Biomedical subjects

Guanghui Wu

Publications and source records attributed to Guanghui Wu.

7 recordsLinked to original sources

PubMed2004

Escherichia coli Hmp, an "oxygen-binding flavohaemoprotein", produces superoxide anion and self-destructs.

Escherichia coli Hmp is a homologue of Ralstonia eutropha FHP, the first reported bacterial flavohaemoglobin, and functions in NO detoxification. Photolysis of CO-ligated Hmp in the presence of oxygen gave a photodissociable oxy species with k(on) 2.82x10(7) M(-1) s(-1) and k(off) 4.49x10(3) s(-1). The dissociation constant of the primary O(2) compound was 160 microM (25 degrees C, pH 7.0). In order to detect superoxide formation, ferric horseradish peroxidase was used. Hmp formed the oxy compound within milliseconds, followed by formation of compound III, arising from superoxide formation. The rate of superoxide formation was independent of oxygen concentration between 0.05 and 0.7 mM oxygen, suggesting a K(m) <0.05 mM. During prolonged oxidation of NADH, the spectral signals of Hmp decayed and iron was released in a process prevented by superoxide dismutase or catalase. NADH oxidation by purified Hmp was characterised by progressive slowing of oxygen uptake. Inclusion of NO, superoxide dismutase or catalase during NADH oxidation partially protected oxygen uptake, consistent with the formation, in the absence of NO, of reactive oxygen species that inhibit Hmp function. The results are discussed in relation to the tight control exerted on Hmp synthesis in vivo.

Bacterial Proteins↗

PubMed2004

Role of an inducible single-domain hemoglobin in mediating resistance to nitric oxide and nitrosative stress in Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from Vitreoscilla stercoraria and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of C. jejuni was hypersensitive to nitrosating agents (S-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress, cgb expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the cgb mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the cgb mutant. Based on similar studies in Campylobacter coli, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.

Adaptation, Physiological↗

PubMed2003

Microbial globins.

Globins are an ancient and diverse superfamily of proteins. The globins of microorganisms were relatively ignored for many decades after their discovery by Warburg in the 1930s and rediscovery by Keilin in the 1950s. The relatively recent focus on them has been fuelled by recognition of their structural diversity and fine-tuning to fulfill (probably) discrete functions but particularly by the finding that a major role of certain globins is in protection from the stresses caused by exposure to nitric oxide (NO)--itself a molecule that has attracted intense curiosity recently. At least three classes of microbial globin are recognised, all having features of the classical globin protein fold. The first class is typified by the myoglobin-like haemprotein Vgb from the bacterium Vitreoscilla, which has attracted considerable attention because of its ability to improve growth and metabolism for biotechnological gain in a variety of host cells, even though its physiological function is not fully understood. The truncated globins are widely distributed in bacteria, microbial eukaryotes as well as plants and are characterised by being 20-40 residues shorter than Vgb. The polypeptide is folded into a two-over-two helical structure while retaining the essential features of the globin superfamily. Roles in oxygen and NO metabolism have been proposed. The third and best understood class comprises the flavohaemoglobins, which were first discovered and partly characterised in yeast. These are distinguished by the presence of an additional domain with binding sites for FAD and NAD(P)H. Widely distributed in bacteria, these proteins undoubtedly confer protection from NO and nitrosative stresses, probably by direct consumption of NO. However, a bewildering array of enzymatic capabilities and the presence of an active site in the haem pocket reminiscent of peroxidases hint at other functions. A full understanding of microbial globins promises advances in controlling the interactions of pathogenic bacteria with their animal and plant hosts, and manipulations of microbial oxygen transfer with biotechnological applications.

Amino Acid Sequence↗

PubMed2002

Cysteine is exported from the Escherichia coli cytoplasm by CydDC, an ATP-binding cassette-type transporter required for cytochrome assembly.

Assembly of Escherichia coli cytochrome bd and periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type and cydD mutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in the cydD periplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropic cydD phenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmic c-type cytochromes in cydD and wild-type strains but did not restore cytochrome d. Consistent with CydDC being a cysteine exporter, cydD mutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective in orf299, encoding a transporter of the major facilitator superfamily. A cydD orf299 double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm.

ATP-Binding Cassette Transporters↗

PubMed2002

NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp.

Nitric oxide (NO) is a signalling and defence molecule of major importance in biology. The flavohaemoglobin Hmp of Escherichia coli is involved in protective responses to NO. Because hmp gene transcription is repressed by the O(2)-responsive regulator FNR, we investigated whether FNR also senses NO. The [4Fe-4S](2+) cluster of FNR is oxygen labile and controls protein dimerization and site-specific DNA binding. NO reacts anaerobically with the Fe-S cluster of purified FNR, generating spectral changes consistent with formation of a dinitrosyl-iron-cysteine complex. NO-inactivated FNR can be reconstituted, suggesting physiological relevance. FNR binds at an FNR box within the hmp promoter (P(hmp)). FNR samples inactivated by either O(2) or NO bind specifically to P(hmp), but with lower affinity. Dose-dependent up-regulation of P(hmp) in vivo by NO concentrations of pathophysiological relevance is abolished by fnr mutation, and NO also modulates expression from model FNR-regulated promoters. Thus, FNR can respond to not only O(2), but also NO, with major implications for global gene regulation in bacteria. We propose an NO-mediated mechanism of hmp regulation by which E.coli responds to NO challenge.

Anaerobiosis↗

PubMed2002

[Construction and expression of recombinant retrovirus vector carrying HBV vector].

BACKGROUND: To explore the possibility of using retrovirus vector to carry HBV vector, and to prove that replication defective HBV could be normally packaged. METHODS: Two kinds of full length of mutant HBV gene, which express dominant negative mutants, were inserted into retrovirus vector. After recombinant retroviruses were harvested, they were used to infect Hep G2 and 2.2.15 cell line. Then the expression of HBV core antigen in the Hep G2 cell was examined by immune fluorescence, and the existence of recombinant HB virion in the culture medium was examined by PCR. RESULTS: High titer of recombinant retroviruses were obtained in the culture medium of transfected PA317 cell line. Core antigen was detectable in the recombinant retrovirus infected Hep G2 cell. Recombinant HB virion was detectable in the culture medium of recombinant retrovirus infected 2.2.15 cell. CONCLUSIONS: The results suggested that recombinant retrovirus could carry HBV vector and express HBV products. When structural protein is offered by wt-HBV, the recombinant retrovirus may function as HBV vector, therefore it could be used in anti?HBV gene therapy.

Genetic Therapy↗

PubMed1997

The cydR gene product, required for regulation of cytochrome bd expression in the obligate aerobe Azotobacter vinelandii, is an Fnr-like protein.

The cytochrome bd complex in the obligately aerobic diazotroph Azotobacter vinelandii is an oxidase, which, in vivo, has a low affinity for oxygen and is required for respiratory protection of nitrogenase. Mutations caused by insertion of Tn5-B20 upstream of the structural genes (cydAB) for cytochrome bd result in over-expression of this oxidase and, for unexplained reasons, inability of the organism to grow microaerobically. Cloning and sequencing of this upstream region revealed a gene, cydR. The deduced amino acid sequence of CydR indicates that it is a new member of the Fnr Class of regulators and that it represses cydAB expression. Refined mapping data for three insertions in cydR are presented. The cloned cydR gene complemented anaerobic growth of Escherichia coli fnr mutants and strongly enhanced expression of a narG-lacZ fusion in an E. coli fnr mutant.

Amino Acid Sequence↗