Understanding the mechanism by which tetraspanins regulate the 'molecular scissor' ADAM10

跨膜金属蛋白酶ADAM10是一种普遍表达的‘分子剪刀’，它将细胞外区域从其底物上切割出来，包括Notch、淀粉样前体蛋白和钙粘附素。ADAM10可以被视为胚胎发育的‘主调节器’。ADAM10还通过其在阿尔茨海默氏症、癌症、金黄色葡萄球菌感染等疾病以及心脏病发作、中风和哮喘等炎症性疾病中的作用影响人类健康。因此，ADAM10的治疗靶向具有巨大的潜力。然而，目前实现这一潜力是不可能的，因为将ADAM10靶向体内每一个细胞会产生毒性。我们最近的研究表明，我们可以解决这个问题。我们和其他人最近鉴定了六种TspanC8蛋白，我们称之为TspanC8s，它们促进ADAM10对特定底物的切割。因此，未来特定TspanC8/ADAM10复合体的治疗靶点可能适用于某些疾病，同时将全球靶向ADAM10的毒副作用降至最低。该项目的目的是确定TspanC8中的Tspan15如何特定地促进ADAM10对N-钙粘附素黏附分子的切割。N-钙粘附素起着“分子魔术贴”的作用，对维持心脏跳动中的组织结构至关重要，并调节神经元突触的形成和癌细胞的转移。我们假设Tspan15通过调节ADAM10亚细胞定位和/或使其采用特定构象来促进N-钙粘蛋白的切割。为了解决这一假设，我们将使用包括我们新的Tspan15和ADAM10基因敲除的CRISPR/Cas9细胞的细胞系模型。我们的主要目标如下：1)了解Tspan15如何定位ADAM10到N-钙粘素。我们将使用先进的荧光显微镜来确定Tspan15/ADAM10定位到N-钙粘素的程度，并与其他TspanC8/ADAM10复合体进行比较。我们将使用蛋白质组学和免疫共沉淀确定促进Tspan15定位的细胞内运输蛋白。我们将通过在敲除后没有ADAM10的情况下以及在不能与运输蛋白结合的Tspan15突变体的存在下评估ADAM10的切割来证明它们的重要性。2)确定Tspan15是否诱导独特的ADAM10构象。我们将使用一组构象ADAM10单抗通过流式细胞术研究ADAM10与Tspan15复合体中的ADAM10构象，并与其他五个TspanC8/ADAM10复合体进行比较。我们将使用我们开发的一种新的膜蛋白包埋方法来纯化天然状态的膜蛋白，从而获得Tspan15/ADAM10复合体的结构信息，这也是与其他TspanC8/ADAM10复合体相比较的。包裹的TspanC8/ADAM10结构将通过分析超速离心、小角X射线散射、负染透射电子显微镜和冷冻电子显微镜来确定。3)研究Tspan15单抗的功能效应。我们将使用Western blotting和N-钙粘素依赖的细胞迁移功能分析来确定我们的12个新的Tspan15单抗如何影响ADAM10对N-钙粘素的裂解。这项工作的发现将帮助我们了解其他TspanC8是如何调节其他底物的ADAM10裂解的，并使我们能够第一次评估TspanC8抗体如何在治疗环境中发挥作用。

The transmembrane metalloproteinase ADAM10 is a ubiquitously expressed 'molecular scissor' that cleaves the extracellular regions from its substrates, which include Notch, amyloid precursor protein and cadherins. ADAM10 can be regarded as a 'master regulator' of embryonic development. ADAM10 also impacts on human health via its role in diseases such as Alzheimer's, cancer, Staphylococcus aureus infection and inflammatory diseases including heart attack, stroke and asthma. As such, therapeutic targeting of ADAM10 has huge potential. However, realising this potential is currently impossible due to the toxicity that would result from targeting ADAM10 on every cell in the body. Our recent research demonstrates how we can solve this problem. We and others have recently identified six tetraspanin proteins, which we termed TspanC8s, which promote ADAM10 cleavage of specific substrates. Therefore future therapeutic targetting of specific TspanC8/ADAM10 complexes may be applicable to certain diseases, whilst minimising the toxic side effects of global ADAM10 targetting.The aim of this project is to determine how one of the TspanC8s, Tspan15, specifically promotes ADAM10 cleavage of the N-cadherin adhesion molecule. N-cadherin acts as 'molecular velcro' and is essential for maintaining tissue architecture in the beating heart, and regulates neuronal synapse formation and cancer cell metastasis. We hypothesise that Tspan15 promotes cleavage of N-cadherin by regulating ADAM10 subcellular localisation and/or causing it to adopt a specific conformation.To address this hypothesis, we will use cell line models that include our new Tspan15- and ADAM10-knockout CRISPR/Cas9 cells. The main objectives are as follows:1) To discover how Tspan15 localises ADAM10 to N-cadherin.We will use advanced fluorescent microscopy to determine the extent of Tspan15/ADAM10 localisation to N-cadherin, in comparison to other TspanC8/ADAM10 complexes. We will identify the intracellular trafficking proteins that promote Tspan15 localisation using proteomics and co-immunoprecipitation. We will demonstrate their importance by assessing ADAM10 cleavage of N-cadherin in their absence following knockdown, and in the presence of Tspan15 mutants that cannot bind to the trafficking proteins.2) To determine whether Tspan15 induces a distinct ADAM10 conformation.We will investigate ADAM10 conformation in complex with Tspan15 by flow cytometry using a panel of conformational ADAM10 monoclonal antibodies, and compare with the other five TspanC8/ADAM10 complexes. We will obtain structural information on the Tspan15/ADAM10 complex, again compared to the other TspanC8/ADAM10 complexes, using a novel membrane protein encapsulation method that we have developed for the purification of membrane proteins in their native state. Encapsulated TspanC8/ADAM10 structures will be determined by analytical ultracentrifugation, small-angle X-ray scattering, negative stain transmission electron microscopy and cryo-electron microscopy.3) To investigate the functional effects of Tspan15 monoclonal antibodies.We will determine how each of our 12 new Tspan15 monoclonal antibodies affects ADAM10 cleavage of N-cadherin using western blotting and an N-cadherin-dependent functional assay for cell migration.The findings from this work will help us to understand how other TspanC8s regulate ADAM10 cleavage of other substrates, and allow us to assess for the first time how TspanC8 antibodies might act in a therapeutic setting.