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

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.

There is increasing evidence that regions of membrane contact between intracellular organelles, termed contact sites, play an important role in cholesterol transport. However, our understanding of the molecular processes that regulate their formation is still very limited and their role in homeostasis in vivo has barely been explored. We have recently identified that VAMP4 is required for ER-endosome contact site formation and its loss in both cell culture and animal models leads to defects in cholesterol homeostasis. Our current working hypothesis is that VAMP4 is acting in a non-fusogenic manner to tether a sub-class of endosomes on to the ER possibly via a direct interaction with VAP-A/B. The loss of VAMP4 dependent contact sites then causes defects in cholesterol transfer between endosomes and the ER. This in turn leads to reduced esterification and increased synthesis of cholesterol, promoting cholesterol efflux from peripheral tissues, resulting in elevated HDL-cholesterol levels. To elucidate the role of VAMP4 in this important process we will: a) identify and characterise the molecular interactions that drive VAMP4 dependent membrane contact site formation; b) elucidate how these interactions are regulated and c) determine how loss of VAMP4 dependent contact sites alter cholesterol homeostasis. To address these objectives we will use a range of approaches from standard biochemistry through to cutting edge techniques such as proximity based proteomics and correlative light and electron microscopy combined with Photoclick labelling approaches. To make these studies as physiologically relevant as possible we will use cell models that most closely recapitulate in vivo processes such as primary macrophages. The research outlined in this proposal will not only provide molecular and mechanistic insight into the role of VAMP4 in the formation and regulation of ER-endosome contact sites but also their role in cholesterol physiology.

The dysregulation of cholesterol levels are thought to be a key factor in the development of a range of diseases from atherosclerosis to neurodegeneration. If we just take cardiovascular disease into account, it is estimated that it costs the NHS approximately 6.8 billion pounds per year (2014 British Heart Foundation Statistics). Thus, there is a significant need for the development of more effective ways of treating this condition. However, our understanding of the cellular and physiological processes involved in cholesterol biology are far from complete. The aim of this proposal is develop a detailed molecular and mechanistic understanding of how endosomal-ER membrane contact sites mediate cholesterol transport and regulate cholesterol physiology. In the long term, this new information will help underpin the development of new drugs for the treatment of conditions caused by altered cholesterol physiology. There are three main groups in society who will benefit from our research:

Pharmaceutical and biotech companies: The global market for cholesterol lowering drugs is estimated to be approximately 19 billion dollars per year. However, many of the original statin based drugs are coming off patent. Thus, there is increasing interest in the development of novel strategies for lowering cholesterol levels. The work outlined in this proposal will lead to the identification of novel cellular machinery which is either directly involved cellular cholesterol transport or its regulation. These factors, in the long term, could be developed as new targets that could be used to manipulate cholesterol levels. In addition, having a better understanding of the cellular machinery regulating cholesterol levels will help in the identification of novel genetic factors or biomarkers, which could be useful in the development of stratified approaches for treating diseases associated with perturbed cholesterol levels.

Patients: There are a large number of patients who are either unable or are unwilling to take the current cholesterol-lowering drugs due to their adverse side effects. Thus, the development of more specific and targeted therapies should increase the amount of choice patients have and lead to larger numbers of patients taking cholesterol-lowering drugs so reducing the rates of morbidity associated with failing to take this medication.

General Public: Through our planned public engagement activities we hope to foster a better understanding of science, develop trust and highlight the relevance of critical thinking in day to day life. In the longer term, this will hopefully help the public make more informed choices in assessing the potential risks and benefits associated with the application of new technologies for the treatment of disase.