Structural and functional characterisation of the human vitamin c transporters
File(s)
Author(s)
Woubshete, Menebere
Type
Thesis
Abstract
Vitamin C is an essential micronutrient that functions as an antioxidant and a cofactor for several enzymes. Vitamin C has also shown potential as a possible therapeutic for treating several diseases such as cancers and neurodegenerative diseases. Humans and other primates are not able to synthesise vitamin C, so they must obtain it exclusively from their diet. The sodium-dependent vitamin C transporters (SVCT1 and SVCT2) are responsible for the cellular uptake of the reduced form of vitamin C, ascorbic acid. A greater understanding of the molecular mechanisms of the SVCTs would provide insights into their roles and potential with respect to health and disease.
The recent mouse SVCT1 structure provided insights into the molecular determinants of substrate binding and revealed that SVCT1 exists as a homodimer. Based on this structure and that of a related protein, I introduced several individual mutations, such as L65A, F112A, R206A, I439A and L458G, to human SVCT1 (hSVCT1) and explored their effect on expression, membrane trafficking and protein function. Results showed that these residues are important in substrate transport with no influence on expression and membrane trafficking. In addition, truncations at the N- and C-terminal ends of the protein confirmed the importance of the C-terminus in membrane trafficking. Correct dimerisation was also found to be essential in both membrane trafficking and function of hSVCT1.
hSVCT2 differs from hSVCT1 in that, in addition to transporting ascorbic acid, it activates the JAK/STAT pathway. However, this transceptor activity is not well understood as the structure of hSVCT2 has not yet been solved. Here, I performed extensive optimisation of hSVCT2 expression and purification, including changes to the expression construct, buffer conditions and purification resin, enabling the production of stable protein for structural studies. This will provide the basis for the future structural characterisation of hSVCT2 using cryo-EM.
The recent mouse SVCT1 structure provided insights into the molecular determinants of substrate binding and revealed that SVCT1 exists as a homodimer. Based on this structure and that of a related protein, I introduced several individual mutations, such as L65A, F112A, R206A, I439A and L458G, to human SVCT1 (hSVCT1) and explored their effect on expression, membrane trafficking and protein function. Results showed that these residues are important in substrate transport with no influence on expression and membrane trafficking. In addition, truncations at the N- and C-terminal ends of the protein confirmed the importance of the C-terminus in membrane trafficking. Correct dimerisation was also found to be essential in both membrane trafficking and function of hSVCT1.
hSVCT2 differs from hSVCT1 in that, in addition to transporting ascorbic acid, it activates the JAK/STAT pathway. However, this transceptor activity is not well understood as the structure of hSVCT2 has not yet been solved. Here, I performed extensive optimisation of hSVCT2 expression and purification, including changes to the expression construct, buffer conditions and purification resin, enabling the production of stable protein for structural studies. This will provide the basis for the future structural characterisation of hSVCT2 using cryo-EM.
Version
Open Access
Date Issued
2024-10-25
Date Awarded
01/02/2025
License URL
Advisor
Byrne, Bernadette
Sponsor
Imperial College London
Publisher Department
Department of Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)