Regulation of store-operated calcium entry by rhomboid intramembrane proteolysis

我们体内的细胞相互沟通，帮助它们决定如何行动。例如，它们应该分裂，还是迁移，还是变得更加专业化，甚至死亡？细胞间和细胞内信号传递的主要语言之一依赖于改变细胞内钙的浓度。因此，我们有强烈的动机去了解钙信号是如何工作的以及它是如何被调节的：它是许多生物、生物技术和医学相关过程的基础。钙信号的一个关键组成部分是所谓的CRAC通道，它位于细胞周围的膜上，起着看门人的作用，控制钙是否能从钙水平高得多的外部进入。CRAC通道的主要成分是一种名为Orai1的蛋白质（以希腊神话中天堂之门的守护者Orai命名）。我们最近发现Orai1可以被我们研究的另一种蛋白质，一种叫做RHBDL2的酶切割。在这个项目中，我们将利用这一意想不到的最新发现来揭示允许RHBDL2切割Orai1的分子机制及其生物学意义。总的来说，我们希望充分理解这一过程，但首先是在基本层面上。切割Orai是否会减少钙通过CRAC通道打开时的流量，或者它是否会控制CRAC通道打开的位置和时间？我们想知道这个过程是如何发生的，RHBDL2切割Orai1的确切位置，切割Orai1的后果的细节，以及这个过程在人类细胞中的重要性。为此，我们将使用尖端技术，如高分辨率显微镜来观察活细胞中的这些事件，以及CRISPR-Cas9基因编辑，这是一种设计细胞中Orai和RHBDL2基因功能的新方法。一旦我们在基本层面上理解了这个过程，我们就会研究它在人类生物学中的重要性。尽管我们怀疑RHBDL2可能调节许多不同的钙信号传导，但我们将重点关注两种特定的人类细胞类型，这两种细胞类型对我们的健康都非常重要，并且都具有依赖于Orai1的钙信号传导的特征。第一种是人体T细胞，它是我们免疫系统的重要组成部分。它们在体内巡逻，寻找其他有疾病迹象的细胞。T细胞识别癌细胞和被细菌或病毒感染的细胞表面的不寻常的蛋白质分子。当他们找到这些武器时，他们可以引发一系列事件，导致它们被安全销毁和清除。我们要关注的第二种细胞是角质形成细胞。它们是皮肤细胞，不仅在皮肤的屏障功能中起着重要作用，而且在伤口愈合中也起着重要作用。在T细胞和角质形成细胞中，我们的项目将揭示RHBDL2切割Orai1时的作用。这是如何影响钙信号的，这对这两种基本细胞类型的功能的最终影响是什么？Orai1在严重联合免疫缺陷中发生突变，CRAC通道与许多人类疾病（如哮喘和癌症）有关。通过了解RHBDL2如何以及在哪里切割Orai，以及刺激RHBDL2这样做的因素，我们有可能获得信息，从而设计出可以控制这一过程的药物。由于RHBDL2的作用尚未得到解决，这一重要发现可能为改善一系列疾病的治疗铺平道路。最后，生物技术产业也在很大程度上依赖于理解细胞行为是如何被调节的，例如，这样细胞才能被有效地改造以产生有用的蛋白质。钙信号传导的基本意义意味着我们的工作也可能在这些领域提供未来的好处。

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.