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

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.

The transmembrane metalloproteinase ADAM10 is a ubiquitously expressed molecular scissor that cleaves the extracellular regions from its substrates, including Notch, amyloid precursor protein and cadherins. ADAM10 regulates embryonic development and diseases such as Alzheimer's, cancer, inflammatory diseases and Staphylococcus aureus infection. However, realising the therapeutic potential of ADAM10 is currently impossible due to toxicity that would result from global ADAM10 targetting. Our recent research shows how we can solve this problem. We and others have identified six tetraspanin proteins, termed TspanC8s, which promote ADAM10 cleavage of specific substrates. Therefore future targetting of specific TspanC8/ADAM10 complexes may be applicable to certain diseases, with minimal toxicity. However, the molecular mechanisms by which TspanC8s regulate ADAM10 are not known.

The aim of this project is to determine how one TspanC8, Tspan15, specifically promotes ADAM10 cleavage of N-cadherin, an adhesion molecule which maintains heart tissue architecture and regulates neuronal synapses and cancer metastasis. We hypothesise that Tspan15 promotes N-cadherin cleavage by regulating ADAM10 localisation and/or conformation. To address this, we will use cell line models that include our new Tspan15-knockout CRISPR/Cas9 cells, our 12 novel Tspan15 monoclonal antibodies, conformational ADAM10 antibodies, advanced fluorescent microscopy, proteomics and a novel membrane encapsulation technique that we have developed to determine membrane protein structures.

Objectives are as follows:
1) To discover how Tspan15 localises ADAM10 to N-cadherin.
2) To determine whether Tspan15 induces a distinct ADAM10 conformation.
3) To investigate the functional effects of Tspan15 monoclonal antibodies.

Our findings will help us to understand how other TspanC8s regulate ADAM10 cleavage of other substrates, and how TspanC8 antibodies might act in a therapeutic setting.

BBSRC STRATEGIC PRIORITIES. Due to the fundamental role of ADAM10 in development and disease processes, this basic science proposal falls within the BBSRC strategic area of 'Bioscience for health', and the responsive mode priority of 'Healthy aging across the lifecourse'. Indeed, it is focussed on the proteolytic cleavage, or 'shedding', of the extracellular regions of cell surface proteins, which is an emerging mechanism for the regulation of healthy cellular function. The consequences of shedding can include the removal of signalling receptors from the cell surface, the removal of adhesion molecules to weaken cell-cell adhesive contacts, the release of chemokines or growth factors, or the initiation of an intracellular signalling cascade. The transmembrane metalloprotease ADAM10 is a 'molecular scissor' that is responsible for a substantial proportion of shedding from the surface of human cells. At least 40 target proteins have been identified for ADAM10, including proteins involved in embryonic development and development of the central nervous system, and maintenance of blood vessel integrity and a healthy immune response. This proposal aims to determine the mechanism by which ADAM10 substrate specificity is regulated by the TspanC8 subgroup of tetraspanin transmembrane proteins. The findings will therefore yield novel and fundamental insights into normal cell function.

INDUSTRY. ADAM10 is a potential therapeutic drug target for the pharmaceutical industry due to its role in Alzheimer's disease, cancer, Staphylococcus aureus infection and inflammatory diseases such as heart attack, stroke and asthma. However, it is currently unclear how ADAM10 could be targeted to treat specific diseases. Indeed, ADAM10 is a ubiquitous protein with an important role in the function of normal healthy cells, so global inhibition of ADAM10 could yield serious, perhaps fatal, side effects. Our recent research has provided a solution to this problem by showing that specific TspanC8s promote ADAM10 cleavage of distinct substrates. The current project will determine the molecular mechanism by which TspanC8s regulate ADAM10. Moreover, we will show for the first time the effects of TspanC8 monoclonal antibodies on ADAM10 function, enabling us to determine how such reagents might act in a therapeutic setting. The results of our basic research could be used by the pharmaceutical industry to design therapeutic strategies that target specific TspanC8-ADAM10 complexes. This could allow ADAM10 targetting in a cell type and/or substrate-specific manner, so avoiding the toxic side effects of global ADAM10 targetting.

ACADEMIA. Tetraspanins and ADAM10 are emerging as important regulators of other cell surface proteins through their compartmentalisation into microdomains and shedding from the cell surface, respectively. As outlined in our 'Academic beneficiaries' section, new academic researchers are becoming interested in these fields as they discover that their proteins of interest are regulated by tetraspanins and/or ADAM10. Our results will provide important mechanistic and technological breakthroughs in these fields that will be of great benefit to other researchers.

STUDENTS. Dr Tomlinson incorporates his latest tetraspanin and ADAM10 research into lectures given to final year undergraduate and masters students in Birmingham. Most students are very enthusiastic to learn about these emerging areas that are fundamental and yet often completely new to them. As such, several enquire about the possibilities of undertaking their final year research project in the Tomlinson lab. Indeed, aspects of the project, in addition to the spin-off projects that will emerge, are ideally suited to short-term projects which will greatly benefit the students. The post doctoral researcher who undertakes the project will also benefit in developing their supervising skills.