Regulation of store-operated calcium entry by rhomboid intramembrane proteolysis

通过菱形膜内蛋白水解调节钙库操作的钙进入

• 批准号: BB/R016771/1
• 负责人: Matthew Freeman
• 金额: $ 48.56万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR016771%2F1
• 关键词: Regulation; store; operated; calcium; entry

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.

我们身体中的细胞相互交流，以帮助它们决定如何行动。例如，它们是否应该分裂、迁移、变得更加专业化，甚至死亡？细胞之间和细胞内信号传递的主要语言之一取决于改变细胞内钙的浓度。因此，有一个非常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：它是许多生物学、生物技术和医学相关过程的基础。钙信号传导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中，充当看门人，控制钙是否可以从外部进入，其中钙水平要高得多。CRAC通道的主要成分是一种名为Orai 1的蛋白质（以希腊神话中天堂之门的守护者奥赖命名）。我们最近发现Orai 1可以被我们研究的另一种蛋白质切割，这种蛋白质叫做RHBDL 2。在这个项目中，我们将利用这个意想不到的最新发现来揭示允许RHBDL 2切割Orai 1的分子机制，以及它的生物学意义。总的来说，我们希望完全理解这个过程，但首先是在基础水平上。切割奥赖是否会减少CRAC通道打开时的钙流量，或者是否会控制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，RHBDL 2在哪里切割Orai 1，切割Orai 1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奥赖和RHBDL 2基因功能的新方法。一旦我们在基础水平上了解了这一过程，我们将研究其在人类生物学中的重要性。虽然我们怀疑RHBDL 2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，这两种细胞对我们的健康非常重要，并且具有依赖于Orai 1的良好特征的钙信号传导。首先是人类T细胞，这是我们免疫系统的重要组成部分。它们在体内巡逻，寻找其他表现出疾病迹象的细胞。T细胞识别癌细胞和被细菌或病毒感染的细胞表面上不寻常的蛋白质分子。当他们发现这些武器时，他们可以促成一系列事件，最终安全销毁和清除这些武器。第二类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重要作用，而且在伤口愈合中也起重要作用。在T细胞和角质形成细胞中，我们的项目将揭示RHBDL 2在切割Orai 1时的作用。这是如何影响钙信号传导的，以及这对这两种基本细胞类型的功能的最终影响是什么？Orai 1在严重的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RHBDL 2如何以及在何处切割奥赖，以及刺激RHBDL 2这样做的因素，我们将有可能获得可以导致设计可以控制这一过程的药物的信息。由于RHBDL 2的作用尚未得到解决，这一重要发现可能为改善多种疾病的治疗铺平道路。最后，生物技术产业还在很大程度上依赖于了解细胞行为是如何调节的，例如，这样才能有效地改造细胞以生产有用的蛋白质。钙信号的根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。

项目成果

iRhom2 regulates ERBB signalling to promote KRAS-driven oncogenesis

• DOI: 10.1101/2021.08.06.455383
• 发表时间: 2021-08
• 期刊: bioRxiv
• 作者: Boris Sieber;Fangfang Lu;S. Stribbling;A. Grieve;A. Ryan;M. Freeman

iRhom2 regulates ERBB signalling to promote KRAS-driven tumour growth of lung cancer cells.

• DOI: 10.1242/jcs.259949
• 发表时间: 2022-09-01
• 期刊: Journal of cell science
• 影响因子: 4