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At least 1,693 records · Page 94Linked to original sources
PubMed2003

The siderophore system is essential for viability of Aspergillus nidulans: functional analysis of two genes encoding l-ornithine N 5-monooxygenase (sidA) and a non-ribosomal peptide synthetase (sidC).

The filamentous ascomycete A. nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin intracellularly. In this study we report the characterization of two siderophore biosynthetic genes, sidA encoding l-ornithine N(5)-monooxygenase and sidC encoding a non-ribosomal peptide synthetase respectively. Disruption of sidC eliminated synthesis of ferricrocin and deletion of sidA completely blocked siderophore biosynthesis. Siderophore-deficient strains were unable to grow, unless the growth medium was supplemented with siderophores, suggesting that the siderophore system is the major iron assimilatory system of A. nidulans during both iron depleted and iron-replete conditions. Partial restoration of the growth of siderophore-deficient mutants by high concentrations of Fe(2+) (but not Fe(3+)) indicates the presence of an additional ferrous transport system and the absence of an efficient reductive iron assmilatory system. Uptake studies demonstrated that TAFC-bound iron is transferred to cellular ferricrocin whereas ferricrocin is stored after uptake. The siderophore-deficient mutant was able to synthesize ferricrocin from triacetylfusarinine C. Ferricrocin-deficiency caused an increased intracellular labile iron pool, upregulation of antioxidative enzymes and elevated sensitivity to the redox cycler paraquat. This indicates that the lack of this cellular iron storage compound causes oxidative stress. Moreover, ferricrocin biosynthesis was found to be crucial for efficient conidiation.

Amino Acid Sequence↗
PubMed2004

Leukotriene A4 hydrolase: identification of a common carboxylate recognition site for the epoxide hydrolase and aminopeptidase substrates.

Leukotriene (LT) A(4) hydrolase is a bifunctional zinc metalloenzyme, which converts LTA(4) into the neutrophil chemoattractant LTB(4) and also exhibits an anion-dependent aminopeptidase activity. In the x-ray crystal structure of LTA(4) hydrolase, Arg(563) and Lys(565) are found at the entrance of the active center. Here we report that replacement of Arg(563), but not Lys(565), leads to complete abrogation of the epoxide hydrolase activity. However, mutations of Arg(563) do not seem to affect substrate binding strength, because values of K(i) for LTA(4) are almost identical for wild type and (R563K)LTA(4) hydrolase. These results are supported by the 2.3-A crystal structure of (R563A)LTA(4) hydrolase, which does not reveal structural changes that can explain the complete loss of enzyme function. For the aminopeptidase reaction, mutations of Arg(563) reduce the catalytic activity (V(max) = 0.3-20%), whereas mutations of Lys(565) have limited effect on catalysis (V(max) = 58-108%). However, in (K565A)- and (K565M)LTA(4) hydrolase, i.e. mutants lacking a positive charge, values of the Michaelis constant for alanine-p-nitroanilide increase significantly (K(m) = 480-640%). Together, our data indicate that Arg(563) plays an unexpected, critical role in the epoxide hydrolase reaction, presumably in the positioning of the carboxylate tail to ensure perfect substrate alignment along the catalytic elements of the active site. In the aminopeptidase reaction, Arg(563) and Lys(565) seem to cooperate to provide sufficient binding strength and productive alignment of the substrate. In conclusion, Arg(563) and Lys(565) possess distinct roles as carboxylate recognition sites for two chemically different substrates, each of which is turned over in separate enzymatic reactions catalyzed by LTA(4) hydrolase.

Amino Acid Sequence↗
PubMed2001

Eukaryotic peptide deformylases. Nuclear-encoded and chloroplast-targeted enzymes in Arabidopsis.

Arabidopsis (ecotype Columbia-0) genes, AtDEF1 and AtDEF2, represent eukaryotic homologs of the essential prokaryotic gene encoding peptide deformylase. Both deduced proteins contain three conserved protein motifs found in the active site of all eubacterial peptide deformylases, and N-terminal extensions identifiable as chloroplast-targeting sequences. Radiolabeled full-length AtDEF1 was imported and processed by isolated pea (Pisum sativum L. Laxton's Progress No. 9) chloroplasts and AtDEF1 and 2 were immunologically detected in Arabidopsis leaf and chloroplast stromal protein extracts. The partial cDNAs encoding the processed forms of Arabidopsis peptide deformylase 1 and 2 (pAtDEF1 and 2, respectively) were expressed in Escherichia coli and purified using C-terminal hexahistidyl tags. Both recombinant Arabidopsis peptide deformylases had peptide deformylase activity with unique kinetic parameters that differed from those reported for the E. coli enzyme. Actinonin, a specific peptide deformylase inhibitor, was effective in vitro against Arabidopsis peptide deformylase 1 and 2 activity, respectively. Exposure of several plant species including Arabidopsis to actinonin resulted in chlorosis and severe reductions in plant growth and development. The results suggest an essential role for peptide deformylase in protein processing in all plant plastids.

Amidohydrolases↗
PubMed2007

Crystal structures of a bacterial 6-phosphogluconate dehydrogenase reveal aspects of specificity, mechanism and mode of inhibition by analogues of high-energy reaction intermediates.

Crystal structures of recombinant Lactococcus lactis 6-phosphogluconate dehydrogenase (LlPDH) in complex with substrate, cofactor, product and inhibitors have been determined. LlPDH shares significant sequence identity with the enzymes from sheep liver and the protozoan parasite Trypanosoma brucei for which structures have been reported. Comparisons indicate that the key residues in the active site are highly conserved, as are the interactions with the cofactor and the product ribulose 5-phosphate. However, there are differences in the conformation of the substrate 6-phosphogluconate which may reflect distinct states relevant to catalysis. Analysis of the complex formed with the potent inhibitor 4-phospho-d-erythronohydroxamic acid, suggests that this molecule does indeed mimic the high-energy intermediate state that it was designed to. The analysis also identified, as a contaminant by-product of the inhibitor synthesis, 4-phospho-d-erythronamide, which binds in similar fashion. LlPDH can now serve as a model system for structure-based inhibitor design targeting the enzyme from Trypanosoma species.

Amino Acid Sequence↗
PubMed1999

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

ADAM Proteins↗
PubMed2006

Histone deacetylase inhibitor-mediated radiosensitization of human cancer cells: class differences and the potential influence of p53.

Histone deacetylase inhibitors (HDI) are emerging as potentially useful components of the anticancer armamentarium and as useful tools to dissect mechanistic pathways. HDIs that globally inhibit histone deacetylases (HDAC) have radiosensitizing effects, but the relative contribution of specific HDAC classes remains unclear. Newly characterized HDIs are now available that preferentially inhibit specific HDAC classes, including SK7041 (inhibits class I HDACs) and splitomicin (inhibits class III HDACs). We investigated in human cancer cells the relative radiosensitizations that result from blocking specific HDAC classes. We found that trichostatin A (TSA; inhibitor of both class I and II HDACs) was the most effective radiosensitizer, followed by the class I inhibitor SK7041, whereas splitomicin (inhibitor of class III) had least effect. Interestingly, radiosensitization by TSA in cell lines expressing p53 was more pronounced than in isogenic lines lacking p53. Radiosensitization of cells expressing p53 by TSA was reduced by pifithrin-alpha, a small-molecule inhibitor of p53. In contrast, the radiosensitization by TSA of cells expressing low levels of p53 was enhanced by transfection of wild-type p53-expressing vector or pretreatment with leptomycin B, an inhibitor of nuclear export that increased intracellular levels of p53. These effects on radiosensitization were respectively muted or not seen in cells treated with SK7041 or splitomicin. To our knowledge, this may be among the first systematic investigations of the comparative anticancer effects of inhibiting specific classes of HDACs, with results suggesting differences in the degrees of radiosensitization, which in some cell lines may be influenced by p53 expression.

Amides↗
PubMed2000

Sp1 and chromatin environment are important contributors to the formation of repressive chromatin structures on the transfected human adenine nucleotide translocase-2 promoter.

The influence of chromatin on the human adenine nucleotide translocase isoform 2 (ANT2) promoter was investigated in transfected cells treated with the deacetylase inhibitors butyrate and trichostatin A (TSA). Both inhibitors activated the expression of reporter plasmids transfected into HeLa cells, indicating that the promoter was suppressed by hypoacetylated chromatin and activated by hyperacetylation. Inhibitor-dependent activation was traced to the two Sp1-activation elements within the proximal promoter region, indicating that the Sp1 elements are repressed by chromatin structure. Repressive chromatin structures were also formed on the promoter integrated into a stable chromatin environment, as shown by the effects of TSA and butyrate on 14 single-cell-derived NIH3T3 clones bearing the stable integrated ANT2 promoter. Both the basal expression of the luciferase reporter gene and the response to TSA and butyrate varied widely between clones. The range of basal expression (4000-fold) was due partially to variation in the formation of repressive chromatin, since clones with low basal expression were induced by TSA, but those with high basal expression were less effected. These data indicate that chromatin environment surrounding the integrated DNA exerts a strong influence on chromatin-dependent repression of the ANT2 promoter, and that the ability of Sp1 to activate ANT2 expression is compromised in the repressed state.

3T3 Cells↗
PubMed1994

Biosynthesis of 12(S)-hydroxyeicosatetraenoic acid by B16 amelanotic melanoma cells is a determinant of their metastatic potential.

BACKGROUND: We have previously demonstrated that the metastatic potential of tumor cells can be increased by treatment with exogenous 12(S)hydroxyeicosatetraenoic acid [12(S)-HETE], a lipoxygenase metabolite of arachidonic acid. However, the biosynthesis of the authentic lipid mediator by tumor cells, and especially the correlation of its biosynthesis to tumor cell metastatic capacity have not been characterized. In addition, a role for other mono HETEs in influencing tumor cell metastatic behavior has been suggested, but conclusive evidence is lacking. In this study, we analyzed the biosynthesis of mono HETEs from arachidonic acid in tumor cells of different metastatic ability and correlated biosynthesis to metastatic potential. EXPERIMENTAL DESIGN: The biosynthesis of mono HETEs by low and high metastatic subpopulations of B16 amelanotic melanoma (B16a) cells was analyzed by high performance liquid chromatography (HPLC). The identity of biosynthetic 12-HETE was confirmed by gas chromatography/mass spectrometry (GC/MS) and its stereochemical structure assigned by chiral phase HPLC. The effect of a lipoxygenase inhibitor on the biosynthesis of mono HETEs and its effect on metastatic behavior was examined. RESULTS: HPLC analysis revealed that low (LM180) and high (HM340) metastatic B16a cells exhibited different profiles and efficiencies for conversion of arachidonic acid to mono HETEs. LM180 cells produced equal quantities of 12-HETE and 5-HETE. In contrast, HM340 cells synthesized predominantly 12-HETE and small amounts of 15-, 11- and 5-HETEs. At equal concentrations of substrate, four times more 12-HETE was synthesized by HM340 cells than by LM180 cells. The identity of biosynthetic 12-HETE was confirmed by gas chromatography/mass spectrometry and chiral phase HPLC demonstrated that it was the S enantiomer. The biosynthesis of 12(S)-HETE, but not other HETEs, was significantly inhibited by a lipoxygenase inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide. N-benzyl-N-hydroxy-5-phenylpentanamide, in a dose-dependent manner, decreased the adhesion of HM340 cells to murine pulmonary microvessel endothelium in vitro and lung colony formation in vivo. Furthermore, re-introduction of 12(S)-HETE, but not other mono HETEs, to HM340 cells pretreated with N-benzyl-N-hydroxy-5-phenylpentanamide, increased their adhesion to endothelium. CONCLUSIONS: Biosynthesis of 12(S)-HETE by tumor cells is a determinant of their metastatic potential and inhibition of 12(S)-HETE biosynthesis in tumor cells may be a crucial target for intervening in metastasis.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed2003

Prevention of acetic acid-induced colitis by desferrithiocin analogs in a rat model.

Iron contributes significantly to the formation of reactive oxygen species via the Fenton reaction. Therefore, we assessed whether a series of desferrithiocin analogs, both carboxylic acids and hydroxamates, could (1) either promote or diminish the iron-mediated oxidation of ascorbate, (2) quench a model radical species, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+), and (3) when applied topically, prevent acetic acid-induced colitis in rats. Surprisingly, most of the desferrithiocin analogs inhibited the Fenton reaction to an approximately equivalent degree; however, substantial differences were observed in the capacity of the analogs to scavenge the model radical cation. Four carboxylic acid desferrithiocin analogs and their respective N-methylhydroxamates were tested along with desferrioxamine and Rowasa, a currently accepted topical therapeutic agent for inflammatory bowel disease (IBD), in a rodent model of acetic acid-induced colitis. The colonic damage was quantitated by two independent measurements. Although neither radical scavenging nor prevention of Fenton chemistry was a definitive predictor of in vivo efficacy, the overall trend is that desferrithiocin analogs substituted with an N-methylhydroxamate in the place of the carboxylic acid are both better free radical scavengers and more active against acetic acid-induced colitis. These results represent an intriguing alternative avenue to the development of improved IBD therapeutic agents.

Acetic Acid↗