Biology subjects

John, A. E.

Publications and source records attributed to John, A. E..

5 recordsLinked to original sources

bioRxiv2026

Targeting AKAP13 RhoGEF activity ameliorates pro-fibrotic phenotypes driven by the IPF associated AKAP13 risk variant

RationaleIdiopathic pulmonary fibrosis (IPF) is a progressive, incurable scarring disease of the lung. A common genetic variant near AKAP13, a multifunctional scaffold protein that integrates intracellular signalling through its interactions with RhoA and protein kinase A (PKA), has been associated with IPF susceptibility and elevated AKAP13 mRNA expression in lung tissue from patients. However, its contribution to the pathogenesis of IPF remains unclear. ObjectiveThis study investigates how an AKAP13 variant alters epithelial signalling and evaluates the therapeutic potential of targeting AKAP13. Findingsrs62025270-bearing iHBECs exhibited selective upregulation of AKAP13 isoforms, accompanied by increased cell adhesion and reduced proliferation. Transcriptomic profiling revealed upregulated fibrosis-related genes in rs62025270-bearing iHBECs, including SAA1, FGF2, MMP1, CTSB, COL4A1, and CDKN1A. rs62025270-bearing iHBECs also displayed increased RhoA activation and SMAD2 phosphorylation following LPA stimulation. Furthermore, cells harbouring the AKAP13 variant showed reduced intracellular cAMP levels. Pharmacological inhibition of AKAP13 with A13 reversed the pro-adhesive phenotype and reduced RhoA activation in iHBECs. Moreover, in IPF-derived PCLS, A13 suppressed SERPINE1, CCN2, and MMP7 expression, reduced SMAD2 nuclear translocation, and decreased hydroxyproline levels. ConclusionsPresence of an AKAP13 variant disrupts epithelial homeostasis and promotes pro-fibrotic signalling. Inhibition of AKAP13s RhoGEF domain with A13 restores epithelial function and attenuates fibrotic activation, supporting AKAP13 as a therapeutic target in IPF.

genetics↗

bioRxiv2025

Distal mutations in a designed retro-aldolase alter loop dynamics to shift and accelerate the rate-limiting step

Amino-acid residues distant from an enzymes active site are known to influence catalysis, but their mechanistic contributions to the catalytic cycle remain poorly understood. Here, we investigate the structural, functional, and mechanistic impacts of distal and active-site mutations discovered through directed evolution of the computationally designed retro-aldolase RA95. Active-site mutations improve catalytic efficiency by 3,600-fold, while distal mutations alone offer no improvement. When combined with active-site mutations, distal mutations further increase efficiency by 6-fold, demonstrating an epistatic effect. X-ray crystallography and molecular dynamics simulations reveal that distal mutations promote active site opening by altering loop dynamics. Kinetic solvent viscosity effects and electrostatic analysis show that distal mutations accelerate the chemical transformation by 100-fold, shifting the rate-limiting step to product release, which is further accelerated by the increased opening of the active site. These findings highlight the critical role of distal residues in shaping the active-site environment and facilitating the structural dynamics essential for progression through the catalytic cycle.

biochemistry↗

bioRxiv2023

Defining the mechanism of galectin-3-mediated TGF-β1 activation and its role in lung fibrosis.

Integrin-mediated activation of the pro-fibrotic mediator transforming growth factor-{beta}1 (TGF-{beta}1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF individuals, although its mechanism of action is not precisely defined. We hypothesised that galectin-3 potentiates TGF-{beta}1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-{beta}1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L--lysophosphatidic acid sodium salt (LPA)-induced integrin-mediated TGF-{beta}1 activation. Surface plasmon resonance (SPR) analysis confirmed that galectin-3 binds to the v integrins, v{beta}1, v{beta}S and v{beta}6 and also to the TGF{beta}RII subunit in a glycosylation-dependent manner. This galectin-3 binding is heterogeneous and not a 1:1 binding stoichiometry. These binding interactions were blocked by small molecule inhibitors of galectin-3 which target the carbohydrate recognition domain. Binding of galectin-3 to the {beta}1 integrin was validated in vitro by co-immunoprecipitation (Co-IP) in HLFs. In addition, proximity ligation assay (PLA) data indicates that galectin-3 and the {beta}1 integrin colocalize closely (40 nm) on the cell surface of HLFs, that colocalization is increased by TGF-{beta}1 treatment and galectin-3 inhibitors prevented this colocalization. In the absence of TGF-{beta}1 stimulation, such colocalization was detectable only in HLFs isolated from IPF patients suggesting that the proteins are inherently more closely associated in the disease state. Taken together, this data suggests that galectin-3 promotes TGF-{beta}1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-{beta}1 signaling cascade. Declaration of InterestsRGJ reports grants or contracts from AstraZeneca, Biogen, Galecto Biotech, GlaxoSmithKline, Nordic Biosciences, RedX, Plaint, consulting fees from Bristol Myers Squibb, Chiesi, Daewoong Veracyre, Resolution Therapeutics and Pliant, honoraria from Boehringer Ingelheim, Chiesi, Roche, PatientMPower, AstraZeneca, advisory roles with Boehringer Ingelheim, Galapagos and Vicore, non-financial support from NuMedii, and is a Trustee for Action for Pulmonary Fibrosis. AEJ reports grant funding from Galecto Biotech. BG reports CASE student project partnership with Galecto Biotech, honoraria from GlaxoSmithKline and Vertex and grants and fellowships from UKRI MRC and BBSRC, Alpha-1 Foundation, BLF/A+LUK, Wellcome Trust. RJS, ACM, FRZ, JFC are Galecto employees with shares/options in the company. NRP is a Roche employee. GH, RML, PS, SBC, DJS declare no competing interests.

molecular biology↗

bioRxiv2020

Lysyl oxidase-like 2 (LOXL2) is Increased in Asthma and Contributes to Asthmatic Airway Remodelling

Airway smooth muscle cells (ASM) are fundamental to asthma pathogenesis, influencing bronchoconstriction, airway hyper-responsiveness, and airway remodelling. Extracellular matrix (ECM) can influence tissue remodelling pathways, however, to date no study has investigated the effect of ASM ECM stiffness and crosslinking on the development of asthmatic airway remodelling. We hypothesised that TGF{beta} activation by ASM is influenced by ECM in asthma and sought to investigate the mechanisms involved. This study combines in vitro and in vivo approaches: human ASM cells were used in vitro to investigate basal TGF{beta} activation and expression of ECM crosslinking enzymes. Human bronchial biopsies from asthmatic and non-asthmatic donors were used to confirm LOXL2 expression ASM. A chronic ovalbumin model of asthma was used to study the effect of LOXL2 inhibition on airway remodelling. We found that ASM cells from asthmatics activated more TGF{beta} basally than non-asthmatic controls and that diseased cell-derived ECM influences levels of TGF{beta} activated. Our data demonstrate that the ECM crosslinking enzyme LOXL2 is increased in asthmatic ASM cells and in bronchial biopsies. Crucially, we show that LOXL2 inhibition reduces ECM stiffness and TGF{beta} activation in vitro, and can reduce subepithelial collagen deposition and ASM thickness, two features of airway remodelling, in an ovalbumin mouse model of asthma. These data are the first to highlight a role for LOXL2 in the development of asthmatic airway remodelling and suggest that LOXL2 inhibition warrants further investigation as a potential therapy to reduce remodelling of the airways in severe asthma.

cell biology↗

bioRxiv2020

Stretch Regulates Alveologenesis Via Mesenchymal Gαq/11-Mediated TGFβ2 Activation

Alveolar development and repair require tight spatiotemporal regulation of numerous signalling pathways that are influenced by chemical and mechanical stimuli. Mesenchymal cells play key roles in numerous developmental processes. Transforming growth factor-{beta} (TGF{beta}) is essential for alveologenesis and lung repair, and the G protein subunits Gq and G11 (Gq/11) transmit mechanical and chemical signals to activate TGF{beta} in epithelial cells. To understand the role of mesenchymal Gq/11 in lung development, we generated constitutive (Pdgfrb-Cre+/-;Gnaqfl/fl;Gna11-/-) and inducible (Pdgfrb-Cre/ERT2+/-;Gnaqfl/fl;Gna11-/-) mesenchymal Gq/11 deleted mice. Mice with constitutive Gq/11 gene deletion exhibited abnormal alveolar development, with suppressed myofibroblast differentiation, altered mesenchymal cell synthetic function, and reduced lung TGF{beta}2 deposition, as well as kidney abnormalities. Tamoxifen-induced mesenchymal Gq/11 gene deletion in adult mice resulted in emphysema associated with reduced TGF{beta}2 and elastin deposition. Cyclical mechanical stretch-induced TGF{beta} activation required Gq/11 signalling and serine protease activity, but was independent of integrins, suggesting an isoform-specific role for TGF{beta}2. These data highlight a previously undescribed mechanism of cyclical stretch-induced Gq/11-dependent TGF{beta}2 signalling in mesenchymal cells, which is imperative for normal alveologenesis and maintenance of lung homeostasis. Summary statementMesenchymal cell Gq/11 signalling regulates myofibroblast function and stretch-mediated TGF{beta}2 signalling, which are important for alveologenesis and organ homeostasis. These mechanisms are relevant to both developmental and adult lung disease.

developmental biology↗