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dc.contributor.authorSu, Guoping
dc.date.accessioned2014-03-03T21:07:47Z
dc.date.available2014-03-03T21:07:47Z
dc.date.issued2003-12
dc.identifier.othersu_guoping_200312_phd
dc.identifier.urihttp://purl.galileo.usg.edu/uga_etd/su_guoping_200312_phd
dc.identifier.urihttp://hdl.handle.net/10724/21379
dc.description.abstractA programmed –1 frameshift in the HIV-1 protease gene has been previously demonstrated. The sequence encoded in the overlapping –1 reading frame, named pro-fs, has significant similarity to the DNA binding loop of NF-êB, which is also the peptide known to bind thioredoxin (Trx) as part of the process of NF-êB activation. The hypothesis that the putative HIV-1 pro-fs gene product functions by mimicry of NF-êB via interacting with Trx was tested by coimmunoprecipitation and GST-pull down assay in vitro. Both experiments consistently showed that pro-fs binds human Trx in vitro; and the binding affinity is apparently stronger with Trx-wt than with Trx-CS, in which the two Cys residues in the active center of Trx were mutated to Ser. The fact that pro-fs interacts with Trx-CS rules out the possibility that the interaction between pro-fs and Trx is just due to cross-linkage via formation of an intermolecular disulfide bond between the Cys residues of pro-fs (the Sec residues in pro-fs sequence are mutated to Cys for bacterial expression) and the Cys residues in the active site of Trx. Due to the nature of the –1 frameshift mechanism, pro-fs and the HIV-1 protease share the first 20 amino acid residues at the N-terminus. Since both the retroviral protease and NF-êB function as dimers, we investigated whether pro-fs also dimerizes in living mammalian cells by fluorescent resonance energy transfer (FRET) analysis using confocal microscopy. Fluorescence microscopy was utilized to investigate the hypothesis that pro-fs is a nuclear protein, based upon its high pI and mimicry of NF-êB. The results demonstrated that pro-fs localizes in cell nuclei and forms oligomers. FRET analysis was also used to study the interaction between pro-fs and Trx in living cells. The results showed that phorbol 12-myristate 13-acetate (PMA) treatment of the 293T cells induces the nuclear translocation of Trx, and pro-fs binds to both Trx-wt and Trx-CS in PMA-stimulated cells. Together with the results of co-immunoprecipitation and GST-pull down assay, it suggests that Trx-pro-fs binding is a structurally specific interaction that involves multiple amino acid residues in the interactive region.
dc.languageeng
dc.publisheruga
dc.rightspublic
dc.subjectFrameshift
dc.subjectretrovirus
dc.subjectHIV-1
dc.subjectprotease
dc.subjectpro-fs
dc.subjectNF kappa B
dc.subjectthioredoxin
dc.subjectPMA
dc.subjectFRET
dc.subjectconfocal microscopy
dc.subjectcysteine
dc.subjectdimer
dc.subjectoligomer
dc.subjectGST-pull down
dc.subjectco-immunoprecipitation.
dc.titleDemonstration of a molecular interaction between thioredoxin and the pro-fs gene product of HIV-1, a mimic of NF kappa B
dc.typeDissertation
dc.description.degreePhD
dc.description.departmentPharmacy (Pharmaceutics)
dc.description.majorPharmacy (Pharmaceutics)
dc.description.advisorEthan Will Taylor
dc.description.committeeEthan Will Taylor
dc.description.committeeDiane Hartle
dc.description.committeePhillip Greenspan
dc.description.committeeJames Hargrove
dc.description.committeeArthur Grider


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