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Hypoxia and Hypoxia mimics

ABSTRACT: Comparison between hypoxia (1%O2) and cobalt chloride (100 uM) and deferoxamine (100 uM) and Nickel Chloride (100 uM) in Hep3B cells (human hepatocellular carcinoma cells) Keywords = Hep3B Keywords = hypoxia Keywords = cobalt Keywords = deferoxamine Keywords = Nickel Keywords: ordered

ORGANISM(S): Homo sapiens

PROVIDER: GSE1056 | GEO | 2004/02/27

SECONDARY ACCESSION(S): PRJNA87133

REPOSITORIES: GEO

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Project description:A novel ArsR-SmtB family transcriptional repressor, KmtR, has been characterised from mycobacteria. Mutants of M. tuberculosis lacking kmtR show elevated expression of Rv2025c encoding a deduced CDF-family metal-exporter. KmtR-dependent repression of the cdf and kmtR operator-promoters was alleviated by nickel and cobalt in minimal medium. Electrophoretic mobility shift assays (EMSA) and fluorescence anisotropy (FA) show binding of purified KmtR to nucleotide sequences containing a region of dyad symmetry from the cdf and kmtR operator-promoters. A relatively large deltar(obs) in FA implies formation of high order apo-KmtR(n)-DNA and multiple complexes were detected by EMSA. Incubation of KmtR with cobalt inhibits DNA-complex assembly and metal-protein binding was confirmed by competition against 4-(2-pyridylazo)-resorcinol. KmtR is the second, to NmtR, characterised ArsR-SmtB sensor of nickel and cobalt from M. tuberculosis suggesting special significance for these ions in this pathogen. KmtR-dependent expression is elevated in complete medium with no increase in response to metals, while NmtR retains a response to nickel and cobalt under these conditions. Mixing equimolar apo-KmtR and apo-NmtR with 0.8 equivalents of nickel or cobalt gave nickel- and cobalt-dependent difference emission spectra similar to nickel(0.8)-KmtR and cobalt(0.8)-KmtR, respectively. Thus, KmtR has tighter affinities for nickel and cobalt than NmtR consistent with basal levels of these metals being sensed by KmtR but not NmtR in complete medium. More than a thousand genes encoding ArsR-SmtB related proteins are listed in databases and a proportion can be predicted to detect metals through known allosteric sites. KmtR has none of the previously defined sites. Substitution of His(88), Glu(101), His(102), His(110) or His(111) with Gln generated KmtR-variants that repress the cdf and kmtR operator-promoters even in elevated nickel and cobalt, revealing a new sensory site. Importantly, ArsR-SmtB sequence groupings do not correspond with the different sensory-motifs revealing that only the latter should be used to predict metal-sensing. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-49

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Hypoxia and Hypoxia mimics

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