Dr Mark Young

Publication

• Date
• Type

Research

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 [3], 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 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 proteins do the P2X7 C-terminal domain 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] and cryoEM structure of rat P2X7 [9]. 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 discover ‘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). I also collaborate with Professor Kenneth Harris (School of Chemistry) using powder X-ray diffraction for the structural study of biologically relevant molecules.

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. Pasqualetto G et al. (2018) The molecular determinants of small-molecule ligand binding at P2X receptors. Frontiers in Pharmacology 9 , 58.
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
9. McCarthy AE et al. (2019) Full-Length P2X₇ Structures Reveal How Palmitoylation Prevents Channel Desensitization. Cell 2019 Oct 17;179(3):659-670

Current collaborators

Cardiff University

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

Mikota PLC

• Alex Mühlhölzl

Postgraduate research students

• Miss Marika Zuanon
• Mr Ryan Coates

Teaching

I teach in the Biochemistry/Protein Engineering subject areas in Years 2, 3 and 4. Subject areas include quantitative biochemistry, protein structure determination, protein purification, protein detection, enzyme regulation, structure-based drug design, membrane protein structure and synthetic biology chassis organisms.

I offer research projects in Years 3 and 4 looking at the structure-function relationship of plasma membrane ion channels and their roles in heath and disease.

I also teach Biochemistry and Exercise Physiology in the Platform for Clinical Science (Medical School).

Biography

Supervisions

PhD project available:

How does P2X7 receptor activation couple to downstream signalling?

P2X7 receptors are ion channels that are found on the surface of immune cells, where they respond to the binding of extracellular ATP, a damage signal released by dying cells in infection and injury. ATP binding to P2X7 receptors leads to calcium influx and initiates a downstream signalling cascade, resulting in the formation of large pores in the plasma membrane, changes in cell shape, protein secretion, kinase activation and changes in gene expression. Both ion channel function and downstream signalling are highly regulated by the 30 amino-acid N-terminal and 240 amino-acid C-terminal intracellular domains of the receptor (NTD and CTD), at least in part via interaction with lipids in the plasma membrane, but we know very little about their interactions with other biomolecules, or which parts of them are involved in signalling pathways. The recent publication of the cryoEM structure of full-length rat P2X7 shows that both the NTD and the cysteine-rich N-terminal portion of the CTD (C-Cys anchor) are palmitoylated, anchoring these domains to the inner leaflet of the plasma membrane, and that the C-terminal portion of the CTD (ballast) forms a globular domain containing binding sites for dinuclear zinc and GDP/GTP, raising intriguing questions about its potential function.

The focus of this project will be to understand the role of P2X7 intracellular domains in downstream signalling, using a combination of molecular modelling, site-directed mutagenesis, protein expression, protein purification and functional assays. Particular focus will be given to understanding the role of lipids in regulating receptor function, and the role of the ballast domain in coupling to intracellular signalling cascades. We will also attempt to express and purify full-length P2X7 receptors (or their intracellular domains) for 3D-structure study using cryoEM or X-ray crystallography.