The regulation of gene expression at the level of transcription is an important mechanism for maintaining homeostasis. The peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear hormone receptor superfamily that is involved in transcriptionally modulating such pathways as lipid and fatty acid metabolism. This receptor binds to enhancer elements, peroxisome proliferator-responsive elements (PPREs), upstream of a variety of target genes including those involved in β-oxidation of fatty acids, in the peroxisome and mitochondria, and ketogenesis. One such element has been identified upstream of the rat mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase (mtHMG-CoAS) gene. This enzyme has been shown to be one of the key regulatory points of ketogenesis.
To learn how PPARα mediated transcriptional regulation occurs, this receptor was used as bait in a yeast dihybrid screen and was found to interact with human mtHMG-CoAS. Reproduction of this interaction 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 was performed by solid phase capture assays, using GST fusion proteins, and by co-immunoprecipitations. It was also ascertained that the synthase enzyme interacts with the retinoid X receptor α (RXRα). The hamster cytoplasmic form of the enzyme was chosen as a control and showed no binding capabilities to either nuclear hormone receptor. Interestingly, the mitochondrial enzyme contains a motif, LXXLL that has previously been shown to be important for binding between a transcriptional coactivator and a receptor. A site-directed mutant of the mitochondrial synthase sequence from
LASLL to LASVL was made. The mutant showed a reduced ability to interact with both nuclear receptors. Consequently, the LXXLL motif is responsible, at least in part, for the interaction between mtHMG-CoAS and both PPARα and RXRα. The cytoplasmic synthase does not contain the motif; its corresponding sequence is LASVL. The effect ofthe mitochondrial synthase within a cell was then determined by transient transfection assays. It was discovered that on the HMG PPRE the mitochondrial synthase potentiated PPARα mediated transactivation while on the AOx PPRE the enzyme inhibited it. Thus, mtHMG-CoAS modulates PPARα activity in a PPRE dependent manner. The LXXVL mitochondrial mutant inhibited PPARα transactivation on both the HMG and AOx PPRE. Therefore, the mutant acts as a
dominant-negative inhibitor of α mediated activity. The cytoplasmic control enzyme had no effect on either PPRE. To determine if the mitochondrial enzyme could be detected within the nucleus where it appeared to be modulating PPARα mediated transcription, localization studies were performed with the use of immunofluorescence. Immunofluorescence was done by utilizing hemagglutinin (HA) epitope tagged fusions of the mitochondrial, mutant and cytoplasmic enzymes. When the HA tag was placed at the carboxyl terminus of mtHMG-CoAS, the enzyme was localized to the mitochondria with no apparent nuclear staining. This is consistent with previous localization studies done with the rat mtHMG-CoAS. Also, the mutant enzyme, with the HA tag at the carboxyl terminus, was only detected in the mitochondria. However, under certain conditions, when PPARα and the mitochondrial synthase were co-transfected, the mitochondrial enzyme was detected within both the mitochondria and the nucleus. The mutant, on the other hand, when co-transfected with PPARα was found to remain non-nuclear. The HA tagged cytoplasmic control enzyme was also non-nuclear. Therefore, mtHMG-CoAS can be detected within the nucleus, it binds, due to at least in part a LXXLL motif, to nuclear receptors, and it is capable of modulating transcription in a PPRE dependent manner. Ketogenesis becomes an important mechanism for fuel production during starvation, prolonged exercise and diabetes. Perhaps, during extreme circumstances such as starvation, mtHMG-CoAS is involved in autoregulation which allows for an amplification of the transcription of its own gene. Also, mtHMG-CoAS appears to inhibit the transcription of the AOx enzyme which is involved in peroxisomal fatty acid β-oxidation. Again, perhaps under extreme conditions, the β-oxidation of fatty acids is concentrated within the mitochondria which allows for the production of acetyl-CoA that can subsequently be converted to ketone bodies that can then be utilized for fuel. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23148 |
| Date | 08 1900 |
| Creators | Meertens |
| Contributors | Capone, John, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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