Titel: Structural Basis of Membrane Transport and Pyrophosphorylation of Thiamine in Humans
Sprache: Englisch
Autor*in: Gabriel, Florian
Erscheinungsdatum: 2024
Tag der mündlichen Prüfung: 2025-01-31
Zusammenfassung: 
Thiamine (vitamin B1) is an essential coenzyme for prokaryotic and eukaryotic cells alike. In conjunction with thiamine-dependent enzymes, the vitamin catalyses reactions of central metabolic pathways, like the citric acid cycle and the pentose phosphate pathway. Metazoans, including humans, have lost the ability to synthesise thiamine de novo. Hence, every cell in these organisms needs to import the vitamin over its plasma membrane. In humans, this membrane transport is mainly mediated by the solute carrier proteins SLC19A2 and SLC19A3. Perturbation of this transport system has severe effects on human health. Loss of function mutations in SLC19A2 and SLC19A3 cause detrimental diseases. Furthermore, the inhibition of SLC19A3 by the Janus kinase inhibitor fedratinib is associated with severe neurological side effects in the form of Wernicke’s encephalopathy. Despite the physiological and pharmacological importance of thiamine transport in humans, the underlying molecular machinery remained poorly understood on a structural and biophysical level. In this Dissertation, I present high-resolution cryo-EM structures of the human SLC19A3 (hSLC19A3) in complex with its endogenous substrate thiamine, as well as the known thiamine uptake inhibitors fedratinib, amprolium, and hydroxychloroquine. The structure determination was enabled by the discovery of three unique SLC19A3-specific nanobodies. The nanobodies allowed me to determine the structure of hSLC19A3 in its outward- and inward-open conformational states. Using thermal shift and cellular uptake assays, I explored the wider drug interaction spectrum of hSLC19A3. This brought about the discovery of seven inhibitors of the human thiamine transporter. Based on the determined cryo-EM structures, we performed computational docking to screen medications for their propensity to block thiamine transport in humans. Hit compounds from this virtual screen were validated experimentally on a biophysical, functional, and structural level. The biological function of thiamine as a coenzyme in the cell is critically dependent on its pyrophosphorylation by the enzyme TPK1. In this Dissertation, I present a cryo-EM structure of the human TPK1 at a resolution of 2.1 Å. This work showcases the ability of cryo-EM to determine small protein structures at resolutions useful for clear biochemical interpretation and structure-based drug design.
URL: https://ediss.sub.uni-hamburg.de/handle/ediss/11473
URN: urn:nbn:de:gbv:18-ediss-125572
Dokumenttyp: Dissertation
Betreuer*in: Wilson, Daniel
Löw, Christian
Enthalten in den Sammlungen:Elektronische Dissertationen und Habilitationen

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