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Leticia M. Toledo-Sherman Leroi Desouza Christopher M. Hosfield Linda Liao Kelly Boutillier Paul Taylor Shane Climie Linda McBroom-Cerajewski Michael F. Moran 《Clinical proteomics》2004,1(1):45-67
Methotrexate has been a clinical agent used in cancer, immunosuppression, rheumatoid arthritis and other highly proliferative
diseases for many years, yet its underlying molecular mechanism of action in these therapeutic areas is still unclear. We
present a chemical proteomics approach that uses ultra-sensitive mass spectrometry coupled to an inverse protein-ligand docking
computational technique to unravel the mechanism of action of this drug. Using methotrexate tethered to a solid support we
were able to isolate a signficant number of proteins. We effectively captured a large portion of the de novo purine metaolome and propose a pathway architecture similar to that seen in signaling pathways and consistent with substrate
channeling. More importantly, we were able to capture protein targets that could potentially provide new insights into the
mechanism of action of methotrexate in rheumatoid arthritis and immunosuppression. The application of this approach to other
drugs and drug candidates may facilitate the prediction of unknown and secondary therapeutic target proteins and those related
to the side effects and toxicity. These results demonstrate that this proteomics technology could play an important role in
drug discovery and development since it allows monitoring of the interactions between a drug and the protein content of a
cell. 相似文献
44.
Hosfield CM Moldoveanu T Davies PL Elce JS Jia Z 《The Journal of biological chemistry》2001,276(10):7404-7407
The ubiquitous calpain isoforms (mu- and m-calpain) are Ca(2+)-dependent cysteine proteases that require surprisingly high Ca(2+) concentrations for activation in vitro ( approximately 50 and approximately 300 microm, respectively). The molecular basis of such a high requirement for Ca(2+) in vitro is not known. In this study, we substantially reduced the concentration of Ca(2+) required for the activation of m-calpain in vitro through the specific disruption of interdomain interactions by structure-guided site-directed mutagenesis. Several interdomain electrostatic interactions involving lysine residues in domain II and acidic residues in the C(2)-like domain III were disrupted, and the effects of these mutations on activity and Ca(2+) sensitivity were analyzed. The mutation to serine of Glu-504, a residue that is conserved in both mu- and m-calpain and interacts most notably with Lys-234, reduced the in vitro Ca(2+) requirement for activity by almost 50%. The mutation of Lys-234 to serine or glutamic acid resulted in a similar reduction. These are the first reported cases in which point mutations have been able to reduce the Ca(2+) requirement of calpain. The structures of the mutants in the absence of Ca(2+) were shown by x-ray crystallography to be unchanged from the wild type, demonstrating that the increase in Ca(2+) sensitivity was not attributable to conformational change prior to activation. The conservation of sequence between mu-calpain, m-calpain, and calpain 3 in this region suggests that the results can be extended to all of these isoforms. Whereas the primary Ca(2+) binding is assumed to occur at EF-hands in domains IV and VI, these results show that domain II-domain III salt bridges are important in the process of the Ca(2+)-induced activation of calpain and that they influence the overall Ca(2+) requirement of the enzyme. 相似文献
45.
Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication 总被引:1,自引:0,他引:1
Chapados BR Chai Q Hosfield DJ Qiu J Shen B Tainer JA 《Journal of molecular biology》2001,307(2):541-556
DNA replication and cellular survival requires efficient removal of RNA primers during lagging strand DNA synthesis. In eukaryotes, RNA primer removal is initiated by type 2 RNase H, which specifically cleaves the RNA portion of an RNA-DNA/DNA hybrid duplex. This conserved type 2 RNase H family of replicative enzymes shares little sequence similarity with the well-characterized prokaryotic type 1 RNase H enzymes, yet both possess similar enzymatic properties. Crystal structures and structure-based mutational analysis of RNase HII from Archaeoglobus fulgidus, both with and without a bound metal ion, identify the active site for type 2 RNase H enzymes that provides the general nuclease activity necessary for catalysis. The two-domain architecture of type 2 RNase H creates a positively charged binding groove and links the unique C-terminal helix-loop-helix cap domain to the active site catalytic domain. This architectural arrangement apparently couples directional A-form duplex binding, by a hydrogen-bonding Arg-Lys phosphate ruler motif, to substrate-discrimination, by a tyrosine finger motif, thereby providing substrate-specific catalytic activity. Combined kinetic and mutational analyses of structurally implicated substrate binding residues validate this binding mode. These structural and mutational results together suggest a molecular mechanism for type 2 RNase H enzymes for the specific recognition and cleavage of RNA in the RNA-DNA junction within hybrid duplexes, which reconciles the broad substrate binding affinity with the catalytic specificity observed in biochemical assays. In combination with a recent independent structural analysis, these results furthermore identify testable molecular hypotheses for the activity and function of the type 2 RNase H family of enzymes, including structural complementarity, substrate-mediated conformational changes and coordination with subsequent FEN-1 activity. 相似文献
46.
Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation 总被引:7,自引:0,他引:7
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The combination of thiol protease activity and calmodulin-like EF-hands is a feature unique to the calpains. The regulatory mechanisms governing calpain activity are complex, and the nature of the Ca(2+)-induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 A crystal structure of m-calpain in the Ca(2+)-free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca(2+). It also reveals an unusual thiol protease fold, which is associated with Ca(2+)-binding domains through heterodimerization and a C(2)-like beta-sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca(2+)-binding must induce conformational changes that re-orient the protease domains to form a functional active site. The alpha-helical N-terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca(2+)-sensitivity through association with the regulatory subunit. This Ca(2+)-dependent activation mechanism is clearly distinct from those of classical proteases. 相似文献
47.
Schofield DJ Pope AR Clementel V Buckell J Chapple SDj Clarke KF Conquer JS Crofts AM Crowther SR Dyson MR Flack G Griffin GJ Hooks Y Howat WJ Kolb-Kokocinski A Kunze S Martin CD Maslen GL Mitchell JN O'Sullivan M Perera RL Roake W Shadbolt SP Vincent KJ Warford A Wilson WE Xie J Young JL McCafferty J 《Genome biology》2007,8(11):R254-18
We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation. 相似文献
48.
Background
In a number of species males damage females during copulation, but the reasons for this remain unclear. It may be that males are trying to manipulate female mating behaviour or their life histories. Alternatively, damage may be a side-effect of male-male competition. In the black scavenger or dung fly Sepsis cynipsea (Diptera: Sepsidae) mating reduces female survival, apparently because males wound females during copulation. However, this damage does not seem to relate to attempted manipulation of female reproduction by males. Here we tested the hypothesis that harming females during mating is an incidental by-product of characters favoured during pre-copulatory male-male competition. We assessed whether males and their sons vary genetically in their ability to obtain matings and harm females, and whether more successful males were also more damaging. We did this by ranking males' mating success in paired competitions across several females whose longevity under starvation was subsequently measured. 相似文献49.
50.
Application of ultrasound for pregnancy diagnosis has been tested and evaluated in 15 Iranian camels (Camelus dromedarius), all of which ultimately calved. Transabdominal examinations were unsuccessful, while intrapelvic application resulted in the reception of sounds characteristic for foetal life, similar to those found in other domestic animals. Signals of foetal heart, pulse of umbilical vessels and uterine artery as well as foetal movement could be recognized as distinct sounds and have been recorded for further studies. An attempt was made to verify the findings of the ultrasonic diagnosis through rectal palpation. The ultrasonic technique resulted in 12 correct and three incorrect diagnoses. 相似文献