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Understanding the mechanism by which the ‘molecular scissor’ ADAM10 and tetraspanins regulate lymphocyte function

Principal Supervisor: Dr Farhat Khanim

Co-supervisors: Dr Mike Tomlinson

PhD project title: Understanding the mechanism by which the ‘molecular scissor’ ADAM10 and tetraspanins regulate
lymphocyte function

University of Registration: Birmingham

Project outline:

Background. The transmembrane metalloprotease ADAM10 is a ‘molecular scissor’ which is ubiquitously expressed and has important roles in health and disease by cleaving the extracellular domains from its transmembrane target proteins. Whilst the majority of studies on ADAM10 have been performed in mouse models or in neuronal systems, relatively little work has been done using human cells and even less is known about the role of ADAM10 in immune cells.  On lymphocytes, which are critical to an effective immune response, ADAM10 has recently emerged as an important regulator of growth and differentiation through cleavage of the Notch cell fate regulators, NKG2D natural killer cell ligands and the CD23 immunoglobulin E receptor. In addition, expression of ADAM10 has been shown to be aberrant in myeloma, a cancer of mature B lymphocytes (plasma cells). However, the mechanism by which ADAM10 activity is regulated remains unclear, and its substrates on lymphocytes are relatively poorly characterised.

Recent discoveries that have lead up to this project. The Tomlinson group have recently proposed that ADAM10 should not be regarded as a single molecular scissor, but as six different scissors with different substrate specificities depending on which one of six tetraspanins it is associated with [1]. The tetraspanins are a superfamily of 33 membrane proteins that possess four transmembrane regions and control the subcellular localisation and function of proteins such as ADAM10 with which they interact. The Tomlinson group have recently initiated a collaboration with Prof Stefan Lichtenhaler (University of Munich, Germany), who has developed a novel proteomic technology to identify ADAM10 substrates: ‘secretome protein identification with click sugars’ (SPECS) [2]. The Khanim group work on normal immune cells as well as with myeloma, leukaemia and lymphoma models. They have successfully established protocols for performing CRISPR gene knockouts in these cells.

This project will combine the expertises of the two groups with the aim to understand the role of ADAM10 and tetraspanins in human immune cell function.

Hypothesis. We hypothesise that specific tetraspanins promote ADAM10 shedding of specific substrates on lymphocytes thereby regulating immune cell function, and that this activity is altered in lymphocyte tumours.  

Objectives. The present PhD project will focus on the role of ADAM10/tetraspanins in lymphocyte biology and will use human cell line models and primary cells to address the following objectives;

1. To determine whether ADAM10 is important for growth/differentiation/function of lymphocytes using an ADAM10 inhibitor and CRISPR/Cas9 knockout

2. To identify ADAM10 substrates on lymphocytes using the novel SPECS proteomic technology, developed in Munich, and to establish this in Birmingham

3. To discover which ADAM10 substrates are important for lymphocyte growth/ differentiation/function using CRISPR/Cas9 knockout

4. To identify which tetraspanins regulate ADAM10 cleavage of the important lymphocyte substrates

The aims complement a BBSRC Project Grant recently awarded to Mike Tomlinson (PI), Tim Dafforn (Co-I) and Carol Murphy (Co-I) entitled, Understanding the mechanism by which tetraspanins regulate the ‘molecular scissor’ ADAM10 (£501,611) which focuses on epithelial cell biology (start in April 2017). 

Key references

[1] Matthews AL, Noy PJ, Reyat JS and Tomlinson MG (2016). Regulation of A Disintegrin and Metalloproteinase (ADAM) family sheddases: the emerging role of tetraspanins and rhomboids. Platelets. In press.

[2] Kuhn PH, Colombo AV, Schusser B, Dreymueller D, Wetzel S, Schepers U, Herber J, Ludwig A, Kremmer E, Montag D, Müller U, Schweizer M, Saftig P, Bräse S, Lichtenthaler SF (2016). Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function. Elife, Jan 23;5. pii: e12748.

BBSRC Strategic Research Priority: World Class Bioscience - Molecular Mechanisms

Techniques that will be undertaken during the project:

  • Isolation of primary human lymphocytes
  • Culture of lymphocyte cell lines
  • Flow cytometry
  • Real-time quantitative PCR
  • Western blotting using the Odyssey Infrared Imaging System
  • CRISPR/Cas9 genome editing
  • Fluorescent microscopy including dSTORM super-resolution imaging
  • Biophysical characterisation of ADAM10/tetraspanin complexes
  • Mass spectrometry-based proteomics

Contact: Dr Farhat Khanim, University of Birmingham