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Vacuolar proton-translocating ATPase (holoATPase and free membrane sector) was isolated from bovine chromaffin granules by blue native polyacrylamide gel electrophoresis. A 5-fold excess of membrane sector over holoenzyme was determined in isolated chromaffin granule membranes. M9.2, a novel extremely hydrophobic 9.2-kDa protein comprising 80 amino acids, was detected in the membrane sector. It shows sequence and structural similarity to Vma21p, a yeast protein required for assembly of vacuolar ATPase. A second membrane sector-associated protein (M8-9) was identified and characterized by amino-terminal protein sequencing.
The 2[4Fe-4S] ferredoxin from Chromatium vinosum arises as one prominent member of a recently defined family of proteins found in very diverse bacteria. The potentiometric circular dichroism titrations of the protein and of several molecular variants generated by site-directed mutagenesis have established that the reduction potentials of the two clusters differ widely by almost 200 mV. This large difference has been confirmed by electrochemical methods, and each redox transition has been assigned to one of the clusters. The unusually low potential center is surprisingly the one that displays a conventional CX1X2CX3X4C (Xn, variable amino acid) binding motif and a structural environment similar to that of clusters having less negative potentials. A comparison with other ferredoxins has highlighted factors contributing to the reduction potential of [4Fe-4S] clusters in proteins. (i) The loop between the coordinating cysteines 40 and 49 and the C terminus alpha-helix of C. vinosum ferredoxin cause a negative, but relatively moderate, shift of approximately 60 mV for the nearby cluster. (ii) Very negative potentials, below -600 mV, correlate with the presence of a bulky side chain in position X4 of the coordinating triad of cysteines. These findings set the framework in which previous observations on ferredoxins can be better understood. They also shed light onto the possible occurrence and properties of very low potential [4Fe-4S] clusters in less well characterized proteins.
The crystal structure of the bovine Rieske iron-sulfur protein indicates a sulfur atom (S-1) of the iron-sulfur cluster and the sulfur atom (Sgamma) of a cysteine residue that coordinates one of the iron atoms form hydrogen bonds with the hydroxyl groups of Ser-163 and Tyr-165, respectively. We have altered the equivalent Ser-183 and Tyr-185 in the Saccharomyces cerevisiae Rieske iron-sulfur protein by site-directed mutagenesis of the iron-sulfur protein gene to examine how these hydrogen bonds affect the midpoint potential of the iron-sulfur cluster and how changes in the midpoint potential affect the activity of the enzyme. Eliminating the hydrogen bond from the hydroxyl group of Ser-183 to S-1 of the cluster lowers the midpoint potential of the cluster by 130 mV, and eliminating the hydrogen bond from the hydroxyl group of Tyr-185 to Sgamma of Cys-159 lowers the midpoint potential by 65 mV. Eliminating both hydrogen bonds has an approximately additive effect, lowering the midpoint potential by 180 mV. Thus, these hydrogen bonds contribute significantly to the positive midpoint potential of the cluster but are not essential for its assembly. The activity of the bc1 complex decreases with the decrease in midpoint potential, confirming that oxidation of ubiquinol by the iron-sulfur protein is the rate-limiting partial reaction in the bc1 complex, and that the rate of this reaction is extensively influenced by the midpoint potential of the iron-sulfur cluster.
[Poster-Abstract] Formel zur Beurteilung der Blut-Liquor-Schrankenfunktion bei älteren Patienten
(1998)
The iron-sulfur proteins of the cytochrome bc1 complexes of Schizosaccharomyces pombe and Saccharomyces cerevisiae contain the three amino acid motif RX( downward arrow)(F/L/I)XX(T/S/G)XXXX (downward arrow) that is typical for proteins that are cleaved sequentially in two steps by matrix processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). Despite the presence of this recognition sequence the S. pombe iron-sulfur protein is processed only once during import into mitochondria, whereas the S. cerevisiae protein is processed in two steps. Import of S. pombe iron-sulfur protein in which the putative MIP or MPP recognition sites are eliminated by site-directed mutagenesis and import of iron-sulfur protein into mitochondria from yeast mutants that lack MIP activity indicate that one step processing of the S. pombe iron-sulfur protein is independent of those sites and of MIP activity. Sequencing of the mature protein obtained after import in vitro and of the endogenous iron-sulfur protein isolated from mitochondrial membranes by preparative 2D-electrophoresis shows that MPP recognizes a second site in the presequence and processing occurs between residues 43 and 44. If proline-20 of the S. pombe presequence is changed into a serine, a second cleavage step is induced. Conversely, if serine-24 of the S. cerevisiae presequence is changed to a proline, the first cleavage step that is normally catalyzed by MPP is blocked, causing precursor iron-sulfur protein to accumulate. Together these results indicate that a single amino acid change in the presequence is responsible for one-step processing in S. pombe versus two-step processing in S. cerevisiae.
Using apoE phenotyping by immunoblotting and apoE genotyping we identified four heterozygous carriers of a rare apolipoprotein (apo) E2 variant, apoE2 (Arg136 → Cys). ApoE2 (Arg136 → Cys) was not distinct from apoE2 (Arg158 → Cys) by phenotyping, but produced a unique pattern of bands on CfoI restriction typing of a 244 bp apoE gene fragment. Two of the four apoE2 (Arg136 → Cys)/3 heterozygotes had elevated triglycerides, two were normolipidemic. The composition of very low density lipoproteins (VLDL) was normal in each of the four apoE2 (Arg136 → Cys) carriers, regardless of the triglyceride concentrations. None of the apoE2 (Arg136 → Cys) carriers displayed a broad β-band and none revealed β-migrating particles in the VLDL. The two hypertriglyceridemic carriers of apoE2 (Arg136 → Cys) were, therefore, classified as having type IV rather than type III hyperlipoproteinemia. LDL receptor binding activities were studied using recombinant apoE loaded to dimyristoylphosphatidylcholine (DMPC) vesicles and to VLDL and from an apoE-deficient individual. LDL receptor binding of apoE2 (Arg136 → Cys) was 14% of apoE3 and was thus higher than that of apoE2 (Arg158 → Cys). Both apoE2 (Arg136 → Cys) and apoE2 (Arg158 → Cys) displayed substantial heparin binding (61 and 53% of apoE3, respectively). As the dominant apoE variants known so far are characterized by more pronounced reductions of heparin binding, we suggest that apoE2 (Arg136 → Cys) is not associated with dominant expression of type III hyperlipoproteinemia. These findings lend support to the concept that apoE variants predisposing to dominant type III hyperlipoproteinemia differ from recessive mutations by a more severe defect in heparin binding.—März, W., M. M. Hoffmann, H. Scharnagl, E. Fisher, M. Chen, M. Nauck, G. Feussner, and H. Wieland. Apolipoprotein E2 (Arg136 → Cys) mutation in the receptor binding domain of apoE is not associated with dominant type III hyperlipoproteinemia.
A gene trap strategy has been used to identify genes that are repressed in cells transformed by an activated epidermal growth factor (EGF)/EGF receptor signal transduction pathway. EGF receptor-expressing NIH3T3 cells (HER1 cells) were infected with a retrovirus containing coding sequences for the human CD2 antigen and for secreted alkaline phosphatase in the U3 region. By selecting for and against CD2 expression, we obtained clones in which the gene trap had integrated into genes selectively repressed by EGF. Two of these clones encoded for the secreted extracellular matrix proteins TIMP3 and COL1A2. We show here that both genes are downstream targets of RAS and are specifically repressed by EGF-induced transformation. Moreover, this strategy tags tumor suppressor genes in their normal chromosomal location, thereby improving target-specific screens for antineoplastic drugs.
The human hemopoietic cell kinase (HCK) is a member of the src family of protein tyrosine kinases specifically expressed in myeloid cells and to a minor extent in B-lymphoid cells. HCK expression is up-regulated at the transcriptional level during myeloid differentiation of hematopoietic cells. To elucidate the molecular basis of the differential HCK gene expression, the genomic region containing the HCK promoter was isolated and functionally characterized. A DNA fragment containing 101 base pairs of the 5′-flanking sequence showed strong promoter activity in the macrophage cell line RAW264 but was inactive in the non-monocytic cell lines HUT-78 and NIH-3T3. Site-directed mutagenesis of the proximal promoter region showed that two GC-rich sequence elements are essential for transcriptional activity in myeloid cells. Electrophoretic mobility shift analysis using nuclear extracts obtained from RAW264 cells and from the promonocytic cell line U-937 revealed the formation of at least three distinct protein-DNA complexes at each of these sites, one of which was found to contain the transcription factor Sp1. Expression of a reporter gene linked to the −101HCK promoter region was up-regulated by Sp1, but not by other members of the Sp1 family of transcription factors, in Drosophila Schneider cells. A synergistic effect onHCK promoter activity was observed at high concentrations of Sp1. Our results show that Sp1 plays an essential role in the regulation of the differential gene expression of the HCKgene.
Objectives: The possible etiologic relevance of occupational factors such as cadmium, cutting oils, diesel fuel and fumes, herbicides, polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls, soot, tar, mineral oil, and solvents to prostate cancer was studied.
Methods: A case-referent study design was used to recruit 192 subjects with histologically confirmed prostate cancer and 210 referents who had prostate cancer histologically excluded either in one of two urologic practices (Hamburg and Frankfurt) or in the urological policlinic of the Frankfurt University. Data were gathered with a self-administered questionnaire and analyzed using logistic regression to control for age, region, and cigarette smoking. A job-exposure matrix was used for assigning exposure. For the calculation of dose-years, the duration of contact with specific substances was weighted by the intensity and probability of exposure according to a job-exposure matrix.
Results: The analysis of dose-years yielded a statistically significant association between occupational exposure to diesel fuel or fumes and prostate cancer (odds ratio 3.7, 95% confidence interval 1.4-9.8, for subjects exposed to more than 25 dose-years in a comparison with subjects never exposed). For the other substances, no statistically significant differences in exposure were found between the cases and referents. When only jobs with a high exposure probability were used to classify the participants as exposed, only exposure to PAH was significantly associated with prostate cancer.
Conclusion: In keeping with results from other studies, this study provides further evidence that exposure to diesel fuel or fumes - possibly mediated through PAH - may be associated with the development of prostate cancer.