Regulation of store-operated calcium entry by rhomboid intramembrane proteolysis

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Cells in our bodies communicate with each other to help them decide how to behave. For example, should they divide, or migrate, or become more specialised, or even die? One of the main languages of signalling between and within cells depends on altering the concentration of calcium inside the cell. There is therefore a very strong incentive to understand how calcium signalling works and how it is regulated: it underlies many biological, biotechnological and medically relevant processes. A key component of calcium signalling is the so-called CRAC channel, which sits in the membrane surrounding the cell and acts as a gatekeeper, controlling whether calcium can enter from outside, where calcium levels are much higher. The main constituent of the CRAC channel is a protein called Orai1 (named after the Orai, which are the keepers to heaven's gate in Greek mythology). We have recently discovered that Orai1 can be cut by another protein we study, an enzyme called RHBDL2. In this project we will capitalise on this unexpected recent discovery to reveal both the molecular mechanisms that allow RHBDL2 to cut Orai1, and also its biological significance.Overall, we want to understand this process fully, but first at a fundamental level. Does cutting Orai decrease calcium flow through the CRAC channel upon opening, or does it control where and when the CRAC channel can open? We want to know how this process occurs, exactly where RHBDL2 cuts Orai1, the details of the consequence of cutting Orai1, and the importance of this process in human cells. To do so, we will use cutting-edge technologies such as high resolution microscopy to see these events in living cells, and CRISPR-Cas9 gene editing, a new way to engineer the function of Orai and RHBDL2 genes in cells.Once we understand the process at a fundamental level, we then will investigate its importance in human biology. Although we suspect that RHBDL2 may regulate many different instances of calcium signalling, we will focus on two specific human cell types, both of which are very important to our health and have well characterised calcium signalling that relies on Orai1. The first are human T cells, which are essential components of our immune system. They patrol the body for other cells that display signs of disease. T cells recognise unusual protein molecules on the surface of cancer cells and cells that have been infected by bacteria or viruses. When they find them, they can induce a programme of events that leads to their safe destruction and clearance. The second type of cell that we will focus upon is keratinocytes. They are skin cells and have an essential role not just in the barrier function of skin, but also in wound healing. In both T cells and keratinocytes our project will reveal what the role is of RHBDL2 when it cuts Orai1. How does this affect calcium signalling and what is the ultimate effect of this on the function of these two essential cell types?Orai1 is mutated in severe combined immunodeficiency and the CRAC channel is implicated in many human diseases such as asthma and cancer. By understanding how and where RHBDL2 cuts Orai, and the factors that stimulate RHBDL2 to do so, it is possible that we will gain information that can lead to the design of drugs that can control this process. As the role of RHBDL2 has not yet been addressed, this important discovery may pave the way to improvements in the treatment of a wide array of diseases. Finally, the biotechnology industry also depends greatly on understanding how cellular behaviour is regulated, for example so that cells can be efficiently engineered to produce useful proteins. The fundamental significance of calcium signalling means that our work may also provide future benefits in those areas.