Elucidating the role of SNAREs in membrane contact site formation and cholesterol homeostasis

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The human body is made of billions of cells and each cell is surrounded by a membrane made from lipids and proteins. In addition, each cell also contains numerous specialised compartments called organelles, which are also surrounded by membranes. A key lipid found in many membranes is cholesterol. Cholesterol can be obtained from the food that we eat or is made in a cellular compartment called the endoplasmic reticulum. Imbalances in cholesterol levels are toxic to cells so its levels are tightly regulated. However, this process can go wrong and defects in cholesterol regulation are associated with a range of diseases from atherosclerosis through to neurodegeneration. The aim of this research proposal is to work out how cholesterol is transported within cells and determine how this process regulates cholesterol levels throughout the body. Preliminary work performed by our research team has identified that a protein called VAMP4 is important for this process. When we disrupt the function of VAMP4 in cells and in mice we see that the levels of cholesterol are significantly altered. Using a technique called electron microscopy we can directly look at organelles within cells and see how they interact with each other. In cells lacking VAMP4, we can see that an organelle called an endosome is not properly bound to the endoplasmic reticulum. In addition, when cells are made to make too much VAMP4 we see that more of the endoplasmic reticulum is bound to endosomes. Taken all of these results in consideration, it is our current model that VAMP4 is working to bring endosomes and the endoplasmic reticulum in very close proximity, which then allows cholesterol to move between these organelles. At present, it is unclear how this process is regulated so we plan to use a range of biochemical techniques, including mass spectrometry to identify which proteins interact with VAMP4 and co-ordinate this process. To determine how loss of VAMP4 leads to global changes in cholesterol levels we plan to investigate the function of VAMP4 in macrophages, which is a cell type with an important role in cholesterol biology. In summary, the research outlined in this proposal will increase our understanding of important cellular pathways, which regulate cholesterol levels and in the long term provide a foundation for understanding how nutrition and ageing effect cholesterol physiology.