Show simple item record

dc.contributor.authorSu, Guoping
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.subjectNF kappa B
dc.subjectconfocal microscopy
dc.subjectGST-pull down
dc.titleDemonstration of a molecular interaction between thioredoxin and the pro-fs gene product of HIV-1, a mimic of NF kappa B
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

Files in this item


There are no files associated with this item.

This item appears in the following Collection(s)

Show simple item record