Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 9 de 9
Filter
Add more filters










Database
Language
Publication year range
1.
J Inorg Biochem ; 192: 98-106, 2019 03.
Article in English | MEDLINE | ID: mdl-30616070

ABSTRACT

Escherichia coli ZraP (zinc resistance associated protein) is the major Zn containing soluble protein under Zn stress conditions. ZraP is the accessory protein of a bacterial two-component, Zn2+ sensitive signal transduction system ZraSR. ZraP has also been reported to act as a Zn2+ dependent molecular chaperone. An explanation why ZraP is the major Zn protein under the stress condition of Zn2+ overload (0.2 mM) has remained elusive. We have recombinantly produced E. coli ZraP and measured Zn2+ and Cu2+ affinity in-vitro using Isothermal Titration Calorimetry. ZraP has a significantly higher affinity for Cu2+ than for Zn2+. Mutation of the conserved Cys102 to Ala or Ser resulted in a change of the oligomeric state of the protein. Mutation of the conserved His107 to Ala did not affect the zinc binding affinity or the oligomeric state of the protein. Deletion of the ZraP coding gene from the E. coli genome resulted in a phenotype with tolerance to very high zinc concentrations (up to 2.5 mM) that were lethal to wild type E. coli. These results exclude a direct role for ZraP in Zn2+ tolerance in E. coli.


Subject(s)
Drug Tolerance/genetics , Escherichia coli Proteins , Escherichia coli , Stress, Physiological/drug effects , Zinc/pharmacology , Amino Acid Substitution , Copper/pharmacology , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Deletion , Mutation, Missense , Stress, Physiological/genetics
2.
J Vis Exp ; (93): e51611, 2014 Nov 26.
Article in English | MEDLINE | ID: mdl-25490157

ABSTRACT

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state. The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor. A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.


Subject(s)
Coenzymes/analysis , Electron Spin Resonance Spectroscopy/methods , Hydrogenase/analysis , Iron-Sulfur Proteins/analysis , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae/enzymology , Coenzymes/chemistry , Coenzymes/metabolism , Conductometry/methods , Dithionite/chemistry , Ferricyanides/chemistry , Hydrogenase/chemistry , Hydrogenase/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Oxidation-Reduction , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism
3.
PLoS One ; 8(2): e55549, 2013.
Article in English | MEDLINE | ID: mdl-23405168

ABSTRACT

BACKGROUND: The highly homologous [4Fe-4S] containing fumarases FumA and FumB, sharing 90% amino acid sequence identity, from Escherichia coli are differentially regulated, which suggests a difference in their physiological function. The ratio of FumB over FumA expression levels increases by one to two orders of magnitude upon change from aerobic to anaerobic growth conditions. METHODOLOGY/PRINCIPAL FINDINGS: To understand this difference in terms of structure-function relations, catalytic and thermodynamic properties were determined for the two enzymes obtained from homologous overexpression systems. FumA and FumB are essentially identical in their Michaelis-Menten kinetics of the reversible fumarate to L-malate conversion; however, FumB has a significantly greater catalytic efficiency for the conversion of D-tartrate to oxaloacetate consistent with the requirement of the fumB gene for growth on D-tartrate. Reduction potentials of the [4Fe-4S](2+) Lewis acid active centre were determined in mediated bulk titrations in the presence of added substrate and were found to be approximately -290 mV for both FumA and FumB. CONCLUSIONS/SIGNIFICANCE: This study contradicts previously published claims that FumA and FumB exhibit different catalytic preferences for the natural substrates L-malate and fumarate. FumA and FumB differ significantly only in the catalytic efficiency for the conversion of D-tartrate, a supposedly non-natural substrate. The reduction potential of the substrate-bound [4Fe-4S] active centre is, contrary to previously reported values, close to the cellular redox potential.


Subject(s)
Escherichia coli/enzymology , Fumarate Hydratase/metabolism , Iron-Sulfur Proteins/metabolism , Iron/metabolism , Sulfur/metabolism , Amino Acid Sequence , Catalysis , Electron Spin Resonance Spectroscopy , Enzyme Stability , Fumarate Hydratase/chemistry , Iron-Sulfur Proteins/chemistry , Isoenzymes , Kinetics , Molecular Sequence Data , Oxaloacetic Acid/metabolism , Oxidation-Reduction , Oxygen/metabolism , Sequence Homology, Amino Acid , Tartrates/metabolism
4.
J Biol Inorg Chem ; 17(6): 975-85, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22739810

ABSTRACT

Storage of iron in a nontoxic and bioavailable form is essential for many forms of life. Three subfamilies of the ferritin-like superfamily, namely, ferritin, bacterioferritin, and Dps (DNA-binding proteins from starved cells), are able to store iron. Although the function of these iron-storage proteins is constitutive to many organisms to sustain life, the genome of some organisms appears not to encode any of these proteins. In an attempt to identify new iron-storage systems, we have found and characterized a new member of the ferritin-like superfamily of proteins, which unlike the multimeric storage system of ferritin, bacterioferritin, and Dps is monomeric in the absence of iron. Monomers catalyze oxidation of Fe(II) and they store the Fe(III) product as they assemble to form structures comparable to those of 24-meric ferritin. We propose that this mechanism is an alternative method of iron storage by the ferritin-like superfamily of proteins in organisms that lack the regular preassociated 24-meric/12-meric ferritins.


Subject(s)
Ferritins/metabolism , Pyrococcus furiosus/metabolism , Amino Acid Sequence , Ferritins/chemistry , Ferritins/genetics , Molecular Sequence Data , Polymerase Chain Reaction , Pyrococcus furiosus/genetics
5.
Hum Gene Ther ; 21(7): 795-805, 2010 Jul.
Article in English | MEDLINE | ID: mdl-19947826

ABSTRACT

Prostate cancer is a leading cause of death among men in Western countries. Whereas the survival rate approaches 100% for patients with localized cancer, the results of treatment in patients with metastasized prostate cancer at diagnosis are much less successful. The patients are usually presented with a variety of treatment options, but therapeutic interventions in prostate cancer are associated with frequent adverse side effects. Gene therapy and oncolytic virus therapy may constitute new strategies. Already a wide variety of preclinical studies has demonstrated the therapeutic potential of such approaches, with oncolytic prostate-specific adenoviruses as the most prominent vector. The state of the art and future prospects of gene therapy in prostate cancer are reviewed, with a focus on adenoviral vectors. We summarize advances in adenovirus technology for prostate cancer treatment and highlight areas where further developments are necessary.


Subject(s)
Adenoviridae/genetics , Genetic Therapy , Genetic Vectors/genetics , Prostatic Neoplasms/therapy , Genetic Therapy/methods , Genetic Therapy/trends , Humans , Male
6.
Microbiology (Reading) ; 155(Pt 9): 3015-3020, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19520720

ABSTRACT

The hyperthermophilic archaeon Pyrococcus furiosus is a strict anaerobe. It is therefore not expected to use the oxidative tricarboxylic acid (TCA) cycle for energy transduction. Nonetheless, its genome encodes more putative TCA cycle enzymes than the closely related Pyrococcus horikoshii and Pyrococcus abyssi, including an aconitase (PF0201). Furthermore, a two-subunit fumarase (PF1755 and PF1754) is encoded on the Pyr. furiosus genome. In the present study, these three genes were heterologously overexpressed in Escherichia coli to enable characterization of the enzymes. PF1755 and PF1754 were shown to form a [4Fe-4S]-cluster-containing heterodimeric enzyme, able to catalyse the reversible hydratation of fumarate. The aconitase PF0201 also contained an Fe-S cluster, and catalysed the conversion from citrate to isocitrate. The fumarase belongs to the class of two-subunit, [4Fe-4S]-cluster-containing fumarate hydratases exemplified by MmcBC from Pelotomaculum thermopropionicum; the aconitase belongs to the aconitase A family. Aconitase probably plays a role in amino acid synthesis when the organism grows on carbohydrates. However, the function of the seemingly metabolically isolated fumarase in Pyr. furiosus has yet to be established.


Subject(s)
Aconitate Hydratase/metabolism , Fumarate Hydratase/metabolism , Iron-Sulfur Proteins/metabolism , Pyrococcus furiosus/enzymology , Aconitate Hydratase/chemistry , Aconitate Hydratase/genetics , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Citric Acid Cycle , Escherichia coli/metabolism , Fumarate Hydratase/chemistry , Fumarate Hydratase/genetics , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Molecular Sequence Data , Pyrococcus furiosus/genetics , Sequence Alignment , Sequence Analysis, Protein , Spectrum Analysis
7.
Prostate ; 67(8): 829-39, 2007 Jun 01.
Article in English | MEDLINE | ID: mdl-17394196

ABSTRACT

BACKGROUND: Specificity of transgene expression is important for safety during gene therapeutical applications. For prostate cancer, transcriptional targeting has been applied but was hampered by loss of specificity and low activity. We constructed a small chimeric promoter for high and prostate-specific transgene expression from adenoviral vectors. METHODS: A chimeric promoter, composed of the prostate-specific antigen (PSA) enhancer and the rat probasin promoter, was cloned into an adenoviral vector and its activity was compared to vectors containing conventional prostate-specific promoters and the constitutive Cytomegalovirus (CMV) promoter in in vitro and in vivo prostate cancer models. RESULTS: The chimeric PSA-probasin promoter was the most active prostate-specific promoter reaching up to 20% of CMV promoter activity while maintaining prostate-specificity. CONCLUSIONS: The chimeric PSA-probasin promoter is a small promoter that can be utilized in viral vectors for high prostate-specific transgene expression.


Subject(s)
Adenocarcinoma/therapy , Genetic Therapy/methods , Promoter Regions, Genetic , Prostatic Neoplasms/therapy , Transgenes , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenoviridae/genetics , Androgen-Binding Protein/genetics , Animals , Cell Line, Tumor , Cytomegalovirus/genetics , Genetic Vectors/genetics , Humans , Male , Mice , Mice, Nude , Microscopy, Fluorescence , Plasmids/genetics , Prostate-Specific Antigen/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Recombinant Fusion Proteins/genetics
8.
Mol Ther ; 10(2): 355-64, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15294182

ABSTRACT

TARP (T cell receptor gamma-chain alternate reading frame protein) is a protein that in males is uniquely expressed in prostate epithelial cells and prostate cancer cells. We have previously shown that the transcriptional activity of a chimeric sequence comprising the TARP promoter (TARPp) and the PSA enhancer (PSAe) is strictly controlled by testosterone and highly restricted to cells of prostate origin. Here we report that a chimeric sequence comprising TARPp and the PSMA enhancer (PSMAe) is highly active in testosterone-deprived prostate cancer cells, while a regulatory sequence comprising PSAe, PSMAe, and TARPp (PPT) has high prostate-specific activity both in the presence and in the absence of testosterone. Therefore, the PPT sequence may, in a gene therapy setting, be beneficial to prostate cancer patients that have been treated with androgen withdrawal. A recombinant adenovirus vector with the PPT sequence, shielded from interfering adenoviral sequences by the mouse H19 insulator, yields high and prostate-specific transgene expression both in cell cultures and when prostate cancer, PC-346C, tumors were grown orthotopically in nude mice. Intravenous virus administration reveals both higher activity and higher selectivity for the insulator-shielded PPT sequence than for the immediate-early CMV promoter. Therefore, we believe that an adenovirus with therapeutic gene expression controlled by an insulator-shielded PPT sequence is a promising candidate for gene therapy of prostate cancer.


Subject(s)
Adenoviridae/genetics , Genetic Therapy/methods , Neoplasms, Hormone-Dependent/metabolism , Nuclear Proteins/genetics , Promoter Regions, Genetic/genetics , Prostatic Neoplasms/metabolism , Animals , Cell Line, Tumor , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Neoplastic , Genes, Reporter/genetics , Genetic Vectors/genetics , Humans , Insulator Elements/genetics , Luciferases/analysis , Luciferases/genetics , Male , Mice , Neoplasms, Hormone-Dependent/genetics , Neoplasms, Hormone-Dependent/therapy , Prostatic Neoplasms/genetics , Prostatic Neoplasms/therapy , Testosterone/metabolism
9.
Genes Cells ; 7(3): 333-42, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11918676

ABSTRACT

BACKGROUND: Fanconi anaemia (FA) is an autosomal recessive chromosomal instability disorder. Six distinct FA disease genes have been identified, the products of which function in an integrated pathway that is thought to support a nuclear caretaker function. Comparison of FA gene characteristics in different species may help to unravel the molecular function of the FA pathway. RESULTS: We have cloned the murine homologue of the Fanconi anaemia complementation group G gene, FANCG/XRCC9. The murine Fancg protein shows an 83% similarity to the human protein sequence, and has a predicted molecular weight of 68.5 kDa. Expression of mouse Fancg in human FA-G lymphoblasts fully corrects their cross-linker hypersensitivity. At mRNA and protein levels we detected the co-expression of Fancg and Fanca in murine tissues. In addition, mouse Fancg and Fanca proteins co-purify by immunoprecipitation. Upon transfection into Fanca-deficient mouse embryonic fibroblasts EGFP-Fancg chimeric protein was detectable in the nucleus. CONCLUSIONS: We identified a murine cDNA, Fancg, which cross-complements the cellular defect of human FA-G cells and thus represents a true homologue of human FANCG. Spleen, thymus and testis showed the highest Fancg expression levels. Although Fancg and Fanca are able to form a complex, this interaction is not required for Fancg to accumulate in the nuclear compartment.


Subject(s)
DNA-Binding Proteins/metabolism , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Amino Acid Sequence , Animals , DNA-Binding Proteins/genetics , Fanconi Anemia Complementation Group A Protein , Fanconi Anemia Complementation Group G Protein , Fibroblasts/metabolism , Green Fluorescent Proteins , Luminescent Proteins , Mice , Molecular Sequence Data , Proteins/metabolism , RNA, Messenger , Sequence Alignment
SELECTION OF CITATIONS
SEARCH DETAIL
...