Dr Mark Young

Introduction

P2X receptors are ATP-gated ion channels which play key roles in a variety of physiological processes such as synaptic transmission, taste sensation and smooth muscle control.  They function in cells as trimers, with two transmembrane domains per monomer and large, glycosylated extracellular domains [1]. Several crystal structures of P2X receptors have recently been published (including those of zebrafish P2X4 in the apo- and ATP-bound state [2]), transforming our understanding of their structure-function relationship, but more structures, particularly of the human subtypes, are needed, and this represents a significant challenge because eukaryotic membrane proteins are hard to express and purify in the large quantities required for structural study.

P2X receptors are important drug targets primarily because of their involvement in pain following nerve damage and inflammation.  In a rat model of neuropathic pain, tactile allodynia was abolished when levels of the P2X4 receptor subtype were knocked down using antisense oligonucleotides [3].  It is therefore likely that drugs which target P2X4 may have significant analgesic properties, but the current lack of human P2X4-selective antagonists is hampering research into its specific roles.

P2X receptors are also involved in inflammation. The P2X7 receptor subtype is expressed in immune cells; and in knock-out mice lacking P2X7, chronic inflammatory pain was abolished, while acute pain responses remained unchanged [4]. P2X7 is unique among the P2X receptors in that its activation leads to the release of pro-inflammatory cytokines, and prolonged activation causes cell death [5]. The properties of P2X7 are regulated by its long intracellular C-terminal domain, which couples ion channel activation to downstream signalling, and it has recently been suggested that targeting P2X7-mediated downstream signalling might represent a good strategy to develop more selective anti-inflammatory drugs [6].

Aims

Exploring the expression of P2X receptors in other eukaryotic systems

We have successfully expressed P2X receptors in baculovirus-infected insect cells [7] (collaboration with Dr Mark Parker, Case Western, USA). Insect cell expression was successfully used in solving the structure of zebrafish P2X4 [2], so it represents a proven system for the expression of P2X receptors. We are developing the fruit fly Drosophila melanogaster as an expression system for membrane proteins which permits both structural and functional studies (collaboration with Dr Wynand Van der Goes Van Naters, School of Biosciences). We have also recently developed a collaboration with Dr Simon Scofield (School of Biosciences) to develop plant expression systems for P2X receptors.

P2X7 downstream signalling pathways

Upon activation by extracellular ATP, P2X7 couples via its long C-terminal domain to several intracellular signalling pathways, leading to recruitment of the NRLP3 inflammasome and release of pro-inflammatory cytokines [5]. This signalling has important consequences in pain and inflammation, particularly in conditions of chronic inflammation such as arthritis, Alzheimer’s disease and age-related macular degeneration (AMD). In collaboration with Professor Jayakrishna Ambati (University of Virginia, USA), we were able to show that disrupting P2X7 signalling with a small-molecule modulator abolished the death of retinal pigment epithelial cells seen in AMD, offering hope that P2X7 may be a target for this incurable disease [8]. Although many downstream components of the signalling pathways have been characterised, the molecular basis of the first step in this process remains unknown. What is the 3D-structure of the P2X7 C-terminal domain, which proteins does it interact with, and how are these interactions regulated?

Structure-based drug design using molecular models of human P2X receptors

While we do not have high-resolution structures for either human P2X4 or human P2X7, we have constructed molecular models based upon the ATP-bound crystal structure of zebrafish P2X4 [2]. In collaboration with Dr Andrea Brancale (School of Pharmacy) we have used these molecular models to perform in silico docking of a range of drug-like compounds into the ATP-binding site, selecting those which give the best fit for functional assays using calcium uptake and electrophysiology. In this way we hope to find ‘hit’ compounds which modulate P2X receptor function, which can then be further modified to develop potent and selective drugs, which may be of significant therapeutic benefit in conditions of pain and inflammation.

Protein technology

As part of the Protein Technology Hub, I am working with Mikota PLC to develop purification and assay procedures for slipper limpet haemocyanin and collagen. Slipper limpets are an invasive species in the UK which destroy marine habitats; finding a commercial use for them would incentivise their removal and aid ecosystem recovery (http://www.cardiff.ac.uk/news/view/987729-life-saving-limpets).

1. Grimes L and Young MT (2015) Purinergic P2X receptors: structural and functional features depicted by X-ray and molecular modelling studies. Curr Med Chem 22, 783-98.
2. Hattori M and Gouaux E (2012) Molecular mechanism of ATP binding and ion channel activation in P2X receptors. Nature 485, 207-212
3. Tsuda M et al. (2003) P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 424, 778-783
4. Chessell IP et al. (2005) Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain 114, 386-396
5. Sluyter R (2017) The P2X7 Receptor. Adv Exp Med Biol. doi: 10.1007/5584_2017_59
6. Sorge RE et al. (2012) Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity. Nat Med 18, 595-599
7. Valente M et al. (2011) Expression, purification, electron microscopy, N-glycosylation mutagenesis and molecular modelling of human P2X4 and Dictyostelium discoideum P2XA. Biochim Biophys Acta 1808, 2859-2866
8. Fowler BJ et al. (2014) Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity. Science 346, 1000-1003

Current collaborators

Cardiff University

• Dr Wynand van der Goes Van Naters
• Dr Andrea Brancale
• Dr Colin Berry
• Dr Pierre Rizkallah
• Professor Kenneth Harris

University of Bath

• Dr Amanda MacKenzie

Mikota PLC

• Alex Mühlhölzl

Postgraduate research students

• Mr Ahmad Jaradat
• Mr Mender Mender
• Miss Marika Zuanon
• Mr Ryan Coates

I became interested in studying membrane protein structure-function relationships during my Biochemistry degree at the University of Bristol (1994-1997).  I stayed on in Bristol for my Ph.D. and first postdoc (1997-2003) under the guidance of Prof. Mike Tanner, where I explored the interaction between the red blood cell anion transporter (band 3) and its accessory subunit, glycophorin A (GPA).

A growing interest in ion channels led me to take up postdocs with Profs. Annmarie Surprenant and Alan North at the Universities of Sheffield (2003-2005) and Manchester (2005-2007), where I worked on P2X receptor structure-function relationships. During this time, the focus of my research shifted towards the direct structural study of P2X receptors.  With the aid of an Advanced Training Fellowship (2007-2010) and the mentorship of Prof. Bob Ford (University of Manchester), I determined the structure of human P2X4 at a resolution of 21Å, using electron microscopy of single protein particles and 3D reconstruction.

In September 2009 I took up the Evans-Huber Fellowship at Cardiff University, which has enabled me to set up my own research lab, where I continue to study the 3D structure and downstream signalling functions of mammalian P2X receptors, as well as looking to develop new expression systems for mammalian membrane proteins. I became a Lecturer (Teaching and Research) in September 2012. I was promoted to Senior Lecturer in 2015, and became the Academic Lead of the new Protein Technology Research Hub in 2016.