Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos 英文参考文献.docVIP

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Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos 英文参考文献.doc

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Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos 英文参考文献

Activity-BasedLabelingofMatrixMetalloproteinasesin LivingVertebrateEmbryos JonathanY.Keow1,EricD.Pond1,JustinS.Cisar2,BenjaminF.Cravatt2,BryanD.Crawford1* 1Department ofBiology,UniversityofNewBrunswick,Fredericton,NewBrunswick,Canada, 2Department ofChemicalPhysiology,TheScripps ResearchInstitute,La Jolla,California,UnitedStatesofAmerica Abstract Extracellularmatrix(ECM)remodelingisaphysiologicallyanddevelopmentallyessentialprocessmediatedbyafamilyof zinc-dependent extracellular proteases called matrix metalloproteinases (MMPs). In addition to complex transcriptional control,MMPsaresubjecttoextensivepost-translationalregulation.Becauseofthis,classicalbiochemical,molecularand histologicaltechniquesthatdetecttheexpressionofspecificgeneproductsprovideusefulbutlimiteddataregardingthe biologically relevant activity of MMPs. Using benzophenone-bearing hydroxamate-based probes that interact with the catalytic zinc ion in MMPs, active proteases can be covalently ‘tagged’ by UV cross-linking. This approach has been successfullyusedtotagMMP-2invitrointissueculturesupernatants,andweshowherethatthisprobetagsproteinswith mobilities consistent with known MMPs and detectable gelatinolytic activity in homogenates of zebrafish embryos. Furthermore,becauseofthetransparencyofthezebrafishembryo,UV-photocroslinkingcanbeaccomplishedinvivo ,and rhodamated benzophenone probe is detected in striking spatial patterns consistent with known distributions of active matrixremodelinginembryos.Finally,inmetamorphosingXenopustadpoles,thisprobecanbeusedtobiotinylateactive MMP-2 by injecting it and cross-linking it in vivo, allowing the protein to be subsequently extracted and biochemically identified. Citation:KeowJY,PondED,CisarJS,CravattBF,CrawfordBD(2012)Activity-BasedLabelingofMatrixMetalloproteinasesinLivingVertebrateEmbryos.PLoS ONE7(8):e43434.doi:10.1371/journal.pone.0043434 Editor:MatthewBogyo,StanfordUniversity,UnitedStatesofAmerica ReceivedSeptember17,2011;AcceptedJuly23,2012;Publi