Polymerisation of α 1 -antitrypsin within hepatocytes is considered central to the pathogenesis of α 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 α 1 -antitrypsin is instead recognized as terminally-misfolded and degraded. It is unclear whether this contributes to Z α 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 α 1 -antitrypsin variants indicated that cellular responses to the Z mutation were surprisingly similar to those seen with the Null HongKong variant (NHK), which can only misfold terminally and cannot polymerise. A minor set of proteins showed increases associated with Z and not NHK α 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 α 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 α 1 -antitrypsin deficiency. Molecular therapies may prove most efficacious for associated liver disease if they address terminal misfolding as well as polymerisation.

Polymerisation of α1-antitrypsin within hepatocytes is considered central to pathogenesis in α1-antitrypsin deficiency-associated liver fibrosis, most commonly seen in homozygotes for the Z (p.Glu342Lys) allele. Polymerisation proceeds via self-association of monomeric intermediate states. In parallel, >50% of synthesised Z α1-antitrypsin is instead recognized as terminally-misfolded and degraded. It is unclear whether this contributes to Z α1-antitrypsin deficiency-associated liver disease. We characterised the relationships between polymer formation, terminal misfolding and their cellular consequences, using ion-mobility and label-free proteomics mass spectrometry, light and electron microscopy, and cellular assays. IM-MS findings supported the relationship between polymerisation propensity of different variants and expansion of the protein consistent with opening of its β-sheet A. Proteomic analyses of well-established CHO cell models of hepatocyte handling of α1-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 α1-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 terminal misfolding tendency of the expressed α1-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 α1-antitrypsin deficiency. Molecular therapies may prove most efficacious for associated liver disease if they address terminal misfolding as well as polymerisation consequences. ### Competing Interest Statement The authors have declared no competing interest.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.

Severe α 1 -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α 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 α 1 -antitrypsin. The lead compound blocks Z α 1 -antitrypsin polymerisation in vitro , reduces intracellular polymerisation and increases the secretion of Z α 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 α 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.

This case report deals with two patients with lacrimal sac swellings. Case 1 presented with bilateral sac swelling and Case 2 with a unilateral presentation. Dacrocystorhinostomy (DCR) followed by biopsies of both sacs in Case 1 revealed inflammatory polyps of the sac mucosa, identical in appearance to typical nasal allergic inflammatory polyps. The biopsies were accompanied by typical allergic mucin, featuring tiered mucin layers between which were numerous eosinophils, accompanied by Charcot-Leyden crystals. The histology of the dacryocystectomy specimen for Case 2 showed identical histopathological changes with the additional feature of prominent numbers of Immunoglobulin G (IgG)4-positive plasma cells in the stroma of the lacrimal sac inflammatory polyps. These features extend the sites affected by allergic inflammatory polyps and allergic mucin and possible pathogenesis is discussed.

"Surgical technique: Lateral canthal tendon relieving incisions in lower lid reconstruction." Orbit, 37(1), pp. 76–77

Many serpinopathies, including alpha-1 antitrypsin (A1AT) deficiency, are associated with the formation of unbranched polymer chains of mutant serpins. In vivo, this deficiency is the result of mutations that cause kinetic or thermodynamic destabilization of the molecule. However, polymerization can also be induced in vitro from mutant or wild-type serpins under destabilizing conditions. The characteristics of the resulting polymers are dependent upon induction conditions. Due to their relationship to disease, serpin polymers, mainly those formed from A1AT, have been widely studied. Here, we describe Förster resonance energy transfer (FRET) and gel-based approaches for their characterization.

Severe alpha‐1‐antitrypsin deficiency ( AATD ) is most frequently associated with the alpha‐1‐antitrypsin ( AAT ) Z variant (E342K). ZZ homozygotes exhibit accumulation of AAT as polymers in the endoplasmic reticulum of hepatocytes. This protein deposition can lead to liver disease, with the resulting low circulating levels of AAT predisposing to early‐onset emphysema due to dysregulation of elastinolytic activity in the lungs. An increasing number of rare AAT alleles have been identified in patients with severe AATD , typically in combination with the Z allele. Here we report a new mutation (E75V) in a patient with severe plasma deficiency, which we designate Trento. In contrast to the Z mutant, Trento AAT was secreted efficiently when expressed in cellular models but showed compromised conformational stability. Polyacrylamide gel electrophoresis ( PAGE ) and ELISA ‐based analyses of the secreted protein revealed the presence of oligomeric species with electrophoretic and immunorecognition profiles different from those of Z and S (E264V) AAT polymers, including reduced recognition by conformational monoclonal antibodies 2C1 and 4B12. This altered recognition was not due to direct effects on the epitope of the 2C1 monoclonal antibody which we localized between helices E and F. Structural analyses indicate the likely basis for polymer formation is the loss of a highly conserved stabilizing interaction between helix C and the posthelix I loop. These results highlight this region as important for maintaining native state stability and, when compromised, results in the formation of pathological polymers that are different from those produced by Z and S AAT .

A 56 year old retired theatre nurse who has metastatic breast carcinoma complains of reduced vision in the left eye since being started on Docetaxel (Taxotere), an oral anti-mitotic chemotherapeutic agent. Best corrected visual acuity was 6/18 in the left eye, and 6/9-1 in the right. Clinical examination and subsequent examination with optical coherence tomography and Intravenous Fluorescein angiography revealed evidence suggestive of drug related maculopathy. No abnormal findings were present in the right eye. As far as we are aware, this is the first reported case of maculopathy following commencement of Docetaxel with the absence of macular oedema.

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A — on the opposite face of the molecule — more liable to adopt an ‘open’ state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin–enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the ‘open’ state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation.

zetto M. Interferon treatment for multiple sclerosis: autoimmune complications may be lethal. Neurology 1998; 50; 570–571. 4. Pietrosi G, Mandala L, Vizzini GB et al. Fulminant hepatic failure and autoimmune disorders in patient with multiple sclerosis on interferon beta 1a: a fatal combination? Transpl. Int. 2008; 21; 502–504. 5. Wallack EM, Callon R. Liver injury associated with the beta-interferons for MS. Neurology 2004; 63; 1142–1143. 6. Yoshida EM, Rasmussen SL, Steinbrecher UP et al. Fulminant liver failure during interferon beta treatment of multiple sclerosis. Neurology 2001; 56; 1416.

Misfolding, polymerisation and defective secretion of functional α1-antitrypsin underlie the predispositions to severe liver and lung disease in α1-antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterised it relative to the wild-type (M) and common severe Z (Glu342Lys) variant. The index case is a homozygous individual of consangineous parentage. Absolute levels of circulating α1-antitrypsin were in the moderate deficiency range but the biochemical phenotype could not be clearly classified by standard methods. Moreover the majority was polymerised, i.e. functionally inactive, and the purified monomer was only 37% active relative to the wild-type ‘M’ variant. Together these resulted in 85–95% loss-of-function, a similarly severe functional deficiency to that of ZZ homozygotes. Biochemical, biophysical and computational studies further defined the molecular basis of this functional deficiency. These demonstrated that native Ala336Pro α1-antitrypsin could adopt the polymerogenic intermediate conformation and polymerised more readily not only than M α1-antitrypsin but also the severe Z variant. Nevertheless folding was far less impaired in Ala336Pro α1-antitrypsin than in the Z variant. The data therefore indicate partitions between the contribution of the ‘breach’ (site of Z mutation) and ‘shutter’ (Ala336Pro) regions of strand 5A to folding and to polymerisation mechanisms. Moreover the findings demonstrate that in these variants, folding efficiency does not correlate directly with the tendency to polymerise in vitro or in vivo. They therefore differentiate generalised misfolding from polymerisation tendencies in missense variants of α1-antitrypsin. Clinically they further support the need to quantify loss-of-function in α1-antitrypsin deficiency to individualise patient care.

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states.

Native mass spectrometry (MS) methods permit the study of multiple protein species within solution equilibria, whereas ion mobility (IM)-MS can report on conformational behavior of specific states. We used IM-MS to study a conformationally labile protein (α1 -antitrypsin) that undergoes pathological polymerization in the context of point mutations. The folded, native state of the Z-variant remains highly polymerogenic in physiological conditions despite only minor thermodynamic destabilization relative to the wild-type variant. Various data implicate kinetic instability (conformational lability within a native state ensemble) as the basis of Z α1 -antitrypsin polymerogenicity. We show the ability of IM-MS to track such disease-relevant conformational behavior in detail by studying the effects of peptide binding on α1 -antitrypsin conformation and dynamics. IM-MS is, therefore, an ideal platform for the screening of compounds that result in therapeutically beneficial kinetic stabilization of native α1 -antitrypsin. Our findings are confirmed with high-resolution X-ray crystallographic and nuclear magnetic resonance spectroscopic studies of the same event, which together dissect structural changes from dynamic effects caused by peptide binding at a residue-specific level. IM-MS methods, therefore, have great potential for further study of biologically relevant thermodynamic and kinetic instability of proteins and provide rapid and multidimensional characterization of ligand interactions of therapeutic interest.

α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10−14). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.

Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate-and therefore polymerize-more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the "breach" region and "shutter" region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care.

Mutant Z alpha(1)-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z alpha(1)-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z alpha(1)-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked alpha(1)-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. Asingle-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12(KDEL)) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z alpha(1)-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z alpha(1)-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognizeda discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z alpha(1)-antitrypsin.