La mitogaligine est une protéine codée par galig, un nouveau gène cytotoxique. Les résultats précédemment obtenus indiquent que cette protéine, adressée aux mitochondries, induit la fuite vers le cytoplasme d'un effecteur pro-apoptotique mitochondrial, le cytochromec. Cette nouvelle étude suggère tout d'abord, que l'interaction directe de la mitogaligine avec la cardiolipine, un phospholipide spécifique des mitochondries, puisse être à l'origine de la destabilisation des membranes mitochondriales, et expliquer ainsi la fuite de cytochrome c. Ce faisceau de résultats indique que la cytotoxicité de galig est associée à l'action de la mitogaligine sur les mitochrondries. Dans une seconde partie, la mort cellulaire induite par la mitogaligine a été caractérisée. Bien que les mitochondries soient précocemment altérées, il apparaît que la mitogaligine puisse également être adressée au noyau des cellules, et induire la mort cellulaire à partir de ce compartiment. Ces résultats suggèrent que l'activité cytotoxique de la mitogaligine puisse être régulée par son adressage subcellulaire.

Graduation date: 2001

Graduation date: 2000

Thesis (Ph. D.)--Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution, 1983. / Includes bibliographical references (p. 252-270).

Thesis (Ph. D.)--Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, 1981. / Vita. Includes bibliographical references (p. 239-262).

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1981. / Supervised by John J. Stegeman. Vita. Includes bibliographical references (leaves 239-262).

Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of drugs and endogenous compounds in human liver and intestine. CYP3A4 gene expression is mainly regulated by Pregnane X receptor (PXR) which is a ligand-dependent nuclear receptor. It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. In this study, I reported that NF-κB activation by LPS and TNF-α plays a pivotal role in the suppression of CYP3A4 through interactions of NF-κB with PXR/RXR complex. Inhibition of NF-κB by NF-κB specific suppressor SRIκBα reversed the suppressive effects of LPS and TNF-α. Furthermore, I showed that NF-κB p65 disrupted the association of PXR/RXRα complex with DNA sequences as determined by EMSA and chromatin immunoprecipitation assays. NF-κB p65 directly interacted with DNA binding domain of RXRα and DNA binding domain, hinge domain and ligand-binding domain of PXR and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by PXR/RXRα complex. This mechanism of suppression by NF-κB activation may be extended to other nuclear receptor-regulated systems where RXRα is a dimerization partner. Many genes regulated by PXR and AhR are important for phase I, II and III drug metabolism. In this study I reported a crosstalk between PXR and AhR pathways. AhR physically and functionally interacted with PXR and enhanced the PXR transcriptional activity, and the interaction repressed the AhR transcriptional activity. AhR also physically interacted with RXRα. The synergistic induction of Gsta1 in the liver of mice by PCN and TCDD might assume a different mechanism. The results suggested the metabolism kinetics of mixture drugs was different from and more complicated than that of single compound. Using a HepG2 cell-based PXR-driven CYP3A4-Luciferase assay, I reported that E/F domain of PXR was responsible for ligand-dependant activation. A/B domain was necessary for co-activating the ligand-dependent activation and D domain was suppressive. High doses of Valerian Root extraction were PXR-dependent CYP3A4 inducers. Green tea polyphenols, aflatoxin B1, CuSO4 and MnCl2 enhanced the PXR transcription activity activated by rifampicin. The results suggested PXR-mediated drug metabolism kinetics altered on xenobiotic exposure.

Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of drugs and endogenous compounds in human liver and intestine. CYP3A4 gene expression is mainly regulated by Pregnane X receptor (PXR) which is a ligand-dependent nuclear receptor. It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. In this study, I reported that NF-κB activation by LPS and TNF-α plays a pivotal role in the suppression of CYP3A4 through interactions of NF-κB with PXR/RXR complex. Inhibition of NF-κB by NF-κB specific suppressor SRIκBα reversed the suppressive effects of LPS and TNF-α. Furthermore, I showed that NF-κB p65 disrupted the association of PXR/RXRα complex with DNA sequences as determined by EMSA and chromatin immunoprecipitation assays. NF-κB p65 directly interacted with DNA binding domain of RXRα and DNA binding domain, hinge domain and ligand-binding domain of PXR and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by PXR/RXRα complex. This mechanism of suppression by NF-κB activation may be extended to other nuclear receptor-regulated systems where RXRα is a dimerization partner. Many genes regulated by PXR and AhR are important for phase I, II and III drug metabolism. In this study I reported a crosstalk between PXR and AhR pathways. AhR physically and functionally interacted with PXR and enhanced the PXR transcriptional activity, and the interaction repressed the AhR transcriptional activity. AhR also physically interacted with RXRα. The synergistic induction of Gsta1 in the liver of mice by PCN and TCDD might assume a different mechanism. The results suggested the metabolism kinetics of mixture drugs was different from and more complicated than that of single compound. Using a HepG2 cell-based PXR-driven CYP3A4-Luciferase assay, I reported that E/F domain of PXR was responsible for ligand-dependant activation. A/B domain was necessary for co-activating the ligand-dependent activation and D domain was suppressive. High doses of Valerian Root extraction were PXR-dependent CYP3A4 inducers. Green tea polyphenols, aflatoxin B1, CuSO4 and MnCl2 enhanced the PXR transcription activity activated by rifampicin. The results suggested PXR-mediated drug metabolism kinetics altered on xenobiotic exposure.

An integrative biophysical and bioanalytical approach to studying the Fe distribution in isolated mitochondria was developed. This procedure involved large-scale growths, the inclusion of a chelator in isolation buffers and an anaerobic isolation protocol. Electron microscopy confirmed that mitochondrial membranes were intact and that samples were largely devoid of contaminants. The Fe-ome-the sum of all Fe species in mitochondria--was studied using a combination of EPR, Mossbauer Spectroscopy, Electron Absorption, ICP-MS and Protein analysis. Isolated mitochondria were packed prior to analysis to improve the S/N ratio. The residual buffer content of sample pellets was determined by use of a radio-labeled buffer. There was essentially no difference in the packing efficiency of mitochondria isolated from respiring and fermenting cells. The determined packing factor, 0.80, was used to calculate concentrations of individual species in neat mitochondria. The Fe-omes of mitochondria isolated from cells grown on respiring, respirofermenting and fermenting media were determined. Neat mitochondria contained ~ 750 mM Fe, regardless of whether the cells had been grown on respiring or fermenting media. The Fe distribution of respirofermenting samples (which can undergo respiration and fermentation simultaneously) was nearly identical to that of respiring mitochondria. Fermenting samples had a very different Fe-distribution. Nearly 40 % of the iron in respiring mitochondria was present in respiratory complexes including cytochrome c, cytochrome bc1, succinate dehydrogenase, and cytochrome c oxidase. Fermenting mitochondria contain an Fe-ome dominated by non-protein centers. Approximately 80 % of the Fe was present as a combination of nonheme HS Fe2+, nonheme Fe3+ and Fe3+ nanoparticles. These centers were present in roughly equal amounts. The remaining 20 % of the Fe was present as respiratory complexes which have concentrations ~ 1/2 to 1/3 that of respiring mitochondria. A model is presented in which the nonheme HS Fe2+ species serves as a feedstock for Fe/S and heme biosynthesis. When the cell is growing on respiring media, this metabolic reservoir diminishes as respiratory complexes are constantly synthesized. Under fermentative growth, the metabolic pool increases due to the reduced demand for respiration-related prosthetic groups.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.