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dc.contributor.authorLewallen, Daniel M.
dc.contributor.authorSteckler, Caitlin J.
dc.contributor.authorKnuckley, Bryan
dc.contributor.authorChalmers, Michael J.
dc.contributor.authorThompson, Paul R
dc.date2022-08-11T08:11:00.000
dc.date.accessioned2022-08-23T17:28:12Z
dc.date.available2022-08-23T17:28:12Z
dc.date.issued2012-06-01
dc.date.submitted2015-05-22
dc.identifier.citationMol Biosyst. 2012 Jun;8(6):1701-6. doi: 10.1039/c2mb25053e. Epub 2012 Mar 28. <a href="http://dx.doi.org/10.1039/c2mb25053e">Link to article on publisher's site</a>
dc.identifier.issn1742-2051 (Linking)
dc.identifier.doi10.1039/c2mb25053e
dc.identifier.urihttp://hdl.handle.net/20.500.14038/50026
dc.description<p>At the time of publication, Paul Thompson was not yet affiliated with UMass Medical School.</p>
dc.description.abstractThe bacterial effector VopS from Vibrio parahaemolyticus modifies host Rho GTPases to prevent downstream signalling, which leads to cell rounding and eventually apoptosis. While previous studies have used [alpha-(32)P] ATP for studying this enzyme, we sought to develop a non-radioactive chemical probe of VopS function. To guide these studies, the kinetic parameters were determined for a variety of nucleotides and the results indicated that the C6 position of adenosine was amenable to modification. Since Fl-ATP is a commercially available ATP analogue that is fluorescently tagged at the C6 position, we tested it as a VopS substrate, and the results show that VopS uses Fl-ATP to label Cdc42 in vitro and in MCF7 whole cell extracts. The utility of this probe was further demonstrated by immunoprecipitating Fl-ATP labeled Cdc42 as well as several novel substrate proteins. The proteins, which were identified by LC-MS/MS, include the small GTPases Rac1 and Cdc42 as well as several proteins that are potential VopS substrates and may be important for V. parahaemolyticus pathology. In total, these studies identify Fl-ATP as a valuable chemical probe of protein AMPylation.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=22456874&dopt=Abstract">Link to Article in PubMed</a>
dc.relation.urlhttp://dx.doi.org/10.1039/c2mb25053e
dc.subjectAdenosine Triphosphate
dc.subjectBacterial Proteins
dc.subjectCell Line, Tumor
dc.subjectFluorescent Dyes
dc.subjectHumans
dc.subjectImmunoprecipitation
dc.subjectKinetics
dc.subjectLimit of Detection
dc.subjectSignal Transduction
dc.subjectVibrio parahaemolyticus
dc.subjectcdc42 GTP-Binding Protein
dc.subjectBiochemistry
dc.subjectEnzymes and Coenzymes
dc.subjectMedicinal-Pharmaceutical Chemistry
dc.subjectTherapeutics
dc.titleProbing adenylation: using a fluorescently labelled ATP probe to directly label and immunoprecipitate VopS substrates.
dc.typeJournal Article
dc.source.journaltitleMolecular bioSystems
dc.source.volume8
dc.source.issue6
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/thompson/36
dc.identifier.contextkey7135702
html.description.abstract<p>The bacterial effector VopS from Vibrio parahaemolyticus modifies host Rho GTPases to prevent downstream signalling, which leads to cell rounding and eventually apoptosis. While previous studies have used [alpha-(32)P] ATP for studying this enzyme, we sought to develop a non-radioactive chemical probe of VopS function. To guide these studies, the kinetic parameters were determined for a variety of nucleotides and the results indicated that the C6 position of adenosine was amenable to modification. Since Fl-ATP is a commercially available ATP analogue that is fluorescently tagged at the C6 position, we tested it as a VopS substrate, and the results show that VopS uses Fl-ATP to label Cdc42 in vitro and in MCF7 whole cell extracts. The utility of this probe was further demonstrated by immunoprecipitating Fl-ATP labeled Cdc42 as well as several novel substrate proteins. The proteins, which were identified by LC-MS/MS, include the small GTPases Rac1 and Cdc42 as well as several proteins that are potential VopS substrates and may be important for V. parahaemolyticus pathology. In total, these studies identify Fl-ATP as a valuable chemical probe of protein AMPylation.</p>
dc.identifier.submissionpaththompson/36
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology
dc.source.pages1701-6


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