ABSTRACT
Group A streptococcus (GAS, Streptococcus pyogenes) is a strict human pathogen that causes severe, invasive diseases. GAS does not produce catalase, but has an ability to resist killing by reactive oxygen species (ROS) through novel mechanisms. The peroxide response regulator (PerR), a member of ferric uptake regulator (Fur) family, plays a key role for GAS to cope with oxidative stress by regulating the expression of multiple genes. Our previous studies have found that expression of an iron-binding protein, Dpr, is under the direct control of PerR. To elucidate the molecular interactions of PerR with its cognate promoter, we have carried out structural studies on PerR and PerR-DNA complex. By combining crystallography and small-angle X-ray scattering (SAXS), we confirmed that the determined PerR crystal structure reflects its conformation in solution. Through mutagenesis and biochemical analysis, we have identified DNA-binding residues suggesting that PerR binds to the dpr promoter at the per box through a winged-helix motif. Furthermore, we have performed SAXS analysis and resolved the molecular architecture of PerR-DNA complex, in which two 30 bp DNA fragments wrap around two PerR homodimers by interacting with the adjacent positively-charged winged-helix motifs. Overall, we provide structural insights into molecular recognition of DNA by PerR and define the hollow structural arrangement of PerR-30bpDNA complex, which displays a unique topology distinct from currently proposed DNA-binding models for Fur family regulators.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , DNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial/physiology , Models, Molecular , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Streptococcus pyogenes/metabolism , Crystallography , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial/genetics , Mutagenesis , Protein Conformation , Reactive Oxygen Species/metabolism , Scattering, Small AngleABSTRACT
Quercetin is a bioflavonoid that exhibits several biological functions in vitro and in vivo. Quercetin 3-O-methyl ether (Q3) is a natural product reported to have pharmaceutical activities, including antioxidative and anticancer activities. However, little is known about the mechanism by which it protects cells from oxidative stress. This study was designed to investigate the mechanisms by which Q3 protects against Cu(2+)-induced cytotoxicity. Exposure to Cu(2+) resulted in the death of mouse liver FL83B cells, characterized by apparent apoptotic features, including DNA fragmentation and increased nuclear condensation. Q3 markedly suppressed Cu(2+)-induced apoptosis and mitochondrial dysfunction, characterized by reduced mitochondrial membrane potential, caspase-3 activation, and PARP cleavage, in Cu(2+)-exposed cells. The involvement of PI3K, Akt, Erk, FOXO3A, and Mn-superoxide dismutase (MnSOD) was shown to be critical to the survival of Q3-treated FL83B cells. The liver of both larval and adult zebrafish showed severe damage after exposure to Cu(2+) at a concentration of 5µM. Hepatic damage induced by Cu(2+) was reduced by cotreatment with Q3. Survival of Cu(2+)-exposed larval zebrafish was significantly increased by cotreatment with 15µM Q3. Our results indicated that Cu(2+)-induced apoptosis in FL83B cells occurred via the generation of ROS, upregulation and phosphorylation of Erk, overexpression of 14-3-3, inactivation of Akt, and the downregulation of FOXO3A and MnSOD. Hence, these results also demonstrated that Q3 plays a protective role against oxidative damage in zebrafish liver and remarked the potential of Q3 to be used as an antioxidant for hepatocytes.
