Biomedical subjects

Jeffrey Green

Publications and source records attributed to Jeffrey Green.

43 records · Page 3Linked to original sources

PubMed2002

Lifetime exposure to a soluble TGF-beta antagonist protects mice against metastasis without adverse side effects.

TGF-betas play diverse and complex roles in many biological processes. In tumorigenesis, they can function either as tumor suppressors or as pro-oncogenic factors, depending on the stage of the disease. We have developed transgenic mice expressing a TGF-beta antagonist of the soluble type II TGF-beta receptor:Fc fusion protein class, under the regulation of the mammary-selective MMTV-LTR promoter/enhancer. Biologically significant levels of antagonist were detectable in the serum and most tissues of this mouse line. The mice were resistant to the development of metastases at multiple organ sites when compared with wild-type controls, both in a tail vein metastasis assay using isogenic melanoma cells and in crosses with the MMTV-neu transgenic mouse model of metastatic breast cancer. Importantly, metastasis from endogenous mammary tumors was suppressed without any enhancement of primary tumorigenesis. Furthermore, aged transgenic mice did not exhibit the severe pathology characteristic of TGF-beta null mice, despite lifetime exposure to the antagonist. The data suggest that in vivo the antagonist may selectively neutralize the undesirable TGF-beta associated with metastasis, while sparing the regulatory roles of TGF-betas in normal tissues. Thus this soluble TGF-beta antagonist has potential for long-term clinical use in the prevention of metastasis.

Animals↗

PubMed2001

Miscoordination of the iron-sulfur clusters of the anaerobic transcription factor, FNR, allows simple repression but not activation.

The FNR protein of Escherichia coli regulates target genes in response to anaerobiosis. Environmental oxygen is sensed by the acquisition of oxygen-labile [4Fe-4S] clusters that promote dimerization, DNA binding, and productive interactions with RNA polymerase. Three N-terminal cysteine residues (Cys(20), Cys(23), and Cys(29)) and Cys(122) act as ligands for the FNR iron sulfur clusters. An FNR variant, FNR-C20S, that retains only trace activity in vivo can acquire [4Fe-4S] clusters in vitro that enhance site-specific DNA binding. Second site substitutions in activating regions AR1, AR2, and AR3 restore in vivo activity to FNR-C20S, suggesting that the impairment in FNR-C20S activity is due to a failure to communicate with RNA polymerase effectively. Here we show that FNR-C20S can repress a simple FNR-regulated promoter in vivo and that it can form productive heterodimers with an FNR variant with altered DNA binding specificity, FNR-E209V. Transcription studies with FNR-E209V.FNR-C20S heterodimers indicate that the presence of a miscoordinated iron-sulfur cluster (FNR-C20S) in the downstream (but not the upstream) subunit of the FNR dimer impairs activation from a class II promoter and that this impairment can be overcome by amino acid substitutions known to unmask AR2 or improve AR3 in the affected subunit.

Bacterial Proteins↗

PubMed2000

YeiL, the third member of the CRP-FNR family in Escherichia coli.

The yeiL open reading frame located at 48.5 min (2254 kb) in the nfo-fruA region of the Escherichia coli chromosome was predicted to encode a CRP and FNR paralogue capable of forming inter- or intra-molecular disulphide bonds and incorporating one iron-sulphur centre per 25 kDa subunit. Purified MBP-YeiL (a maltose-binding-protein-YeiL fusion protein) was a high-molecular-mass oligomer or aggregate which released unstable monomers (68 kDa) under reducing conditions. The MBP-YeiL protein contained substoichiometric amounts of iron and acid-labile sulphide, and an average of one disulphide bond per monomer. The iron and sulphide contents increased consistent with the acquisition of one [4Fe-4S] cluster per monomer after anaerobic NifS-catalysed reconstitution. By analogy with FNR and FLP (the FNR-like protein of Lactobacillus casei) it was suggested that the transcription-regulatory activity of YeiL might be modulated by a sensory iron-sulphur cluster and/or by reversible disulphide bond formation. A yeiL-lacZ transcriptional fusion showed that aerobic yeiL expression increases at least sixfold during stationary phase, requires RpoS, and is positively autoregulated by YeiL, positively activated by Lrp (and IHF in the absence of FNR) and negatively regulated by FNR. A regulatory link between the synthesis of YeiK (a potential nucleoside hydrolase) and YeiL was inferred by showing that the yeiK and yeiL genes are divergently transcribed from overlapping promoters. A 10-15% deficiency in aerobic growth yield and an enhanced loss of viability under nitrogen starvation conditions were detected with a yeiL::kan(R) mutant, suggesting that YeiL might function as a post-exponential-phase nitrogen-starvation regulator.

ATP-Binding Cassette Transporters↗

PubMed1998

A novel regulatory switch mediated by the FNR-like protein of Lactobacillus casei.

FNR (regulator for fumarate and nitrate reduction) and CRP (cAMP receptor protein) are global regulators which regulate the transcription of overlapping modulons of target genes in response to anaerobiosis and carbon source in Escherichia coli. An ORF, designated flp because it encodes an FNR-like protein of the FNR-CRP family, has been found in Lactobacillus casei. The product of the flp coding region (FLP) was overproduced in E. coli, purified and crystallized. FLP is a homodimeric protein in which each subunit can form an intramolecular disulphide bond. The isolated protein also contains non-stoichiometric amounts of Cu and Zn. Although the DNA recognition helix of FLP resembles that of FNR, the flp gene failed to complement the anaerobic respiratory deficiency of an fnr mutant when expressed in E. coli and it neither activated nor interfered with transcription from FNR- or CRP-dependent promoters in E. coli. Site-specific DNA binding by oxidized FLP (the form containing intrasubunit disulphide bonds) was abolished by reduction. The interconversion between disulphide and dithiol forms thus provides the basis for a novel redox-mediated transcriptional switch. Two non-identical FLP-binding sites, distinct from FNR- and CRP-binding sites, were identified in the meIR region of E. coli by gel-retardation analysis. A further eight FLP-binding sites were selected from a random library. A synthetic oligonucleotide conforming to a putative FLP site consensus, CA/CTGA-N4-TCAG/TG (the most significant bases are underlined), was retarded by FLP. Functional tests showed that FLP represses the aerobic transcription of a semi-synthetic promoter in E. coli. A C5S variant of FLP lacking the ability to form intramolecular disulphide bonds was unable to bind to FLP sites and failed to repress transcription in vivo.

Aerobiosis↗

PubMed1997

The molecular basis for the differential regulation of the hlyE-encoded haemolysin of Escherichia coli by FNR and HlyX lies in the improved activating region 1 contact of HlyX.

The regulator of fumarate and nitrate reduction (FNR) protein of Escherichia coli is an oxygen-responsive transcription regulator that acts mainly to activate the transcription of genes associated with anaerobic energy generation during periods of oxygen starvation. The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae encodes an FNR homologue, HlyX, which can complement the anaerobic respiratory deficiencies of an fnr mutant. However, FNR and HlyX have distinct but overlapping regulons because during anaerobic incubation, hlyX-expressing E. coli K-12 strains produce an otherwise latent haemolysin. The gene encoding the 'latent' haemolysin has been designated hlyE and analysis of the promoter region by DNase I footprinting reveals the presence of an FNR- (HlyX-) binding site. Anaerobic expression of an hlyE::lacZ reporter was 6.5-fold higher in hlyX compared to fnr-expressing cells. Both FNR and HlyX recruited RNA polymerase to the hlyE promoter but formed different ternary complexes. One major transcript (tsp1) initiating at 78.5 bp downstream of the FNR-binding site and four minor transcripts initiating at 73.5 (tsp2), 71.5 (tsp3), 63.5 (tsp4) and 62.5 (tsp5) bp from the FNR site were detected. From the position of the FNR box relative to the transcript starts, hlyE is expressed from a Class I FNR-regulated promoter. Substitution of selected FNR amino acids with the residues found in the equivalent positions in HlyX indicated that Activating Region 1 (AR1) of FNR forms a surface encompassing beta to beta 11 and that the AR1 contact at Class I promoters is different to that at Class II promoters, although the same surface is involved. The FNR variant, FNR-A225T, combined the properties of FNR (good activation from Class II promoters) and HlyX (good activation of Class I promoters) and conferred the haemolytic phenotype.

Actinobacillus pleuropneumoniae↗

PubMed1997

FNR-dependent repression of ndh gene expression requires two upstream FNR-binding sites.

The ndh gene of Escherichia coli encodes a non-proton-translocating NADH dehydrogenase (NdhII) that is anaerobically repressed by the global transcription regulator, FNR. FNR binds at two sites (centred at -50.5 and -94.5) in the ndh promoter but the mechanism of FNR-mediated repression appears not to be due to promoter occlusion. This mechanism has been investigated using an aerobically active derivative of FNR, FNR* (FNR-D154A), with ndh promoters containing altered FNR-binding sites. FNR* repressed ndh gene expression both aerobically and anaerobically in vivo. Gel retardation analysis and DNase I footprinting with purified FNR* protein confirmed that FNR interacts at two sites in the ndh promoter, and that FNR and RNA polymerase (RNAP) can bind simultaneously. Studies with three altered ndh promoters, each containing an impaired or improved FNR-site, indicated that both FNR-sites are needed for efficient repression in vivo. The alpha-subunit of RNAP interacted with two regions (centred at -105 and -46), each overlapping one of the FNR-sites in the ndh promoter. Footprints of the FNR*-RNAP-ndh ternary complex indicated that FNR*-binding at -50.5 prevents the alpha-subunit of RNAP from docking with the DNA just upstream of the -35 element. Binding of a second FNR* molecule at the -105 site likewise prevents binding of the alpha-subunit at its alternative site, thus providing a plausible mechanism for FNR-mediated repression based on displacement of the alpha-subunit of RNAP.

Aerobiosis↗

PubMed1997

Regulation of the ndh gene of Escherichia coli by integration host factor and a novel regulator, Arr.

The ndh gene of Escherichia coli encodes the non-proton-translocating NADH dehydrogenase II. Expression of the ndh gene is subject to a complex network of regulatory controls at the transcriptional level. Under anaerobic conditions ndh is repressed by the regulator of fumarate and nitrate reduction (FNR). However, in the absence of FNR, ndh expression is activated by the amino acid response regulator (Arr) during anaerobic growth in rich medium. Expression of the ndh gene varies during the growth cycle in response to the intracellular concentration of the heat-stable DNA-binding protein, Fis. In this work two additional heat-stable proteins, integration host factor (IHF) and the histone-like protein HU were found to interact with the ndh promoter. IHF was shown to bind at three sites centred at +26, -17 and -58 in the ndh promoter (Kd = 10(-8) M), to prevent open-complex formation and to repress ndh transcription in vitro. Studies with an ndh-lacZ fusion confirmed that IHF represses ndh expression in vivo. Two putative binding sites for Arr, which overlap the two FNR boxes in the ndh promoter, were identified. Studies with the FNR-activated and amino-acid-inducible asparaginase II gene (ansB) showed that IHF and a component of the Arr-containing fraction (but not HU) interact with the corresponding ansB promoter.

Amino Acid Sequence↗