Dr Mark Young

(he/him)

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 and cryoEM structures of P2X receptors are now available (including those of rat P2X7 in the apo- and ATP-bound state [2]), transforming our understanding of their structure-function relationship [3], and enabling us to use structure-based approaches to discover and model the binding of novel small-molecule modulators [4,5], as potential drugs to treat inflammatory disease. 

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 [6]. 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 [7]. The properties of P2X7 are regulated by its 200 amino acid intracellular C-terminal 'ballast' domain, which is thought to couple ion channel activation to downstream signalling [2], and it has been suggested that targeting P2X7-mediated downstream signalling might represent a good strategy to develop more selective anti-inflammatory drugs [8].

Aims

How does P2X7 activation couple to downstream signalling pathways?

Upon activation by extracellular ATP, P2X7 couples via its long C-terminal ballast domain to several intracellular signalling pathways, leading to recruitment of the NRLP3 inflammasome and release of pro-inflammatory cytokines [7]. 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). We are currently exploring the role of the ballast domain in P2X7-dependent signalling, using a combination of cell biology, biochemical assays and computational approaches (collaboration with Dr Georgina Menzies, Cardiff).

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

We have constructed molecular models of human P2X receptors and 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 (collaboration with Prof Andrea Brancale). In this way we have discovered novel compounds that modulate P2X7 [5] and investigated the determinatns of ligand binding at P2X4 [4]. We hope to develop this work to find compounds which then be further modified to develop potent and selective drugs, which may be of significant therapeutic benefit in conditions of pain and inflammation.

Structure and dynamics of slipper limpet hemocyanin

As part of the Protein Technology Hub, I have worked with Mikota PLC to investigate the 3D-structure of slipper limpet hemocyanin (oxygen carrier protein in the blood) using cryoEM [10]. 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). We are also interested in how oxygen binds to hemocyanin, and are investigating this using molecular dynamics simulations (collaboration with Dr Georgina Menzies)

References

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. McCarthy AE, Yoshioka C and Mansoor SE (2019) Full-Length P2X₇ Structures Reveal How Palmitoylation Prevents Channel Desensitization.Cell 179, 659-70.
3. Pasqualetto G et al. (2018) The molecular determinants of small-molecule ligand binding at P2X receptors. Frontiers in Pharmacology 9 , 58.
4. Pasqualetto G et al. (2023) Identification of the molecular determinants of antagonist potency in the allosteric binding pocket of human P2X4. Front Pharmacol 14, 1101023.
5. Pasqualetto G et al. (2023) Identification of a novel P2X7 antagonist using structure-based virtual screening. Front Pharmacol 13, 1094607.
6. Chessell IP et al. (2005) Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain 114, 386-396
7. Sluyter R (2017) The P2X7 Receptor. Adv Exp Med Biol. doi: 10.1007/5584_2017_59
8. Sorge RE et al. (2012) Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity. Nat Med 18, 595-599
9. Fowler BJ et al. (2014) Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity. Science 346, 1000-1003
10. Pasqualetto G et al (2023) CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin. PLoS One. 18(6):e0287294.

Cardiff University

• Dr Georgina Menzies
• Professor Kenneth Harris

Mikota PLC

• Alex Mühlhölzl

Postgraduate research students

• Lloyd Allen
• Christopher Slack

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 projects available:

1. Investigating the role of P2X7 receptor intracellular domains in cell 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 little about their interactions with other biomolecules, or which parts of them are involved in signalling pathways. 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. Recent work suggests that the ballast domain can bind to calcium-calmodulin, and that this may affect its conformation.

The focus of this project is to investigate the role of P2X7 intracellular domains in downstream signalling, using a combination of molecular modelling, site-directed mutagenesis, protein expression, protein purification, functional assays and direct structural studies. Particular focus will be given to understanding the role the ballast domain in coupling to intracellular signalling cascades.

2. Assaying the function and antimicrobial activity of slipper limpet hemocyanin

Antimicrobial resistance (AMR) is reaching a crisis point, with recent estimates suggesting that by 2050, 10 million people annually will die of from infection by pathogens with AMR. To tackle this crisis both novel antimicrobial therapeutics and better diagnostic tools are needed. Since developing countries will be disproportionately affected by AMR it is also essential that these novel therapeutics can be easily and cheaply produced in bulk.

Hemocyanins are oxygen transport proteins present in the hemolymph (blood) of arthropods and molluscs. In addition to their normal physiological role, molluscan hemocyanins are involved in the host innate immune response. Damage to hemocyanins by either protease treatment or partial denaturation gives rise to phenoloxidase activity, important in the synthesis of the antimicrobial pigment melanin. Proteolysis has also been demonstrated to liberate peptides with antimicrobial activity.

We have recently started investigating the structure and functional properties of a novel hemocyanin from the slipper limpet (Crepidula fornicata) which may be a source of novel antimicrobial peptides; these peptides may represent a new set of tools for countering the growing problem of AMR. Recent preliminary data indicates that the full-length hemocyanin protein, and a protease-treated preparation, shows antibiofilm activity against Staphylococcus aureus.

In this project, we propose to explore this potential antimicrobial and antibiofilm activity further. Using a range of clinically important bacterial and fungal species and in vitro and in vivo (Galleria mellonella) methodologies, we will explore the susceptibility of both planktonic and biofilm (single and mixed species) communities to these compounds. The ability of the compounds to alter microbial pathogenicity and interact synergistically with current antibiotics will also be explored. Experiments will also be performed to fully characterise the functional properties of slipper limpet hemocyanin, quantifying oxygen binding, enzyme activity and fractionating protease-treated preparations to isolate single functional units and active peptides.

This project represents an excellent opportunity for a student to work towards developing novel antibiotics, learning and applying techniques in protein biochemistry, enzyme and ligand binding assays, antimicrobial susceptibility assays, biofilm assays and virulence assays.

Current supervision

April Talbot

Lloyd Allen

Alex Moger

Lurui Wan