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Membrane permeability and transport studies of coenzyme A, its precursors and antimetabolites.
Thesis (PhD)--Stellenbosch University, 2020.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2020
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| _version_ | 1867614096606625792 |
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| access_status_str | Open Access |
| author | Mogwera, Koketso Seipelo Precious |
| author2 | Strauss, Erick |
| author_browse | Mogwera, Koketso Seipelo Precious Strauss, Erick |
| author_facet | Strauss, Erick Mogwera, Koketso Seipelo Precious |
| author_sort | Mogwera, Koketso Seipelo Precious |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2020. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/108404 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:46:36.532Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/108404 Membrane permeability and transport studies of coenzyme A, its precursors and antimetabolites. Mogwera, Koketso Seipelo Precious Strauss, Erick De Villiers, Marianne Stellenbosch University. Faculty of Science. Dept. of Biochemistry. Coenzyme A (CoA) -- Biosynthesis Phosphopantetheine Membrane permeability Liposomes Fatty acid-binding proteins UCTD Thesis (PhD)--Stellenbosch University, 2020. 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. AFRIKAANSE OPSOMMING: Koënsiem A (KoA) is 'n kofaktor wat noodsaaklik is vir verskeie metaboliese prosesse, waaronder sentrale energie en vetsuurmetabolisme. Daarbenewens is daar toenemende bewyse dat KoA ook 'n belangrike rol speel in die tempo van neurodegenerasie en veroudering. KoA word gewoonlik gebiosintetiseer vanaf vitamien B5, maar onlangs is aangetoon dat KoA in die dieet ook intrasellulêre KoA-vlakke kan aanvul deur die afbraak van eksogene KoA deur ekto-nukleosied pirofosfatases na 3',5'-ADP en 4'-fosfopanteteïen (P- PantSH), wat gevolglik deur selle opgeneem word. Meer belangrik, deurlaatbaarheidstudies het aangetoon dat hierdie opname plaasvind deur passiewe diffusie, 'n waarneming wat verder bevestig is deur voedingsaanvullingsstudies in Drosophila S2-selle wat 'n mutasie bevat wat tot die uitputting KoA-reserwes lei. Verder het die ondersoek na KoA-homeostase in Escherichia coli voorgestel dat hierdie bakterie P-PantSH onomkeerbaar uitvoer om intrasellulêre KoA-vlakke te reguleer. Dit dui op 'n wisselwerking tussen eukariote en hul mikrobioom t.ov. die handhawing van die vlakke van hierdie kofaktor deur die uitruil van P- PantSH as 'n waarskynlike verbindingsmetaboliet in KoA-biosintese. In hierdie studie het ons 'n sistematiese biofisiese benadering gebruik om die determinante van P-PantSH-deurlaatbaarheid te ondersoek, en of dit verband hou met die struktuur van die molekule, of 'n spesifieke membraansamestelling. Dit is bewerkstellig deur 'n nuwe toets te ontwerp wat spesifiek is vir die bepaling van die membraandeurlaatbaarheid van tiol- bevattende metaboliete (soos P-PantSH), waarna hierdie toets gebruik is vir die bestudering van die deurlaatbaarheid van P-PantSH en strukturele analoë daarvan in modelmembrane. Ons resultate dui aan dat die deurlaatbaarheid van P-PantSH afhanklik is van die vloeibaarheid van die betrokke membrane, en dat die manipulering van die membraansamestelling om die membraanvloeibaarheid te beïnvloed 'n nuwe meganisme bied om die handhawing van KoA-vlakke vanuit eksogene bronne te reguleer. Met behulp van dieselfde toets kon ons ook 'n mitochondriale vervoerproteïen bestudeer wat betrokke blyk te wees by die regulering van KoA-vlakke in die mitochondria. ADP-afhanklike uitvloei van P- PantSH is waargeneem vir die vervoerproteïen, en hierdie resultaat vorm 'n voorlopige bewys van die implisiete rol van P-PantSH as 'n verbindingsmetaboliet van KoA-metabolisme. Hierdie bevindings het belangrike implikasies vir die ontwikkeling van medikasie wat KoA- biosintese en -benutting teiken, hetsy in die konteks van menslike siektes vir die behandeling of vertraging van neurodegeneratiewe siektes, of alternatiewelik as antimikrobiese middels vir die behandeling van aansteeklike siektes. Doctoral 2020-02-26T13:14:02Z 2020-04-28T15:12:27Z 2020-02-26T13:14:02Z 2020-04-28T15:12:27Z 2020-04 Thesis http://hdl.handle.net/10019.1/108404 en_ZA Stellenbosch University xvi, 115 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Coenzyme A (CoA) -- Biosynthesis Phosphopantetheine Membrane permeability Liposomes Fatty acid-binding proteins UCTD Mogwera, Koketso Seipelo Precious Membrane permeability and transport studies of coenzyme A, its precursors and antimetabolites. |
| title | Membrane permeability and transport studies of coenzyme A, its precursors and antimetabolites. |
| title_full | Membrane permeability and transport studies of coenzyme A, its precursors and antimetabolites. |
| title_fullStr | Membrane permeability and transport studies of coenzyme A, its precursors and antimetabolites. |
| title_full_unstemmed | Membrane permeability and transport studies of coenzyme A, its precursors and antimetabolites. |
| title_short | Membrane permeability and transport studies of coenzyme A, its precursors and antimetabolites. |
| title_sort | membrane permeability and transport studies of coenzyme a its precursors and antimetabolites |
| topic | Coenzyme A (CoA) -- Biosynthesis Phosphopantetheine Membrane permeability Liposomes Fatty acid-binding proteins UCTD |
| url | http://hdl.handle.net/10019.1/108404 |
| work_keys_str_mv | AT mogwerakoketsoseipeloprecious membranepermeabilityandtransportstudiesofcoenzymeaitsprecursorsandantimetabolites |