community level catabolic profiling. Cultivation-independent analyses were based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from rhizosphere DNA using primers specific for Bacteria, Actinomycetales, or alpha- or beta-Proteobacteria. Several bands of the DGGE patterns were further characterized by sequence analysis. All methods revealed that environmental factors related to season, field site, or year but not to the T4 lysozyme expression of the transgenic plants influenced the rhizosphere communities. For one of the T4 lysozyme-producing cultivars, no deviation in the rhizosphere communities compared to the control lines was observed. For the other, differences were detected at some of the samplings between the rhizosphere community structure and those of one or all other cultivars which were not attributable to T4 lysozyme production but most likely to differences observed in the growth characteristics of this cultivar.     

A novel biosynthetic pathway providing precursors for fatty acid biosynthesis and secondary metabolite formation in myxobacteria

Short chain carboxylic acids are well known as the precursors of fatty acid and polyketide biosynthesis. Iso-fatty acids, which are important for the control of membrane fluidity, are formed from branched chain starter units (isovaleryl-CoA and isobutyryl-CoA), which in turn are derived from the degradation of leucine and valine, respectively. Branched chain carboxylic acids are also employed as starter molecules for the biosynthesis of secondary metabolites, e.g. the therapeutically important anthelmintic agent avermectin or the electron transport inhibitor myxothiazol. During our studies on myxothiazol biosynthesis in the myxobacterium, Stigmatella aurantiaca, we detected a novel biosynthetic route to isovaleric acid. After cloning and inactivation of the branched chain keto acid dehydrogenase complex, which is responsible for the degradation of branched chain amino acids, the strain is still able to produce iso-fatty acids and myxothiazol. Incorporation studies employing deuterated leucine show that it can only serve as precursor in the wild type strain but not in the esg mutant. Feeding experiments using (13)C-labeled precursors show that isovalerate is efficiently made from acetate, giving rise to a labeling pattern in myxothiazol that provides evidence for a novel branch of the mevalonate pathway involving the intermediate 3,3-dimethylacryloyl-CoA. 3,3-Dimethylacrylic acid was synthesized in deuterated form and fed to the esg mutant, resulting in strong incorporation into myxothiazol and iso-fatty acids. Similar experiments employing Myxococcus xanthus revealed that the discovered biosynthetic route described is present in other myxobacteria as well.     

Conformation and stability of alpha-helical membrane proteins. 2. Influence of pH and salts on stability and unfolding of rhodopsin

We studied the stability and pH-induced denaturation of rhodopsin and its photoproducts as a model for alpha-helical membrane proteins. The increased stability of the dark state of rhodopsin as compared to its photoproduct states allows the initiation of unfolding of the protein by light-dependent isomerization of the chromophore. We could therefore characterize the transition from the native to either acid or alkaline denatured states by light-induced Fourier transform infrared difference spectroscopy, UV-visible spectroscopy, and intrinsic tryptophan fluorescence spectroscopy. The results indicate a loss of important tertiary interactions within the protein and between the protein and the retinal chromophore in the denatured state, despite that the secondary structure of the protein is almost fully retained during the transition. We therefore propose that in this denatured state the protein adopts the conformation of a loose bundle of preserved, but only weakly interacting, transmembrane helices with a largely des-oriented and partly solvent-exposed chromophore. We further characterized the influence of salts on the stability of the rhodopsin helix bundle, which was found to follow the Hofmeister series. We found that the effect of sodium chloride may be stabilizing or destabilizing, depending on the intrinsic stability of the examined protein conformation and on salt concentration. In particular, sodium chloride is shown to counteract the formation of the denatured loose bundle state presumably by increasing the lateral pressure on the helix bundle, thereby stabilizing native-like tertiary contacts within the protein.     

Rosetta Stone proteins: "chance and necessity"?

A response to Functional associations of proteins in entire genomes by means of exhaustive detection of gene fusions by AJ Enright, CA Ouzounis. Genome Biology 2000, 2:research0034.1-0034.7     

Rosetta Stone proteins: "chance and necessity"?

A response to Functional associations of proteins in entire genomes by means of exhaustive detection of gene fusions by AJ Enright, CA Ouzounis. Genome Biology 2000, 2:research0034.1-0034.7