OhrRA functions as a redox-responsive system controlling toxinogenesis in Bacillus cereus.

Bacillus cereus OhrR is a member of the subgroup of the MarR (multiple antibiotic resistance) family of transcriptional regulators that use a cysteine-based redox sensing mechanism. OhrA is a thiol-dependent, peroxidase-like protein. The dual OhrRA system triggers B. cereus adaptation in response to redox changes, such as those encountered in the environment of the gastrointestinal tract. Here, we investigated the role of OhrRA in toxinogenesis. Comparative shotgun analysis of exoproteomes from ∆ohrA, ∆ohrR and wild-type cells revealed significant changes in the abundance levels of toxin-related proteins depending on the extracellular redox potential. We complemented these data by measuring the DNA binding activity of reduced and oxidized recombinant OhrR on toxin and putative toxin promoter regions. Furthermore, transcriptomic data and OhrRA-dependent, antiproliferative activity of the B. cereus exoproteome on Caco-2 human epithelial cells were recorded. The results indicate that OhrR controlled toxin gene expression directly or indirectly in a redox- and toxin-dependent manner, and may function as a repressor or an activator. Moreover, we found that OhrR restricts toxin-dependent antiproliferative activity of the B. cereus exoproteome whatever the growth conditions, while the restrictive impact of OhrA occurs only under low ORP anoxic conditions. BIOLOGICAL SIGNIFICANCE: B. cereus is a notorious foodborne pathogen which causes gastroenteritis. Fatal and severe cases have been reported. The pathogenicity of B. cereus is intimately associated with the production of epithelial cell-destructive toxins in the small intestine. The small intestine poses several challenges for a pathogen because it is sliced into various niches with different oxygen concentrations and different redox potentials. We recently showed that the organic hydroperoxide resistance OhrRA system was crucial to the successful adaptation of B. cereus to extreme redox environments such as those encountered in the lumen (high reducing anoxic environment) and on the intestinal epithelium (transient oxic environment). Here we provide evidence that this bacterial system is a major virulence determinant in B. cereus in that it coordinates toxinogenesis in a redox dependent manner. Specifically, our comparative exoproteomic analyses reveal that OhrR strongly restricts B. cereus toxinogenesis under high reducing anoxic conditions while OhrA boosts toxinogenesis. Based on exoproteomic analyses, we further examined the role of OhrR and found that it functions as a redox-dependent transcriptional regulator of toxin and putative toxin genes. These findings provide novel insights into the weapons used by B. cereus to control its toxinogenic potential and, as a result its toxicity against human epithelial cells.

Orthosteric binding of ρ-Da1a, a natural peptide of snake venom interacting selectively with the α1A-adrenoceptor.

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.

Crystallization of recombinant green mamba ρ-Da1a toxin during a lyophilization procedure and its structure determination.

ρ-Da1a toxin from eastern green mamba (Dendroaspis angusticeps) venom is a polypeptide of 65 amino acids with a strong affinity for the G-protein-coupled α(1A)-adrenoceptor. This neurotoxin has been crystallized from resolubilized lyophilized powder, but the best crystals grew spontaneously during lyophilization. The crystals belonged to the trigonal space group P3(1)21, with unit-cell parameters a = b = 37.37, c = 66.05 Å, and diffracted to 1.95 Å resolution. The structure solved by molecular replacement showed strong similarities to green mamba muscarinic toxins.

Autophosphorylated residues involved in the regulation of human chk2 in vitro.

Human checkpoint kinase 2 is a major actor in checkpoint activation through phosphorylation by ataxia telangiectasia mutated in response to DNA double-strand breaks. In the absence of de novo DNA damage, its autoactivation, reported in the event of increased Cds1/checkpoint kinase 2 (Chk2) expression, has been attributed to oligomerization. Here we report a study performed on autoactivated recombinant Chk2 proteins that aims to correlate kinase activity and phosphorylation status. Using a fluorescence-based technique to assay human checkpoint kinase 2 catalytic activity, slight differences in the ability to phosphorylate Cdc25C were observed, depending on the recombinant system used. Using mass spectrometry, the phosphorylation sites were mapped to identify sites potentially involved in the kinase activity. Five phosphorylated positions, at Ser120, Ser260, Thr225, Ser379 and Ser435, were found to be common to bacteria and insect cells expression systems. They were present in addition to the six known phosphorylation sites induced by ionizing radiation (Thr68, Thr432, Thr387, Ser516, Ser33/35 and Ser19) detected by immunoblotting. After phosphatase treatment, Chk2 regained activity via autorephosphorylation. The determination of the five common sites and ionizing-radiation-inducible positions as rephosphorylated confirms that they are potential positive regulators of Chk2 kinase activity. For Escherichia coli's most highly phosphorylated 6His-Chk2, 13 additional phosphorylation sites were assigned, including 7 novel sites on top of recently reported phosphorylation sites.

Boundaries and physical characterization of a new domain shared between mammalian 53BP1 and yeast Rad9 checkpoint proteins.

Eukaryotic cells have evolved DNA damage checkpoints in response to genome damage. They delay the cell cycle and activate repair mechanisms. The kinases at the heart of these pathways and the accessory proteins, which localize to DNA lesions and regulate kinase activation, are conserved from yeast to mammals. For Saccharomyces cerevisiae Rad9, a key adaptor protein in DNA damage checkpoint pathways, no clear human ortholog has yet been described in mammals. Rad9, however, shares localized homology with both human BRCA1 and 53BP1 since they all contain tandem C-terminal BRCT (BRCA1 C-terminal) motifs. 53BP1 is also a key mediator in DNA damage signaling required for cell cycle arrest, which has just been reported to possess a tandem Tudor repeat upstream of the BRCT motifs. Here we show that the major globular domain upstream of yeast Rad9 BRCT domains is structurally extremely similar to the Tudor domains recently resolved for 53BP1 and SMN. By expressing several fragments encompassing the Tudor-related motif and characterizing them using various physical methods, we isolated the independently folded unit for yeast Rad9. As in 53BP1, the domain corresponds to the SMN Tudor motif plus the contiguous HCA predicted structure region at the C terminus. These domains may help to further elucidate the structural and functional features of these two proteins and improve knowledge of the proteins involved in DNA damage.

Long distance charge redistribution upon Cu,Zn-superoxide dismutase reduction: significance for dismutase function.

Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is a ubiquitous enzyme with an essential role in antioxidant defense. To better understand structural factors at the origin of the highly efficient superoxide dismutation mechanism, we analyzed the consequence of copper reduction on the electronic properties of the backbone and individual amino acids by using electrochemistry coupled to Fourier transform infrared spectroscopy. Comparison of data recorded with bovine erythrocyte and recombinant chloroplastic Cu,Zn-SOD from Lycopersicon esculentum, expressed as a functional tetramer in Escherichia coli and (14)N- or fully (15)N-labeled, demonstrated that the infrared changes were dominated by reorganizations of peptide bonds and histidine copper ligands. Two main infrared modes of histidine side chain, markers of metal coordination, were identified by using Cu- and Zn-methylimidazole models: the nu(C(4)C(5))at 1605-1594 cm(-1) or approximately 1586 cm(-1) for Ntau or Npi coordination, and the nu(C(5)Ntau) at approximately 1113-1088 cm(-1). These modes, also identified in Cu,Zn-SOD by using (15)N labeling, showed that the electronic properties of the histidine Ntau ligands of copper are mostly affected upon copper reduction. A striking conclusion of this work is that peptide groups from loops and beta-sheet largely participate in charge redistribution upon copper reduction, and in contrast, electronic properties of polar and charged amino acids of the superoxide access channel remain unaffected. This is notably shown for the strictly conserved Arg-143 by site-directed mutagenesis on chloroplastic Cu,Zn-SOD. Charge compensation by the peptide backbone and preserved electronic properties of the superoxide access channel and docking site upon copper reduction may be the determinant factors for the high reaction kinetics of superoxide with both reduced and oxidized Cu,Zn-SOD.