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Enterohemorrhagic Escherichia coli O157:H7 is responsible for many outbreaks of gastrointestinal illness and hemolytic uremic syndrome worldwide. Monitoring this pathogen in food and water supplies is an important public health issue. Highly conserved genetic markers, which are characteristic for specific strains, can provide direct identification of target pathogens. In this study, we examined a new detection strategy for pathogenic strains of E. coli O157:H7 serotype based on a conserved signature insertion/deletion (CSI) located in the ybiX gene using TaqMan-probe-based quantitative PCR (qPCR). The qPCR assay was linear from 1.0 × 10(2) to 1.0 × 10(7) genome copies and was specific to O157:H7 when tested against a panel of 15 non-O157:H7 E. coli. The assay also maintained detection sensitivity in the presence of competing E. coli K-12, heterologous nontarget DNA spiked in at a 1000-fold and 800-fold excess of target DNA, respectively, demonstrating the assay's ability to detect E. coli O157:H7 in the presence of high levels of background DNA. This study thus validates the use of strain-specific CSIs as a new class of diagnostic marker for pathogen detection.

The interaction of the hydrogenase maturation endopeptidase HycI with its substrate, the precursor of the large subunit, was studied. Replacement of conserved amino-acid residues in HycI, which have been shown to bind a cadmium ion from the crystallization buffer in crystals of HybD (endopeptidase for hydrogenase 2), abolished or strongly reduced processing activity. Atomic absorption spectroscopy of purified HycI and HybD proteins showed the absence of nickel. In vitro processing assays showed that the reaction requires nickel to be bound to the precursor and the protease does not have a function in nickel delivery to the substrate. Radioactive labelling of cells with 63Ni, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]-hydrogenases as a recognition motif.

The maturation of [NiFe]-hydrogenases is a catalysed process in which the activities of at least seven proteins are involved. The last step consists of the endoproteolytic cleavage of the precursor of the large subunit after the [NiFe]-metal centre has been assembled. The amino acid sequence requirements for the endopeptidase HycI involved in the C-terminal processing of HycE, the large subunit of the hydrogenase 3 from Escherichia coli, were investigated. Mutational alteration of the amino acid residues neighbouring the cleavage site showed that proteolysis still occurred when chemically similar amino acids were exchanged. Processing was blocked, however, in a variant in which the methionine at the C-terminal side was replaced by a glutamate residue. Truncation of the precursor from the C-terminal end rendered variants amenable to maturation even when two-thirds of the extension were removed but abolished proteolysis upon further deletion of a cluster of six basic amino acids. A construct in which the C-terminal extension from the large subunit of the hydrogenase 2 was fused to the mature part of the large subunit of hydrogenase 3 was neither processed by HycI nor by HybD, the endopeptidase specific for the large subunit of hydrogenase 2. The maturation endopeptidase, therefore, exhibits a relaxed sequence constraint in recognition of its cleavage site and does not require the entire C-terminal extension. The results point to an interaction of the C-terminus with some domain of the large subunit, rendering a conformation amenable to recognition by the endopeptidase.

An established inverse clinical correlation between serum adiponectin levels and renal cell carcinoma (RCC) aggressiveness exists. We have recently demonstrated that adiponectin suppresses clear cell RCC (ccRCC) progression through interaction with its receptor, adiponectin receptor 1 (AdipoR1). ERp46 has been shown to inhibit adiponectin signaling via interaction with AdipoR1 in HeLa cells. However, the expression of ERp46 in RCC has not been described thus far. The objectives of this study were to investigate ERp46 in RCC, its expression, its effects on RCC growth in a mouse model and whether it interacts with AdipoR1. We demonstrated a higher ERp46/AdipoR1 expression ratio in metastatic compared to non-metastatic ccRCC, as determined by immunohistochemistry of tissue microarrays and subsequent image analysis. When ERp46 was stably knocked down using shRNA or overexpressed in murine RCC RAG cells, RCC growth after subcutaneous injection in BALB/c nude mice was inhibited and accelerated, respectively. In vitro analysis to determine the molecular interaction between AdipoR1 and ERp46 included co-immunoprecipitation using human ccRCC 786-O cells and a bacterial adenylate cyclase-based two hybrid system and demonstrated no sustained AdipoR1-ERp46 interaction. This is the first report to suggest a role for ERp46 as a potential therapeutic target in RCC given its expression profile in human RCC samples and its effect on in vivo RCC growth. Since a stable interaction with AdipoR1 could not be established, we suggest that the tumorigenic properties of ERp46 in RCC cells are not related to an inhibitory modulation of AdipoR1.

The maturation of [NiFe] hydrogenases includes formation of the nickel metallocenter, proteolytic processing of the metal center carrying large subunit, and its assembling with other hydrogenase subunits. The hydrogenase maturating enzyme HYBD from Escherichia coli, a protease of molecular mass 17.5 kDa, specifically cleaves off a 15 amino acid peptide from the C terminus of the precursor of the large subunit of hydrogenase 2 in a nickel-dependent manner. Here we report the crystal structure of HYBD at 2.2 A resolution. It consists of a twisted five-stranded β-sheet surrounded by four and three helices, respectively, on each side. A cadmium ion from the crystallization buffer binds to the proposed nickel-binding site and is penta-coordinated by Glu16, Asp62, His93, and a water molecule in a pseudo-tetragonal arrangement. HYBD is topologically related to members of the metzincins superfamily of zinc endoproteinases, sharing the central β-sheet and three helices. In contrast to the metzincins, the metal-binding site of HYBD is localized at the C-terminal end of the β-sheet. Three helical insertions unique to HYBD pack against one side of the sheet, build up the active site cleft, and provide His93 as ligand to the metal. From this structure, we derive molecular clues into how the protease HYBD is involved in the hydrogenase maturation process.

The hypoxic and glycolytic environment of malignant cells facilitates high levels of reactive oxygen species (ROS) production. Breast, ovarian and prostate cancer etiology is associated with the ROS production, which are thought to play an important role in regulating the cancers cell growth, survival and metastasis. A potent and major cellular, however functional limited characterized source of ROS, is the mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase (GPD2). GPD2 is an essential component of the glycerol-3-phosphate shuttle and necessarily expressed in all tissues. In several cancer cell lines, GPD2 is highly expressed and we estimated it accounts for up to 70% of the total ROS production within the prostate cancer cell line PC3. In this study we have utilized this knowledge, to complete a cell-based high throughput-screening (HTS) assay, aimed to identify small molecule inhibitors of the GPD2 dependent ROS formation in PC3 cell lines. We employed the amplex red hydrogen peroxidase detection assay and adapted it to the HTS format. 133 inhibitory compounds from the Canadian Compound Collection were identified to reduce the ROS production activity within the PC3 cells. In order to validate these hits and to determine their mode of action, we PCR amplified GPD2 form PC3-cell cDNA and purified recombinant human GPD2 from a bacterial overproducing strain to homogeneity by employing Strep-tactin affinity-chromatography. The heterologously expressed enzyme, devoid of its mitochondrial target sequence, displayed glycerol-3-phosphate dependent resazurin, dichlorophenolindophenol, as well as tetrazole/phenazine methosulfate oxidoreductase activity. Follow up enzymatic assays of the primary HTS hits with the artificial electron acceptors identified several GPD2 activity specific inhibitors. Subsequent concentration response studies of the HTS derived inhibitors elucidated their in vitro half maximal inhibitory concentration at the range of 10 μM. In order to map GPD2's ROS production site and determine the inhibitor binding sites within the enzyme, we bioinformatically derived the proteins tertiary structure. Considering that GPD2 dependent ROS formation may drive the prostate cancer proliferation, we aim by exploiting the results of this work to generate novel lead molecules as promising potential anti-proliferating cancer drug candidates. (This work was supported by a grant from the Canadian Institutes of Health Research to Gurmit Singh) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3694.