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

人体由数十亿个细胞组成，每个细胞都被一层由脂质和蛋白质组成的膜所包围。此外，每个细胞还包含许多称为细胞器的专门隔间，它们也被膜包围。在许多细胞膜中发现的一种关键脂质是胆固醇。胆固醇可以从我们吃的食物中获得，也可以在称为内质网的细胞室中产生。胆固醇水平的不平衡对细胞是有毒的，所以它的水平受到严格的调节。然而，这个过程可能出错，胆固醇调节缺陷与一系列疾病有关，从动脉粥样硬化到神经变性。这项研究计划的目的是弄清楚胆固醇是如何在细胞内运输的，并确定这一过程如何调节整个身体的胆固醇水平。我们的研究小组进行的初步工作已经确定，一种名为VAMP4的蛋白质对这一过程很重要。当我们破坏细胞和小鼠中VAMP4的功能时，我们发现胆固醇水平发生了显著变化。使用一种叫做电子显微镜的技术，我们可以直接观察细胞内的细胞器，看看它们是如何相互作用的。在缺乏VAMP4的细胞中，我们可以看到称为内体的细胞器不能正确地与内质网结合。此外，当细胞产生过多的VAMP4时，我们看到更多的内质网与核内体结合。考虑到所有这些结果，这是我们目前的模型，VAMP4正在努力使核内体和内质网非常接近，然后允许胆固醇在这些细胞器之间移动。目前，尚不清楚这一过程是如何调节的，因此我们计划使用一系列生化技术，包括质谱法来确定哪些蛋白质与VAMP4相互作用并协调这一过程。为了确定VAMP4的缺失如何导致胆固醇水平的全局变化，我们计划研究VAMP4在巨噬细胞中的功能，巨噬细胞是一种在胆固醇生物学中起重要作用的细胞类型。总之，本提案中概述的研究将增加我们对调节胆固醇水平的重要细胞途径的理解，并在长期内为理解营养和衰老如何影响胆固醇生理提供基础。

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.