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Investigating tetraspanins as molecular switches

Principal Supervisor: Dr. Michael Tomlinson, School of Biosciences

Co-supervisor: Professor Tim Dafforn, School of Biosciences

PhD project title: Investigating tetraspanins as molecular switches

University of Registration: University of Birmingham

Project outline:

Background: The tetraspanins are a superfamily of 33 four-transmembrane proteins in humans that interact with and regulate the trafficking and clustering of specific ‘partner proteins’. Two partners that have been recently identified in the Tomlinson group are the ‘molecular scissor’ ADAM10 and the store-operated Ca2+ entry channel Orai1. ADAM10 is expressed on all cell types and is essential for embryonic development due to its cleavage and activation of Notch proteins.

ADAM10 has at least 40 other substrates including amyloid precursor protein, cellular prion protein, adhesion molecules, growth factors and their receptors, making ADAM10 a potential therapeutic target for many human diseases. The Tomlinson group have recently proposed that ADAM10 is not a single scissor, but six different scissors with different substrates, depending on which of six regulatory tetraspanins it is in complex with. These tetraspanins are Tspan5, 10, 14, 15, 17 and 33, and are collectively termed the TspanC8s. This raises the possibility of therapeutically targeting just one of the six scissors via specific tetraspanins, which could be used to treat certain diseases without the toxic side effects of targeting ADAM10 on every cell in the body [1].

Store-operated Ca2+ entry through the plasma membrane Ca2+ channel Orai1 is essential for healthy function of most cell types. Indeed, loss of Orai1 results in severe immunodeficiency that requires a bone marrow transplant for survival. The Tomlinson group have shown that Tspan18 interacts with Orai1 in endothelial cells and promotes its trafficking to the cell surface. Endothelial cells lacking Tspan18 have defective Ca2+ signalling and defective release of blood clotting factors, with the result that Tspan18-deficient mice have impaired blood clotting (unpublished).

The mechanisms of activation of ADAM10 scissor function and Orai1 Ca2+ channel opening are not fully understood. We believe that their associated tetraspanins play a role in the activation process. Recently, the first crystal structure of a tetraspanin was reported, showing that tetraspanin CD81 forms a cone-shaped structure with a cholesterol-binding site within the four transmembrane domains. Molecular dynamics simulations suggest that removal of the cholesterol would cause a dramatic conformational change, whereby the main extracellular region would swing upwards [2]. This raises the exciting possibility that tetraspanins function as ‘molecular switches’ to regulate the activity of partners, such as ADAM10 and Orai1, via conformational change.

The Dafforn group have recently developed the styrene maleic acid lipid particle (SMALP) method for encapsulating membrane proteins in their native form, allowing subsequent structural analyses with rapidly emerging technologies such as cyro-electron microscopy [3]. This revolutionary approach is ideally suited to the purification and structural determination of tetraspanins and their partners.

Hypothesis. The activities of tetraspanin partners ADAM10 and Orai1 are regulated by conformational change in their associated tetraspanins.

Objectives 1. To determine whether cholesterol depletion, or tetraspanin cholesterol-binding mutants, activate ADAM10 and Orai1 in cell line models. 2. To determine whether tetraspanins adopt an extended conformation in the absence of cholesterol, and induce a conformational change in ADAM10 and Orai1.

References:

  • Matthews AL…Tomlinson MG (2017). Scissor sisters: regulation of ADAM10 by the TspanC8 tetraspanins. Biochem Soc Trans 45: 719-30.
  • Zimmerman B…Blacklow SC (2016). Crystal structure of a full-length human tetraspanin reveals a cholesterol-binding pocket. Cell 167: 1041-51.
  • Lee SC…Dafforn TR (2016). A method for detergent-free isolation of membrane proteins in their local lipid environment. Nat Protoc 11: 1149-62.  

BBSRC Strategic Research Priority: Bioenergy and Industrial biotechnology


Techniques that will be undertaken during the project:

  • Molecular biology to generate mutant expression constructs
  • Cell culture and transfection
  • Flow cytometry
  • Western blotting using the Odyssey Infrared Imaging System
  • CRISPR/Cas9 genome editing
  • Fluorescent microscopy including dSTORM super-resolution imaging
  • Transcriptional luciferase reporter assays
  • FRET peptide-based ADAM10 activity assay
  • Biophysical characterisation of ADAM10/tetraspanin complexes using SMALPs:

Circular dichroism spectroscopy

Sedimentation velocity analytical ultracentrifugation

Negative stain electron microscopy

Small angle X-ray scattering (SAXS)

Cryo-electron microscopy

Contact: Dr. Michael Tomlinson, School of Biosciences