Browsing by Author "Mogwera, Koketso Seipelo Precious"
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- ItemMembrane permeability and transport studies of coenzyme A, its precursors and antimetabolites.(Stellenbosch : Stellenbosch University, 2020-04) Mogwera, Koketso Seipelo Precious; Strauss, Erick; De Villiers, Marianne; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Coenzyme A (CoA) is a cofactor essential to several metabolic processes, including central energy and fatty acid metabolism. In addition, there is increasing evidence that CoA also plays an important role in the rate of neurodegeneration and aging. CoA is usually biosynthesized from vitamin B5, but recently it was shown that dietary CoA can also replenish intracellular CoA levels through degradation of exogenous CoA by ecto-nucleoside pyrophosphatases into 3',5’-ADP and 4'-phosphopantetheine (P-PantSH), which is subsequently taken up by cells. Importantly, permeability studies suggested that this uptake occurs by passive diffusion, an observation that was further confirmed by nutrient complementation studies in Drosophila S2 cells containing a mutation that results in CoA depletion. Moreover, investigation of CoA homeostasis in Escherichia coli suggested that this bacterium irreversibly exports P-PantSH to regulate intracellular CoA levels. This suggests an interplay between eukaryotes and their microbiome regarding the maintenance of levels of this cofactor through the exchange of P- PantSH as a likely nexus metabolite in CoA biosynthesis. In this study we used a systematic biophysical approach to investigate the determinants of P- PantSH permeability, and whether this relates to the structure of the molecule, or a certain specific membrane composition. This was achieved by devising a new assay specific for determining the membrane permeability of thiolated metabolites (such as P-PantSH), and subsequently applying this assay in studying the permeability of P-PantSH and its structural analogues in model membranes. Our results indicate that the permeability of P-PantSH is dependent on the fluidity of the membranes in question, and that manipulation of membrane composition to affect membrane fluidity offers a new mechanism to regulate the maintenance of CoA levels from exogenous sources. Using the same assay, we were also able to study a mitochondrial solute carrier protein that is apparently involved in the regulation of CoA levels in the mitochondria. ADP-dependent efflux of P-PantSH was observed for the transporter, and this constitutes preliminary evidence of the implied role of P-PantSH as a nexus metabolite of CoA metabolism. These findings have important implications for the development of drugs that target CoA biosynthesis and utilization, whether in the context of human diseases for the treatment or delay of neurodegenerative diseases, or alternatively as antimicrobials for the treatment of infectious diseases.