Regulation of store-operated calcium entry by rhomboid intramembrane proteolysis

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.

Calcium ions regulate most aspects of cellular life. Accordingly, all cells carefully manage calcium levels, and do so through compartmentalisation. Calcium ions are maintained at low levels in the cytosol (~100 nM) and are concentrated and stored in internal organelles such as the endoplasmic reticulum (ER) (~400 uM). The plasma membrane (PM) forms a barrier to high levels of extracellular calcium (~1.2mM). Almost all cells in the animal kingdom use this compartmentalised system to orchestrate myriad cellular events: a sharp rise in cytosolic calcium controls enzymatic activity, protein-protein interactions, gene activation, cell proliferation and apoptosis. Many signalling pathways trigger the release of internal calcium stores from the ER into the cytosol. The consequent decline in ER calcium triggers a flux of calcium through the PM into the cytosol, reinforcing the cytosolic signal and refilling internal calcium stores. A PM calcium release-activated calcium (CRAC) channel controls this 'store-operated calcium entry' (SOCE) pathway. Orai family proteins 1-3 are the pore-forming subunits of the CRAC channel. This proposal builds on our recent discovery that Orai1 (and 2/3) are substrates of the rhomboid intramembrane protease, RHBDL2. Using a multidisciplinary approach, we will investigate the molecular and cellular mechanisms and significance of this newly discovered mode of calcium signalling regulation. We will characterise the protease/substrate interaction and use this to inform the design of specific inhibitors. To place this control process in a wider context we will use hypothesis driven approaches and a proteomic screen to identify potential regulators of RHBDL2 cleavage of Orai1. Finally, we will also explore the physiological role of RHBDL2 as a novel signal regulator in two well characterised primary cell types: T lymphocytes and keratinocytes.

Who will benefit from this research?

1. Academic research scientists
2. Patient groups e.g. immunodeficiency and cancer sufferers
3. Biotech and pharmaceutical industries and the wider economy
4. Dr Adam Grieve, the named postdoctoral research assistant
5. School/medical students, and interested members of the public

Below, we highlight how they will benefit and what will be done to ensure that they have the opportunity to receive this benefit. This is further described in the Pathways to Impact statement.

1. The main immediate impact of this work will be academic. Our research will produce significant advances in several basic biology communities including, very broadly, physiology, cell biology, chemical biology, immunology and pathology. Our research crosses several disciplines and will inform these fields about novel mechanisms for ion channel regulation. This will be ensured by open access publication, conference attendance and data sharing via WWW resources.

2. Defective CRAC channel signalling is a direct cause of severe combined immunodeficiency. It also underpins other diseases such as allergy and certain cancers. Although not directly translational, our project, aimed at discovering the regulation of CRAC channel signalling by a novel enzyme, has obvious health implications by underpinning improvements in healthcare and treatment of disease. As such, it fits within the remit of BBSRC's Strategic Research Priority 3 "Bioscience for Health".

3. Our work is relevant to advances in biotechnological production processes and could provide major economic benefit. Many products of biotech and pharmaceutical industries are secreted proteins, often expressed in animal cells. Rhomboids have demonstrated roles within the secretory pathway, in the shedding and release of factors into the medium. A major component of the cost of production of these factors is the efficiency of this process. Our project will provide information about the central machinery of the secretory system and sheddases. Our novel bioinformatics-led screen, which falls under the remit of Enabling theme 2 of the BBSRC's Strategic Plan "Exploiting new ways of working" may pave the way to improvements to this process. This is because it will provide us with rhomboid protease substrate recognition mechanisms and ideal substrate sequences, which will inform the tailoring of efficient secretion systems that biotech and pharmaceutical industries may be interested in adapting for their purposes. In a similar vein, our discovery of a natural substrate for RHBDL2 will allow us to screen and develop novel inhibitors of this enzyme family. Therefore, we have planned discussions various biotech companies, as described in our Pathways to Impact statement.

4. This project offers many opportunities for Dr Grieve to acquire new skills. The collaborative nature of the research will expose him to new techniques and avenues of research such as calcium imaging, high-end microscopy, patch-clamping and molecular inhibitor design. Plans are in place for career development at workshops and events for science communication. Last, upon completion of this work, incorporated in our plans are applications for Dr Grieve to group leader positions and career development fellowships.

5. More broadly, communication about our work can provide benefits to the wider public by engaging them in thinking about the links between apparently very specialised and obscure research questions, and the possible benefits to society. Dr Grieve will ensure this by learning broader public engagement skills at Cafés Scientifiques and University of Oxford's Scientific Outreach Programme. Both Professor Freeman and Dr Grieve are involved with the Dunn School's public engagment activities. Moreover, the biology of cells is part of the national curriculum, so we can use our expertise to participate in school outreach programmes. Indeed, we often provide work placements for school students.