Biology subjects

Lomas, D. A.

Publications and source records attributed to Lomas, D. A..

7 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

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↗

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↗

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↗

bioRxiv2018

Expanded genetic landscape of chronic obstructive pulmonary disease reveals heterogeneous cell type and phenotype associations

Chronic obstructive pulmonary disease (COPD) is the leading cause of respiratory mortality worldwide. Genetic risk loci provide novel insights into disease pathogenesis. To broaden COPD genetic risk loci discovery and identify cell type and phenotype associations we performed a genome-wide association study in 35,735 cases and 222,076 controls from the UK Biobank and additional studies from the International COPD Genetics Consortium. We identified 82 loci with P value < 5x10-8; 47 were previously described in association with either COPD or population-based lung function. Of the remaining 35 novel loci, 13 were associated with lung function in 79,055 individuals from the SpiroMeta consortium. Using gene expression and regulation data, we identified enrichment for loci in lung tissue, smooth muscle and alveolar type II cells. We found 9 shared genomic regions between COPD and asthma and 5 between COPD and pulmonary fibrosis. COPD genetic risk loci clustered into groups of quantitative imaging features and comorbidity associations. Our analyses provide further support to the genetic susceptibility and heterogeneity of COPD.

genetics↗

bioRxiv2017

An automated high-throughput system for phenotypic screening of chemical libraries on C. elegans and parasitic nematodes

Parasitic nematodes infect hundreds of millions of people and farmed livestock. Further, plant parasitic nematodes result in major crop damage. The pipeline of therapeutic compounds is limited and parasite resistance to the existing anthelmintic compounds is a global threat. We have developed an INVertebrate Automated Phenotyping Platform (INVAPP) for high-throughput, plate-based chemical screening, and an algorithm (Paragon) which allows screening for compounds that have an effect on motility and development of parasitic worms. We have validated its utility by determining the efficacy of a panel of known anthelmintics against model and parasitic nematodes: Caenorhabditis elegans, Haemonchus contortus, Teladorsagia circumcincta, and Trichuris muris. We then applied the system to screen the Pathogen Box chemical library in a blinded fashion and identified known anthelmintics, including tolfenpyrad, auranofin, and mebendazole and 14 compounds previously undescribed as anthelmintics, including benzoxaborole and isoxazole chemotypes. This system offers an effective, high-throughput system for the discovery of novel anthelmintics.

pharmacology and toxicology↗