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

点击翻译按钮获取中文摘要

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.