[
International Worm Meeting,
2021]
Cholesterol is an essential metabolite present in virtually all eukaryotic organisms in which mediates highly relevant biological processes such as the regulation of membrane fluidity and the synthesis of steroid hormones and bile acids. Therefore, proper trafficking of this molecule to different subcellular locations is crucial for cell viability and correct organism functioning. In this sense, non-vesicular cholesterol transport is mediated by a multi-domain membrane protein called STARD3 that binds cholesterol through its cytosolic domain (START). While there is a significant amount of information related to the physiological processes associated with cholesterol metabolism, the regulatory events responsible for STARD3-mediated transport remain unknown. Recently, it has been reported that STARD3 co-localizes and interacts with methionine sulfoxide reductase A, an enzyme that reduces methionine sulfoxide side-chains, suggesting that methionine oxidation could modulate the sterol binding properties of the START domain and cholesterol transport. These findings have been obtained in cultured mammalian cells which lack the complex tissue context present in higher organisms and all its associated biological activities, emphasizing the need for an animal model to understand these biological processes in vivo. We are using the nematode Caenorhabditis elegans to study the molecular events associated with the regulation of cholesterol mobilization mediated by STARD3 and its interaction with MSRA. First, we devised an NMR-based assay to monitor methionine sulfoxide reductase activity in C. elegans extracts and established that these extracts have prominent MSRA activity. Next, we titrated cholesterol to 15N isotopically-enriched START and followed the interaction by 1H-15N HSQC NMR experiments. Our results clearly showed the formation of a high-affinity complex, confirming that cholesterol binding to STARD3 is conserved in C. elegans. We will use this set of tools to characterize with high-resolution the interaction between MSRA and START in vitro and in worm extracts. Complementary genetic and biochemical experiments will allow us to dissect the role of START and MSRA in non-vesicular cholesterol mobilization.