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

跨膜金属蛋白酶ADAM 10是一种广泛表达的“分子酶”，其从其底物（包括Notch、淀粉样前体蛋白和钙粘蛋白）切割细胞外区域。ADAM 10可以被认为是胚胎发育的“主调节器”。ADAM 10还通过其在阿尔茨海默氏症、癌症、金黄色葡萄球菌感染和包括心脏病发作、中风和哮喘在内的炎症性疾病中的作用影响人类健康。因此，靶向治疗ADAM 10具有巨大的潜力。然而，实现这种潜力目前是不可能的，因为靶向体内每个细胞的ADAM 10会产生毒性。我们最近的研究证明了我们如何解决这个问题。我们和其他人最近已经确定了六个四跨膜蛋白，我们称之为TspanC 8 s，促进特定底物的ADAM 10切割。因此，未来的治疗靶向特定的TspanC 8/ADAM 10复合物可能适用于某些疾病，同时最大限度地减少全球ADAM 10 targeting.The项目的目的是确定如何TspanC 8之一，Tspan 15，特异性地促进N-钙粘蛋白粘附分子的ADAM 10切割。N-钙粘蛋白作为“分子尼龙搭扣”，对于维持跳动心脏中的组织结构至关重要，并调节神经元突触形成和癌细胞转移。我们假设Tspan 15通过调节ADAM 10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的新Tspan 15和ADAM 10敲除CRISPR/Cas9细胞的细胞系模型。主要目的如下：1）研究Tspan 15如何将ADAM 10定位于N-cadherin，我们将使用先进的荧光显微镜来确定Tspan 15/ADAM 10定位于N-cadherin的程度，并与其他TspanC 8/ADAM 10复合物进行比较。我们将使用蛋白质组学和免疫共沉淀来鉴定促进Tspan 15定位的细胞内运输蛋白。我们将通过在敲低后不存在ADAM 10和存在不能与运输蛋白结合的Tspan 15突变体的情况下评估ADAM 10对N-钙粘蛋白的切割来证明它们的重要性。2）为了确定Tspan 15是否诱导独特的ADAM 10构象，我们将使用一组构象ADAM 10单克隆抗体通过流式细胞术研究与Tspan 15复合的ADAM 10构象，并与其它5种TspanC 8/ADAM 10复合物进行比较。我们将获得Tspan 15/ADAM 10复合物的结构信息，再次与其他TspanC 8/ADAM 10复合物相比，使用一种新的膜蛋白包封方法，我们已经开发了在其天然状态下的膜蛋白的纯化。包封的TspanC 8/ADAM 10结构将通过分析性超离心、小角X射线散射，3）研究Tspan 15单克隆抗体的功能效应。我们将使用western blotting和N-cadherin-1（N-cadherin-1）检测我们的12种新的Tspan 15单克隆抗体如何影响ADAM 10对N-cadherin的切割。这项工作的发现将帮助我们了解其他TspanC 8如何调节其他底物的ADAM 10切割，并允许我们首次评估TspanC 8抗体如何在治疗环境中起作用。

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.