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Ubiquitination and Degradation of Neuronal Nitric Oxide Synthase.

dc.contributor.authorJenkins, Gary J.en_US
dc.date.accessioned2008-05-08T19:05:05Z
dc.date.availableNO_RESTRICTIONen_US
dc.date.available2008-05-08T19:05:05Z
dc.date.issued2008en_US
dc.date.submitteden_US
dc.identifier.urihttps://hdl.handle.net/2027.42/58432
dc.description.abstractGuanabenz, a clinically used anti-hypertensive agent, inhibits the P450-like enzyme neuronal NO-synthase (nNOS) and enhances its ubiquitination and degradation. To better understand the molecular trigger for nNOS ubiquitination and degradation, we characterized the mechanism of guanabenz inhibition of nNOS and identified the site of ubiquitin attachment to the enzyme. Using purified nNOS and an in vitro system, we found that guanabenz treatment leads to the oxidation of tetrahydrobiopterin by nNOS-derived superoxide. Tetrahydrobiopterin is a known cofactor for NO synthesis by nNOS, binding near the heme and stabilizing the active dimeric structure of the enzyme. Tetrahydrobiopterin was found to reverse the guanabenz-mediated inhibition of nNOS in vitro. Similarly, administration of tetrahydrobiopterin to rats prevented both nNOS inhibition and loss of enzyme after guanabenz treatment, indicating that the loss of tetrahydrobiopterin plays a major role in the effects of guanabenz in vivo. To investigate if the loss of tetrahydrobiopterin was sufficient for eliciting the enhanced turnover of nNOS, we depleted tetrahydrobiopterin in cells by inhibiting GTP cyclohydrolase I with 2,4-diamino-6-hydroxypyrimidine. A 75% decrease in tetrahydrobiopterin levels led to a 2-fold increase in the amount of nNOS-ubiquitin conjugates detected. Consistent with our cellular observations, in vitro ubiquitination and degradation of nNOS by reticulocyte lysate proteins was decreased when tetrahydrobiopterin was added. Thus, tetrahydrobiopterin may serve as an endogenous regulator of nNOS protein levels. Through mutagenesis studies, we were able to localize the ubiquitination site to the calmodulin binding region of nNOS (residues 720-756). Peptide mapping studies using capillary flow liquid chromatography interfaced with a linear ion trap mass spectrometer identified residue 754 as a site for ubiquitin attachment. Furthermore, using methylated ubiquitin and purified nNOS, we determined that mono-ubiquitination of nNOS is sufficient for proteasomal degradation in vitro. Thus, it is possible that alterations of the heme active site structure, in this case through oxidation of tetrahydrobiopterin, are recognized by cellular factors that direct the ubiquitination of a lysine residue in the calmodulin binding region, resulting in the selective proteasomal degradation of nNOS.en_US
dc.format.extent4510009 bytes
dc.format.extent1373 bytes
dc.format.mimetypeapplication/pdf
dc.format.mimetypetext/plain
dc.language.isoen_USen_US
dc.subjectNeuronal Nitric Oxide Synthaseen_US
dc.subjectUbiquitinen_US
dc.subjectUbiquitinationen_US
dc.subjectPost-translational Modificationen_US
dc.subjectPeptide Mappingen_US
dc.subjectMass Spectrometryen_US
dc.titleUbiquitination and Degradation of Neuronal Nitric Oxide Synthase.en_US
dc.typeThesisen_US
dc.description.thesisdegreenamePhDen_US
dc.description.thesisdegreedisciplinePharmacologyen_US
dc.description.thesisdegreegrantorUniversity of Michigan, Horace H. Rackham School of Graduate Studiesen_US
dc.contributor.committeememberOsawa, Yoichien_US
dc.contributor.committeememberHollenberg, Paul F.en_US
dc.contributor.committeememberPratt, William B.en_US
dc.contributor.committeememberRichardson, Rudy J.en_US
dc.subject.hlbtoplevelHealth Sciencesen_US
dc.subject.hlbtoplevelScienceen_US
dc.description.bitstreamurlhttp://deepblue.lib.umich.edu/bitstream/2027.42/58432/1/jenkinsg_1.pdf
dc.owningcollnameDissertations and Theses (Ph.D. and Master's)


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