| Popis: |
Solute carrier transporters (SLCs) mediate the inter- and intra- cellular trafficking of a plethora of substrates and are essential to cell homeostasis. Despite their importance to human physiology and their potential as therapeutic targets, many SLCs are considered orphans as the physiological substrate has not been experimentally determined. Furthermore, SLCs remain understudied and underutilized, partly due to the inherent difficulties in working with SLC transporters. In this thesis, I present the development of the GFP-based thermostability assay (GFP-TS), which enables the detection of ligand-SLC interactions using un-purified material, but with the same high-throughput screening capability as dye-based thermal-shift assays. We highlight how GFP-TS is an excellent complement to native mass spectrometry approaches for analyzing lipid-protein interactions, by demonstrating how specific lipids modulate oligomerization in Na+/H+ exchangers. GFP-TS was combined with other biochemical approaches to show that not all SLC35 members transport nucleotide-sugars, as currently believed. Specifically, we unequivocally demonstrate that SLC35B1 is a strict ADP/ATP exchanger, which is critical for cell homeostasis, as it supplies the endoplasmic reticulum (ER) with ATP. Finally, I outline the progress towards elucidating the function of the synaptic vesicle protein 2A (SV2A), an enigmatic brain SLC transporter that is the receptor for clinically-used anti-epileptic drugs. In summary, my research contributes to the growing body of knowledge of SLC function, and outlines how a simple thermal stability can be utilised for determining ligand- and lipid-interactions of SLC transporters. |
