Biology subjects

Owen, D.

Publications and source records attributed to Owen, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Automating 3DED data processing at eBIC

Three-dimensional electron diffraction (3DED) is an emerging and useful technique for solving molecular structures of small and biological macro-molecules from nanometre-sized crystals. We present our automated data processing workflow for 3DED datasets collected at Diamond Light Sources electron Bio-Imaging Centre (eBIC). For this purpose, we developed a package called AutoED. The processing pipeline includes data collection, analysis of the beam position, metadata gathering, file conversion, and finally data processing using xia2 (which supports both DIALS and XDS). The processing results are captured in a summary report produced by AutoED. Our main goal is to reduce the workload of electron diffraction scientists, but also to enforce good standards already used in macromolecular crystallography (MX). All the collected 3DED datasets are automatically converted into NeXus data format which is considered a Gold Standard for MX. This standardized data format allows for all the relevant metadata about the experiment to be kept together with diffraction images. We also discuss the methods used in AutoED to determine the electron beam position on diffraction images.

biophysics↗

bioRxiv2026

Structural basis of the protein kinase PKN1 HR1 domain oligomerization and differential regulation by RhoA and Rac1

The protein kinase C-related kinase (PKN) family of serine/threonine kinases consists of PKN1, PKN2 and PKN3, all of which are Rho family GTPase effectors. PKNs have three N-terminal Homology Region 1 (HR1) domains (HR1a, HR1b and HR1c), which form antiparallel coiled coils, which in two cases interact with Rho family GTPases, activating the kinase. The PKNs are implicated in several important cellular processes, including cytoskeletal regulation, cell adhesion, gene expression and cell cycle progression, and are also implicated in cancer. Here we have investigated the roles of the HR1 domains in PKN oligomerisation. We show that PKN1 HR1a is a dimer and that the HR1c domain drives further oligomerization. We have mapped the interactions between the HR1 domains and used an integrative approach to model HR1-containing PKN1 dimers. Biophysical analysis shows that RhoA forms a 1:2 complex with HR1a, resulting in a rearrangement of the HR1a dimer, an outcome supported by SAXS models. In contrast, Rac1 binds to monomeric HR1a, suggesting that this GTPase activates PKN1 via a different mechanism. These data provide structural insight into interactions between HR1 domains and the Rho family proteins and their potential consequences for PKN1 activation.

biophysics↗

bioRxiv2025

Nanoscale protein clustering modulates the input-output response of cellular signalling pathways

A key function of the plasma membrane is regulating signal transduction. Proteins involved in the transduction process play a signal-processing role; mapping an input (e.g. number of engaged receptors) to an output (e.g. level of downstream phosphorylation). In many cases, a digital mapping is desirable, i.e. that a cell activates or responds fully once a set input threshold is surpassed. It is believed that the nanoscale organisation of proteins, such as their clustering, modulates this behaviour by altering the frequency of protein-protein interactions. Here, we develop a generalised simulator for dynamic molecular clustering built around agent-based modelling. We show that the clustering properties (e.g. size of clusters, percentage of monomers) tunes the cellular response. This work paves the way for designing potential therapeutic interventions that alter the nanoscale patterning of molecules on the cell surface in order to alter cell behaviour. AUTHOR SUMMARYSignal transduction is the process by which cells convert external signals into internal responses. This is essential for such functions as hormone signalling and immunity. Many membrane proteins form nanoscale clusters on the cell surface, and state-of-the-art imaging methods allow us to see these clusters in detail. We developed an agent-based simulator to explore how clustering of key signaling proteins (such as kinases and phosphatases) can influence cell signalling outputs. Our results suggest that protein clustering can "digitize" signals, pushing cells to respond in an "all-or-nothing" manner, rather than producing a smooth range of responses. This research contributes to a broader understanding of how dynamic reorganization of transmembrane proteins contributes to information processing. This is a central question in systems biology, with relevance to the fields of immunology, neuroscience, and cancer. By highlighting how the geometric properties of protein distributions can alter signalling responses, we may identify novel methods to manipulate cell behavior.

systems biology↗

bioRxiv2025

Kinase interplay centered on two regulatory elements confines the checkpoint protein Lte1 to the bud cell cortical compartment

In budding yeast, Lte1, a component of the spindle position checkpoint, must be confined to the bud for correctly coupling chromosomal segregation across the bud neck with mitotic exit. Bud compartmentalization involves a phospho-dependent program with Ras-GTP acting as the ultimate cortical anchor. Here we have dissected this program by identifying an Lte1 domain participating in Ras binding and two phospho-regulatory motifs, the N- and C-LREs (Lte1 Regulatory Elements). The C-LRE mediates control of Lte1 cortical recruitment by the p21-activated kinase, Cla4. The N-LRE modulates Lte1 binding to Ras more directly and is phosphorylated by the yeast MARK/PAR-1 protein kinases, Kin1/2. Along with previously described CDK phosphorylation sites, the N- and C-LREs are required for Lte1 localization and timely mitotic exit. These studies uncover the coordinated interplay between multiple protein kinases allowing Lte1 to integrate cell polarity and cell cycle cues for strict compartmentalization at the bud cell cortex.

cell biology↗

bioRxiv2024

Untangling structural molecular details of the endocytic adaptor protein CALM upon binding with phosphatidylinositol 4,5-bisphosphate-containing model membranes

Clathrin assembly lymphoid myeloid leukemia protein (CALM) is involved in the formation of clathrin-mediated endocytic coats by virtue of binding many proteins involved in the process, including clathrin itself and AP2 cargo adaptor complex. CALM is able to specifically recognize the inner leaflet of the plasma membrane by binding the membranes phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Here, a biophysical approach, primarily using neutron and X-ray scattering and solid-state NMR experiments, was exploited to investigate CALM interaction with PtdIns(4,5)P2-presenting model membranes. The presented experimental data reveal how the CALM folded domain is partly accommodated within the lipid membrane, directly interacting with PtdIns(4,5)P2 phosphates. Moreover, these data suggest that CALMs amphiphilic N-terminal helix buries into the membrane, not only stabilising the protein docking to the membrane but also providing a mechanism to induce membrane curvature.

biophysics↗

bioRxiv2024

Remote force modulation of the T-cell receptor reveals an NFAT-threshold for CD4+ T cell activation

Abstract/SummaryMechano-modulation of cell surface proteins to influence cell activation has been shown as a promising new advanced therapy for regenerative medicine applications. These strategies rely on the manipulation of mechanosensitive cell surface receptors to initiate intracellular signal transduction. The cell surface receptor of T lymphocytes (TCR), which recognises peptide-MHC molecules central to driving the adaptive immune response, has recently been suggested to be mechano-responsive. Despite this advance, little is known as to whether the TCR can be mechanically modulated to achieve TCR signalling and subsequent T cell activation, and whether these characteristics can be exploited for immunotherapies. Here, we describe a magnetic particle-based platform for mechanical modulation of the TCR and outline how this platform can be utilised to achieve CD4+ T cell activation. We demonstrate that mechanical manipulation of the TCR induces cell surface clustering of the TCR and downstream TCR signalling, leading to eventual TCR downregulation and T cell activation. We investigate the temporal relationship between mechanical modulation of the TCR and subsequent T cell activation, hereby identifying that accumulation of signalling events within the NFAT-pathway is required to reach the threshold required for CD4+ T cell activation, outlining an axis which controls the CD4+ T cell response to external mechanical cues. These findings identify how CD4+ T cells can modulate their function in response to such cues, whilst also outlining a remote-magnetic particle-based platform that may be used for the control of T cell responses.

immunology↗

bioRxiv2023

Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, can invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis. Here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the {beta}1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery preventing cellular spreading and adhesion. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI on the sEV surface. In vitro assays identified collagen VI as playing a key role in cell adhesion and invasion directionality. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate directional invasion, most likely by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction force to enable VSMC movement within the complex vascular ECM network. Figure AbstractVascular smooth muscle cells sense fibronectin via {beta}1 integrin and secrete small extracellular vesicles loaded with collagen VI. These extracellular vesicles are entrapped in the extracellular matrix and induce formation of peripheral focal adhesions presenting adhesion complex ECM proteins including collagen VI, LGALS3BP, EDIL3 and TGFBI. Focal adhesions anchor the extracellular matrix to actin fibrils in the cell. Contraction of the actin fibrils generates the mechanical force for directional cell invasion through the matrix. This figure was created with BioRender (https://biorender.com/). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/551257v3_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1355004org.highwire.dtl.DTLVardef@1186e9forg.highwire.dtl.DTLVardef@106ad30org.highwire.dtl.DTLVardef@1543518_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

ZMYM2 controls transposable element transcription through distinct co-regulatory complexes.

ZMYM2 is a zinc finger transcriptional regulator that plays a key role in promoting and maintaining cell identity. It has been implicated in several diseases such as congenital anomalies of the kidney where its activity is diminished and cancer where it participates in oncogenic fusion protein events. ZMYM2 is thought to function through promoting transcriptional repression and here we provide more evidence to support this designation. Here we studied ZMYM2 function in human cells and demonstrate that ZMYM2 is part of distinct chromatin-bound complexes including the established LSD1-CoREST-HDAC1 corepressor complex. We also identify new functional and physical interactions with ADNP and TRIM28/KAP1. The ZMYM2-TRIM28 complex forms in a SUMO-dependent manner and is associated with repressive chromatin. ZMYM2 and TRIM28 show strong functional similarity and co-regulate a large number of genes. However, there are no strong links between ZMYM2-TRIM28 binding events and nearby individual gene regulation. Instead, ZMYM2-TRIM28 appears to regulate genes in a more regionally defined manner within TADs where it can directly regulate co-associated retrotransposon expression. We find that different types of ZMYM2 binding complex associate with and regulate distinct subclasses of retrotransposons, with ZMYM2-ADNP complexes at SINEs and ZMYM2-TRIM28 complexes at LTR elements. We propose a model whereby ZMYM2 acts directly through retrotransposon regulation, which may then potentially affect the local chromatin environment and associated coding gene expression. Genome browser sessionUCSC browser session containing the peak tracks: http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg19&position=chr1:18,078,462-18,084,961&hide=all&hgct_customText=http://bartzabel.ls.manchester.ac.uk/sharrockslab/yaoyong/ZNF198/index_file_hg19_chipSeq_ZMYM2_final.txt Original ChIP-seq and ATAC-seq data from U2OS cells can be viewed On ArrayExpress at: E-MTAB-12292 (ADNP and TRIM28 ChIP-seq), E-MTAB-12293 (SUMO ChIP-seq) and E-MTAB-12294 (ATAC-seq)

molecular biology↗

bioRxiv2022

Resilient CD8+ T cells maintain a high cytotoxic capacity by balancing ROS via ME1 upregulation

Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignances. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1+ CD8+ T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1+ CD8+ T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysisindependent manner while concurrently curtailing excessive ROS in activated CD8+ T cells; and expands CX3CR1+NKG7+ effector CD8+ T cells with enhanced cytotoxicity. Importantly, transfection of ME1 mRNA promotes tumoricidal activity in CD8+ T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/517988v3_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@12e0f36org.highwire.dtl.DTLVardef@e8d412org.highwire.dtl.DTLVardef@1baf57eorg.highwire.dtl.DTLVardef@14b2b43_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICX3CR1+ and low Dy m identify functional resilient CD8+ T cells. C_LIO_LIResilient CD8+ T cells are highly cytotoxic and have less ROS. C_LIO_LIResilient CD8+ T cells express more ME1 that can balance extra ROS. C_LIO_LIME1 overexpression can promote CTL function of CD8+ T cells. C_LI

immunology↗

bioRxiv2022

Measuring the similarity of SMLM-derived point-clouds

Single-molecule localisation microscopy produces data in the form of point-clouds. Here, we present a tool for assessing the similarity of two such point-clouds, which, unlike measures such as co-localisation, is insensitive to differences that are not preserved between data sets. The presented method can determine whether two point-clouds were generated from the same conditions and can identify from which of two experimental conditions an unseen point-cloud was likely derived.

biophysics↗

bioRxiv2022

Phospholipid tail asymmetry allows cellular adaptation to anoxic environments

Membrane biophysical properties are critical to cell fitness and depend on unsaturated phospholipid acyl tails. These can only be produced in aerobic environments since eukaryotic desaturases require molecular oxygen. This raises the question of how cells maintain bilayer properties in anoxic environments. Here, we demonstrate the existence of an alternative pathway to regulate membrane fluidity that exploits phospholipid acyl-tail length asymmetry, replacing unsaturated species in the membrane lipidome. We show that the fission yeast, S. japonicus, which can grow in aerobic and anaerobic conditions, is capable of utilizing this strategy whereas its sister species, the well-known model organism S. pombe, cannot. The incorporation of asymmetric-tailed phospholipids might be a general adaptation to hypoxic environmental niches. One-Sentence SummaryIn anoxic environments, saturated asymmetric acyl-tailed phospholipids can replace unsaturated ones to maintain membrane physical properties.

cell biology↗

bioRxiv2022

Topology-based fluorescence image analysis for automated cell identification and segmentation

Cell segmentation refers to the body of techniques used to identify cells in images and extract biologically relevant information from them; however, manual segmentation is laborious and subjective. We present Topological Boundary Line Estimation using Recurrence Of Neighbouring Emissions (TOBLERONE), a topological image analysis tool which identifies persistent homological image features as opposed to the geometric analysis commonly employed. We demonstrate that topological data analysis can provide accurate segmentation of arbitrarily-shaped cells, offering a means for automatic and objective data extraction. One cellular feature of particular interest in biology is the plasma membrane, which has been shown to present varying degrees of lipid packing, or membrane order, depending on the function and morphology of the cell type. With the use of environmentally-sensitive dyes, images derived from confocal microscopy can be used to quantify the degree of membrane order. We demonstrate that TOBLERONE is capable of automating this task.

bioinformatics↗

bioRxiv2022

The Cdc42 effector and non-receptor tyrosine kinase, ACK defines a distinct STAT5 transcription signature in a chronic myeloid leukaemia cell model

Activated Cdc42-associated kinase (ACK) is a Rho family effector that is widely implicated in cancer. Here, we describe new roles for ACK in transcriptional regulation mediated by its relationship with the signal transducer and activators of transcription (STAT) family. We show that ACK can interact with STAT3, STAT5A and STAT5B, and augments phosphorylation at the conserved activation tyrosine on these STAT members. ACK stimulates oncogenic STAT nuclear relocation and transcriptional activation. We also identify endogenous relationships between ACK and STAT family members in haematopoietic disease cell lines. In the K562 chronic myeloid leukaemia cell line, we confirm that ACK contributes to the pool of active, nuclear STAT5. By interrogating ACK knock out cells we describe an ACK-driven STAT5 transcriptional signature in K562s. We propose ACK as a contributor to hyperactivated STAT5 signalling in this CML cell line and reveal a new route for therapeutic intervention. O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/494364v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1c4973dorg.highwire.dtl.DTLVardef@1a54ba1org.highwire.dtl.DTLVardef@1083bf9org.highwire.dtl.DTLVardef@1997ad0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Discovery of SARS-CoV-2 Mpro Peptide Inhibitors from Modelling Substrate and Ligand Binding

The main protease (Mpro) of SARS-CoV-2 is central to its viral lifecycle and is a promising drug target, but little is known concerning structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of classical molecular mechanics and quantum mechanical techniques, including automated docking, molecular dynamics (MD) simulations, linear-scaling DFT, QM/MM, and interactive MD in virtual reality, to investigate the molecular features underlying recognition of the natural Mpro substrates. Analyses of the subsite interactions of modelled 11-residue cleavage site peptides, ligands from high-throughput crystallography, and designed covalently binding inhibitors were performed. Modelling studies reveal remarkable conservation of hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular at the P2/S2 sites. The binding modes of the natural substrates, together with extensive interaction analyses of inhibitor and fragment binding to Mpro, reveal new opportunities for inhibition. Building on our initial Mpro-substrate models, computational mutagenesis scanning was employed to design peptides with improved affinity and which inhibit Mpro competitively. The combined results provide new insight useful for the development of Mpro inhibitors.

bioinformatics↗

bioRxiv2021

Cross-platform transcriptional profiling identifies common and distinct molecular pathologies in Lewy Body diseases

Parkinsons disease (PD), Parkinsons disease with dementia (PDD) and dementia with Lewy bodies (DLB) are three clinically, genetically and neuropathologically overlapping neurodegenerative diseases collectively known as the Lewy body diseases (LBDs). A variety of molecular mechanisms have been implicated in PD pathogenesis, but the mechanisms underlying PDD and DLB remain largely unknown, a knowledge gap that presents an impediment to the discovery of disease-modifying therapies. Transcriptomic profiling can contribute to addressing this gap, but remains limited in the LBDs. Here, we applied paired bulk-tissue and single-nucleus RNA-sequencing to anterior cingulate cortex samples derived from 28 individuals, including healthy controls, PD, PDD and DLB cases (n = 7 per group), to transcriptomically profile the LBDs. Using this approach, we (i) found transcriptional alterations in multiple cell types across the LBDs; (ii) discovered evidence for widespread dysregulation of RNA splicing, particularly in PDD and DLB; (iii) identified potential splicing factors, with links to other dementia-related neurodegenerative diseases, coordinating this dysregulation; and (iv) identified transcriptomic commonalities and distinctions between the LBDs that inform understanding of the relationships between these three clinical disorders. Together, these findings have important implications for the design of RNA-targeted therapies for these diseases and highlight a potential molecular "window" of therapeutic opportunity between the initial onset of PD and subsequent development of Lewy body dementia.

neuroscience↗

bioRxiv2020

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mpro inhibitor.

Designing covalent inhibitors is a task of increasing importance in drug discovery. Efficiently designing irreversible inhibitors, though, remains challenging. Here, we present covalentizer, a computational pipeline for creating irreversible inhibitors based on complex structures of targets with known reversible binders. For each ligand, we create a custom-made focused library of covalent analogs. We use covalent docking, to dock these tailored covalent libraries and to find those that can bind covalently to a nearby cysteine while keeping some of the main interactions of the original molecule. We found ~11,000 cysteines in close proximity to a ligand across 8,386 protein-ligand complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In prospective evaluation against a panel of kinases, five out of nine predicted covalent inhibitors showed IC50 between 155 nM - 4.2 M. Application of the protocol to an existing SARS-CoV-1 Mpro reversible inhibitor led to a new acrylamide inhibitor series with low micromolar IC50 against SARS-CoV-2 Mpro. The docking prediction was validated by 11 co-crystal structures. This is a promising lead series for COVID-19 antivirals. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.

biochemistry↗

bioRxiv2020

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease as a Clinical Candidate for the Potential Treatment of COVID-19

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.

pharmacology and toxicology↗

bioRxiv2019

The Kinase Chemogenomic Set (KCGS): An open science resource for kinase vulnerability identification

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

cell biology↗