The CyberCell Database (CCDB): a comprehensive, self-updating, relational database to coordinate and facilitate in silico modeling of Escherichia coli.

The CyberCell Database (CCDB: http://redpoll. pharmacy.ualberta.ca/CCDB) is a comprehensive, web-accessible database designed to support and coordinate international efforts in modeling an Escherichia coli cell on a computer. The CCDB brings together both observed and derived quantitative data from numerous independent sources covering many aspects of the genomic, proteomic and metabolomic character of E.coli (strain K12). The database is self-updating but also supports 'community' annotation, and provides an extensive array of viewing, querying and search options including a powerful, easy-to-use relational data extraction system.

Mutation in mitochondrial complex I ND6 subunit is associated with defective response to hypoxia in human glioma cells.

BACKGROUND: Hypoxia-tolerant human glioma cells reduce oxygen consumption rate in response to oxygen deficit, a defense mechanism that contributes to survival under moderately hypoxic conditions. In contrast, hypoxia-sensitive cells lack this ability. As it has been previously shown that hypoxia-tolerant (M006x, M006xLo, M059K) and -sensitive (M010b) glioma cells express differences in mitochondrial function, we investigated whether mitochondrial DNA-encoded mutations are associated with differences in the initial response to oxygen deficit. RESULTS: The mitochondrial genome was sequenced and 23 mtDNA alterations were identified, one of which was an unreported mutation (T-C transition in base pair 14634) in the hypoxia-sensitive cell line, M010b, that resulted in a single amino acid change in the gene encoding the ND6 subunit of NADH:ubiquinone oxidoreductase (Complex I). The T14634C mutation did not abrogate ND6 protein expression, however, M010b cells were more resistant to rotenone, an agent used to screen for Complex I mutations, and adriamycin, an agent activated by redox cycling. The specific function of mtDNA-encoded, membrane-embedded Complex I ND subunits is not known at present. Current models suggest that the transmembrane arm of Complex I may serve as a conformationally driven proton channel. As cellular respiration is regulated, in part, by proton flux, we used homology-based modeling and computational molecular biology to predict the 3D structure of the wild type and mutated ND6 proteins. These models predict that the T14634C mutation alters the structure and orientation of the trans-membrane helices of the ND6 protein. CONCLUSION: Complex I ND subunits are mutational hot spots in tumor mtDNA. Genetic changes that alter Complex I structure and function may alter a cell's ability to respond to oxygen deficit and consolidate hypoxia rescue mechanisms, and may contribute to resistance to chemotherapeutic agents that require redox cycling for activation.

Structural and functional characterization of a thioredoxin-like protein (Mt0807) from Methanobacterium thermoautotrophicum.

Mt0807 is an 85-residue thiol-redox protein from the anaerobic archaebacterium Methanobacterium thermoautotrophicum. Its small size, its participation in certain redox reactions, and the presence of a "classic" glutareodoxin active-site sequence have led to the suggestion that it might be a glutaredoxin. However, studies by previous workers indicated that it exhibited neither glutaredoxin-like nor thioredoxin-like properties. To clarify the true role of this protein and its structure/functional relationship with a paralogous thioredoxin (Mt0895, 28% sequence identity) and a recently characterized orthologous protein (Mj0307, 51% sequence identity), we undertook a series of biochemical and biophysical studies. Comparative enzymatic assays and thiol titration experiments were combined with NMR structural studies and detailed 3D structure comparisons. Structurally, our results show that Mt0807 has a glutaredoxin-like fold (central four-stranded beta-sheet core surrounded by two helices on one side and a third on the other). However, more detailed comparisons with other members of the thioredoxin superfamily indicate that Mt0807 actually has several key structural and active-site characteristics more common to a thioredoxin. Furthermore, biochemical tests show that Mt0807 actually behaves as true thioredoxin. Comparisons between Mt0807 and its paralogue, Mt0895, indicate these two archaebacterial thioredoxins share very similar folds, but exhibit very different activities and likely serve somewhat different roles. On the basis of its greater relative abundance and significantly stronger redox activity, we believe that Mt0807 is the primary thioredoxin for M. thermoautotrophicum, while Mt0895 plays a minor or supportive role. We also suggest that these two molecules (Mt0807 and Mt0895) may represent a group of ancient proteins that were ancestral to both thioredoxins and glutaredoxins.

Identification of a novel archaebacterial thioredoxin: determination of function through structure.

As part of a high-throughput, structural proteomic project we have used NMR spectroscopy to determine the solution structure and ascertain the function of a previously unknown, conserved protein (MtH895) from the thermophilic archeon Methanobacterium thermoautotrophicum. Our findings indicate that MtH895 contains a central four-stranded beta-sheet core surrounded by two helices on one side and a third on the other. It has an overall fold superficially similar to that of a glutaredoxin. However, detailed analysis of its three-dimensional structure along with molecular docking simulations of its interaction with T7 DNA polymerase (a thioredoxin-specific substrate) and comparisons with other known members of the thioredoxin/glutaredoxin family of proteins strongly suggest that MtH895 is more akin to a thioredoxin. Furthermore, measurement of the pK(a) values of its active site thiols along with direct measurements of the thioredoxin/glutaredoxin activity has confirmed that MtH895 is, indeed, a thioredoxin and exhibits no glutaredoxin activity. We have also identified a group of previously unknown proteins from several other archaebacteria that have significant (34-44%) sequence identity with MtH895. These proteins have unusual active site -CXXC- motifs not found in any known thioredoxin or glutaredoxin. On the basis of the results presented here, we predict that these small proteins are all members of a new class of truncated thioredoxins.