Biology subjects

Irving, J. A.

Publications and source records attributed to Irving, J. A..

8 recordsLinked to original sources

bioRxiv2025

Resolution of the circulating pathogenic phenotype of alpha-1 antitrypsin deficiency following liver transplantation

Alpha-1 antitrypsin is a member of the serpin family of protease inhibitors and normally found in monomeric form at high concentration in the circulation. Certain pathogenic variants of this protein self-assemble into flexible chains (polymers) that undergo toxic accumulation in the liver, underlying the development of liver disease, and a corresponding functional deficiency in circulation results in a susceptibility to chronic damage to the lungs and thereby COPD. A proportion of these polymers are detectable in the circulation. This study aimed to provide data on the dynamics of AAT monomers and polymers in the circulation after correction of a deficiency state by liver transplantation. The evolution of the circulating AAT phenotype was characterised quantitatively over time by analysis of blood samples taken pre- and post-liver transplant from three PiZZ AAT deficiency patients, using a panel of conformer-specific monoclonal antibodies and a novel ELISA with enhanced sensitivity for AAT polymers. Circulating wild-type M AAT was found to increase with a half-time of 29-39 h, attaining a clinically-defined putative protective threshold level within 24-50 h post-transplant. Baseline circulating polymer levels ranged from 5-35 g/mL with a half-time clearance of 3-12 h following transplant, and levels were indistinguishable from reference wild-type plasma 22 months post-surgery. The glycosylation profile and disappearance of detectable circulating polymers of AAT were consistent with their origin in, and canonical secretion by, the liver. To our knowledge this is the first post-transplant evaluation of the hybrid conformational profile of endogenous and donor tissue-derived AAT.

pathology↗

bioRxiv2025

Phospho 394Y LCK flow-cytometry readout predicts dasatinib sensitivity in paediatric T-cell acute lymphoblastic leukaemia

Children with T-cell acute lymphoblastic leukaemia (T-ALL) who relapse or fail induction have poor outcomes. A subset of cases shows glucocorticoid resistance reversible by dasatinib through inhibition of LCK-dependent signalling. To identify a practical biomarker of dasatinib response, we compared in-vitro drug sensitivity with basal phosphorylation of pre-TCR pathway proteins in 28 paediatric T-ALL patient-derived xenografts (PDX). Phospho-flow cytometry quantified LCK (pY394), ZAP70 (pY319) and CD3{zeta} (pY142), normalised to internal controls. Dasatinib IC50 values correlated significantly with pLCK and pZAP70, with pLCK providing the best single-marker performance across clinically relevant thresholds. Logistic, ROC and precision-recall analyses confirmed that pLCK alone achieved excellent classification (AUC [≥] 0.9), while multivariate models added minimal predictive value. Model selection using LASSO and Bayesian Information Criterion further supported pLCK as the dominant predictor. These findings establish pLCK as a robust, scalable biomarker of dasatinib sensitivity suitable for diagnostic integration. A multicentre international validation programme is underway to harmonise pLCK assay protocols, expand testing across biobanks, and assess clinical feasibility in newly diagnosed and relapsed patients within the ALLTogether and HEM-iSMART trials respectively.

cancer biology↗

bioRxiv2025

Both terminal misfolding and polymerisation contribute to disease-relevant phenotypic changes in cell models of α1-antitrypsin deficiency-associated liver disease

Polymerisation of 1-antitrypsin within hepatocytes is considered central to the pathogenesis of 1-antitrypsin deficiency-associated liver fibrosis, most commonly in homozygotes for the Z (p.Glu342Lys) allele. Polymerisation proceeds via self-association of monomeric intermediate states. In parallel, >50% of synthesised Z 1-antitrypsin is instead recognized as terminally-misfolded and degraded. It is unclear whether this contributes to Z 1-antitrypsin deficiency-associated liver disease. We characterised the relationships between polymer formation, terminal misfolding and their cellular consequences, using label-free proteomics mass spectrometry (MS), light and electron microscopy, and cellular assays. Proteomic analyses of well-established CHO cell models of hepatocyte handling of 1-antitrypsin variants indicated that cellular responses to the Z mutation were surprisingly similar to those seen with the NullHongKong variant (NHK), which can only misfold terminally and cannot polymerise. A minor set of proteins showed increases associated with Z and not NHK 1-antitrypsin expression, consistent with a polymer-specific response, characterized by association with increased organellar organization and vesicle-mediated transport. Conversely, proteostatic and pro-fibrotic integrin-associated pathways increased with the degree of terminal misfolding of the expressed 1-antitrypsin variant. Bioenergetic pathway changes indicated concomitant switching from oxidative to glycolytic metabolism. Cell studies further correlated fibrosis-associated behaviours with terminal misfolding rather than polymerisation. Terminal misfolding, as well as polymerisation behaviour, may therefore be important for pro-fibrotic responses including metabolic reprogramming and senescence in Z 1-antitrypsin deficiency. Molecular therapies may prove most efficacious for associated liver disease if they address terminal misfolding as well as polymerisation.

cell biology↗

bioRxiv2025

Visualisation of translating ribosomes reveals the earliest steps of protein misfolding in human disease

The majority of cellular proteins must adopt a particular three-dimensional structure for function1. However, protein folding is a perilous journey due to competing polypeptide misfolding events which result in inactive structures. In this study, we examine the earliest steps of protein misfolding during the biosynthesis of alpha-1-antitrypsin, a secreted plasma protein whose misfolding results in organ disease. Using human cells, we find that, like co-translational protein folding, misfolding, assembly and biosynthesis are interconnected processes. At the molecular level misfolding of alpha-1-antitrypsin is initiated by a molten globule-like folding intermediate formed cotranslationally on the ribosome. The ribosomal complexes subsequently form assemblies by recruiting released proteins, inducing translational arrest. Our data also reveal that a pharmacological chaperone modulates this process. The existence of co- and post-translational (mis)folding and assembly pathways reveals how some proteins form functional complexes, has implications for the pathogenesis of conformational diseases, and suggests novel therapeutic avenues.

biochemistry↗

bioRxiv2024

Systemic Dosing of Virus-derived Serpin Improves Survival and Immunothrombotic Damage in Murine Colitis

Inflammatory bowel disease (IBD) is potentially life-threatening, with risk of bleeding, clotting, infection, sepsis, cancer and toxic megacolon. Systemic and local immune and coagulation dysfunction increase IBD severity. Current treatments are partially effective, but there is no definitive cure. Serine protease cascades activate thrombotic, thrombolytic and complement pathways and are regulated by inhibitors, serpins. Viruses encode proteins evolved from endogenous central regulatory pathways. A purified secreted Myxomavirus-derived serpin, Serp-1, dosed as a systemic anti-inflammatory drug, has proven efficacy in vascular and inflammatory disorders. PEGylated Serp-1 protein (PEGSerp-1) has improved efficacy in lupus and SARS-CoV-2 models. We examined PEGSerp-1 treatment in a mouse Dextran Sodium Sulfate (DSS) colitis model. Prophylactic PEGSerp-1 significantly improved survival in acute severe 4-5% DSS colitis, reducing inflammation and crypt damage in acute 4-5% DSS induced colitis and when dosed as a chronic delayed treatment for recurrent 2% DSS colitis. PEGSerp-1 reduced iNOS+ M1 macrophage invasion, damage to crypt architecture and vascular inflammation with decreased uPAR, fXa, fibrinogen and complement activation. This work supports PEGSerp-1 as a tissue targeting serpin therapeutic.

molecular biology↗

bioRxiv2022

Keratin 8 is a scaffolding and regulatory protein of ERAD complexes

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha 1 antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild type (WT) A1AT with a 13-fold and 4-fold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the s heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.

cell biology↗

bioRxiv2020

Development of a small molecule that corrects misfolding and increases secretion of Z α1-antitrypsin

Severe 1-antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant 1-antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high throughput screen to identify small molecules that bind to, and stabilise Z 1-antitrypsin. The lead compound blocks Z 1-antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z 1-antitrypsin three-fold in mammalian cells including an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerization pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z 1-antitrypsin into the plasma by 7-fold. There was no observable clearance of hepatic inclusions with respect to controls. This study provides proof-of-principle that mutation ameliorating small molecules are a viable approach to treat protein conformational diseases.

pharmacology and toxicology↗

bioRxiv2018

Characterisation of a type II functionally-deficient variant of alpha-1-antitrypsin discovered in the general population

Lung disease in alpha-1-antitrypsin deficiency (AATD) results from dysregulated proteolytic activity, mainly by neutrophil elastase (HNE), in the lung parenchyma. This is the result of a substantial reduction of circulating alpha-1-antitrypsin (AAT) and the presence in the plasma of inactive polymers of AAT. Moreover, some AAT mutants have reduced intrinsic activity toward HNE, as demonstrated for the common Z mutant, as well as for other rarer variants. Here we report the identification and characterisation of the novel AAT reactive centre loop variant Gly349Arg (p.G373R) present in the ExAC database. This AAT variant is secreted at normal levels in cellular models of AATD but shows a severe reduction in anti-HNE activity. Biochemical and molecular dynamics studies suggest it exhibits unfavourable RCL presentation to cognate proteases and compromised insertion of the RCL into {beta}-sheet A. Identification of a fully dysfunctional AAT mutant that does not show a secretory defect underlines the importance of accurate genotyping of patients with pulmonary AATD manifestations regardless of the presence of normal levels of AAT in the circulation. This subtype of disease is reminiscent of dysfunctional phenotypes in antithrombin and C1-inibitor deficiencies so, accordingly, we classify this variant as the first pure functionally-deficient (type II) AATD mutant.

biochemistry↗