Reduction of Z alpha-1 antitrypsin polymers in human iPSC-hepatocytes and mice by LRRK2 inhibitors.

Alpha-1 antitrypsin deficiency (A1ATD) is a life-threatening condition caused by inheritance of the SERPINA1 'Z' genetic variant (PiZ) driving AAT protein misfolding in hepatocytes. There remain no approved medicines for this disease. Here, we report the results of a small molecule screen performed in patient derived iPSC-hepatocytes that identified Leucine-rich repeat kinase-2 (LRRK2) as a potentially new therapeutic target. Of the commercially available LRRK2 inhibitors tested, we identified CZC-25146, a candidate with favorable pharmacokinetic properties, as being capable of reducing polymer load, increasing normal AAT secretion, and reducing inflammatory cytokines in both cells and PiZ mice. Mechanistically, this effect was achieved through induction of autophagy. Our findings support the use of CZC-25146 and LRRK2 inhibitors in hepatic proteinopathy research and their further investigation as novel therapeutic candidates for A1ATD.

A novel pathological mutant reveals the role of torsional flexibility in the serpin breach in adoption of an aggregation-prone intermediate.

Mutants of alpha-1-antitrypsin cause the protein to self-associate and form ordered aggregates ('polymers') that are retained within hepatocytes, resulting in a predisposition to the development of liver disease. The associated reduction in secretion, and for some mutants, impairment of function, leads to a failure to protect lung tissue against proteases released during the inflammatory response and an increased risk of emphysema. We report here a novel deficiency mutation (Gly192Cys), that we name the Sydney variant, identified in a patient in heterozygosity with the Z allele (Glu342Lys). Cellular analysis revealed that the novel variant was mostly retained as insoluble polymers within the endoplasmic reticulum. The basis for this behaviour was investigated using biophysical and structural techniques. The variant showed a 40% reduction in inhibitory activity and a reduced stability as assessed by thermal unfolding experiments. Polymerisation involves adoption of an aggregation-prone intermediate and paradoxically the energy barrier for transition to this state was increased by 16% for the Gly192Cys variant with respect to the wild-type protein. However, with activation to the intermediate state, polymerisation occurred at a 3.8-fold faster rate overall. X-ray crystallography provided two crystal structures of the Gly192Cys variant, revealing perturbation within the 'breach' region with Cys192 in two different orientations: in one structure it faces towards the hydrophobic core while in the second it is solvent-exposed. This orientational heterogeneity was confirmed by PEGylation. These data show the critical role of the torsional freedom imparted by Gly192 in inhibitory activity and stability against polymerisation.

Native and Ion Mobility Mass Spectrometry Characterization of Alpha 1 Antitrypsin Variants and Oligomers.

In this chapter, we describe a method for analyzing both recombinant and plasma-derived alpha 1 antitrypsin and its oligomers by means of native ion mobility mass spectrometry. Our experimental workflow can be applied to other variants of alpha 1 antitrypsin and its oligomers as well as being used to probe their interactions with small molecules in the gas phase.

The under-appreciated world of the serpin family of serine proteinase inhibitors.

In the practice of medicine, many fundamental biological pathways that require tight on/off control, such as inflammation and circulatory homeostasis, are regulated by serine proteinases, but we rarely consider the unique protease inhibitors that, in turn, regulate these proteases. The serpins are a family of proteins with a shared tertiary structure, whose members largely act as serine protease inhibitors, found in all forms of life, ranging from viruses, bacteria, and archaea to plants and animals. These proteins represent up to 2-10% of proteins in the human blood and are the third most common protein family.

X chromosome associations with chronic obstructive pulmonary disease and related phenotypes: an X chromosome-wide association study.

BACKGROUND: The association between genetic variants on the X chromosome to risk of COPD has not been fully explored. We hypothesize that the X chromosome harbors variants important in determining risk of COPD related phenotypes and may drive sex differences in COPD manifestations. METHODS: Using X chromosome data from three COPD-enriched cohorts of adult smokers, we performed X chromosome specific quality control, imputation, and testing for association with COPD case-control status, lung function, and quantitative emphysema. Analyses were performed among all subjects, then stratified by sex, and subsequently combined in meta-analyses. RESULTS: Among 10,193 subjects of non-Hispanic white or European ancestry, a variant near TMSB4X, rs5979771, reached genome-wide significance for association with lung function measured by FEV1/FVC ([Formula: see text] 0.020, SE 0.004, p 4.97 × 10-08), with suggestive evidence of association with FEV1 ([Formula: see text] 0.092, SE 0.018, p 3.40 × 10-07). Sex-stratified analyses revealed X chromosome variants that were differentially trending in one sex, with significantly different effect sizes or directions. CONCLUSIONS: This investigation identified loci influencing lung function, COPD, and emphysema in a comprehensive genetic association meta-analysis of X chromosome genetic markers from multiple COPD-related datasets. Sex differences play an important role in the pathobiology of complex lung disease, including X chromosome variants that demonstrate differential effects by sex and variants that may be relevant through escape from X chromosome inactivation. Comprehensive interrogation of the X chromosome to better understand genetic control of COPD and lung function is important to further understanding of disease pathology. Trial registration Genetic Epidemiology of COPD Study (COPDGene) is registered at ClinicalTrials.gov, NCT00608764 (Active since January 28, 2008). Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints Study (ECLIPSE), GlaxoSmithKline study code SCO104960, is registered at ClinicalTrials.gov, NCT00292552 (Active since February 16, 2006). Genetics of COPD in Norway Study (GenKOLS) holds GlaxoSmithKline study code RES11080, Genetics of Chronic Obstructive Lung Disease.

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 fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the µs 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 and co-immunoprecipitation 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.

Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.

OBJECTIVE: Alpha-1 antitrypsin deficiency (AATD) is a common, potentially lethal inborn disorder caused by mutations in alpha-1 antitrypsin (AAT). Homozygosity for the 'Pi*Z' variant of AAT (Pi*ZZ genotype) causes lung and liver disease, whereas heterozygous 'Pi*Z' carriage (Pi*MZ genotype) predisposes to gallstones and liver fibrosis. The clinical significance of the more common 'Pi*S' variant remains largely undefined and no robust data exist on the prevalence of liver tumours in AATD. DESIGN: Baseline phenotypes of AATD individuals and non-carriers were analysed in 482 380 participants in the UK Biobank. 1104 participants of a multinational cohort (586 Pi*ZZ, 239 Pi*SZ, 279 non-carriers) underwent a comprehensive clinical assessment. Associations were adjusted for age, sex, body mass index, diabetes and alcohol consumption. RESULTS: Among UK Biobank participants, Pi*ZZ individuals displayed the highest liver enzyme values, the highest occurrence of liver fibrosis/cirrhosis (adjusted OR (aOR)=21.7 (8.8-53.7)) and primary liver cancer (aOR=44.5 (10.8-183.6)). Subjects with Pi*MZ genotype had slightly elevated liver enzymes and moderately increased odds for liver fibrosis/cirrhosis (aOR=1.7 (1.2-2.2)) and cholelithiasis (aOR=1.3 (1.2-1.4)). Individuals with homozygous Pi*S mutation (Pi*SS genotype) harboured minimally elevated alanine aminotransferase values, but no other hepatobiliary abnormalities. Pi*SZ participants displayed higher liver enzymes, more frequent liver fibrosis/cirrhosis (aOR=3.1 (1.1-8.2)) and primary liver cancer (aOR=6.6 (1.6-26.9)). The higher fibrosis burden was confirmed in a multinational cohort. Male sex, age ≥50 years, obesity and the presence of diabetes were associated with significant liver fibrosis. CONCLUSION: Our study defines the hepatobiliary phenotype of individuals with the most relevant AATD genotypes including their predisposition to liver tumours, thereby allowing evidence-based advice and individualised hepatological surveillance.

Quantitative 18F-fluorodeoxyglucose positron emission tomography/computed tomography to assess pulmonary inflammation in COPD.

RATIONALE: COPD and smoking are characterised by pulmonary inflammation. 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) imaging may improve knowledge of pulmonary inflammation in COPD patients and aid early development of novel therapies as an imaging biomarker. OBJECTIVES: To evaluate pulmonary inflammation, assessed by FDG uptake, in whole and regional lung in "usual" (smoking-related) COPD patients, alpha-1 antitrypsin deficiency (α1ATD) COPD patients, smokers without COPD and never-smokers using FDG PET/CT. Secondly, to explore cross-sectional associations between FDG PET/CT and systemic inflammatory markers in COPD patients and repeatability of the technique in COPD patients. METHODS: Data from two imaging studies were evaluated. Pulmonary FDG uptake (normalised Ki; nKi) was measured by Patlak graphical analysis in four subject groups: 84 COPD patients, 11 α1ATD-COPD patients, 12 smokers and 10 never-smokers. Within the COPD group, associations between nKi and systemic markers of inflammation were assessed. Repeatability was evaluated in 32 COPD patients comparing nKi values at baseline and at 4-month follow-up. RESULTS: COPD patients, α1ATD-COPD patients and smokers had increased whole lung FDG uptake (nKi) compared with never-smokers (0.0037±0.001, 0.0040±0.001, 0.0040±0.001 versus 0.0028±0.001 mL·cm-3·min-1, respectively, p<0.05 for all). Similar results were observed in upper and middle lung regions. In COPD participants, plasma fibrinogen was associated with whole lung nKi (ß=0.30, p=0.02) in multivariate analysis adjusted for current smoking, forced expiratory volume in 1 s % predicted, systemic neutrophils and C-reactive protein levels. Mean percentage difference in nKi between the baseline and follow-up was 3.2%, and the within subject coefficient of variability was 7.7%. CONCLUSIONS: FDG PET/CT has potential as a noninvasive tool to enable whole lung and regional quantification of FDG uptake to assess smoking- and COPD-related pulmonary inflammation.

Genetic variation in genes regulating skeletal muscle regeneration and tissue remodelling associated with weight loss in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally. COPD patients with cachexia or weight loss have increased risk of death independent of body mass index (BMI) and lung function. We tested the hypothesis genetic variation is associated with weight loss in COPD using a genome-wide association study approach. METHODS: Participants with COPD (N = 4308) from three studies (COPDGene, ECLIPSE, and SPIROMICS) were analysed. Discovery analyses were performed in COPDGene with replication in SPIROMICS and ECLIPSE. In COPDGene, weight loss was defined as self-reported unintentional weight loss > 5% in the past year or low BMI (BMI < 20 kg/m2 ). In ECLIPSE and SPIROMICS, weight loss was calculated using available longitudinal visits. Stratified analyses were performed among African American (AA) and Non-Hispanic White (NHW) participants with COPD. Single variant and gene-based analyses were performed adjusting for confounders. Fine mapping was performed using a Bayesian approach integrating genetic association results with linkage disequilibrium and functional annotation. Significant gene networks were identified by integrating genetic regions associated with weight loss with skeletal muscle protein-protein interaction (PPI) data. RESULTS: At the single variant level, only the rs35368512 variant, intergenic to GRXCR1 and LINC02383, was associated with weight loss (odds ratio = 3.6, 95% confidence interval = 2.3-5.6, P = 3.2 × 10-8 ) among AA COPD participants in COPDGene. At the gene level in COPDGene, EFNA2 and BAIAP2 were significantly associated with weight loss in AA and NHW COPD participants, respectively. The EFNA2 association replicated among AA from SPIROMICS (P = 0.0014), whereas the BAIAP2 association replicated in NHW from ECLIPSE (P = 0.025). The EFNA2 gene encodes the membrane-bound protein ephrin-A2 involved in the regulation of developmental processes and adult tissue homeostasis such as skeletal muscle. The BAIAP2 gene encodes the insulin-responsive protein of mass 53 kD (IRSp53), a negative regulator of myogenic differentiation. Integration of the gene-based findings participants with PPI data revealed networks of genes involved in pathways such as Rho and synapse signalling. CONCLUSIONS: The EFNA2 and BAIAP2 genes were significantly associated with weight loss in COPD participants. Collectively, the integrative network analyses indicated genetic variation associated with weight loss in COPD may influence skeletal muscle regeneration and tissue remodelling.

The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant.

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.

Scaling Concepts in Serpin Polymer Physics.

α1-Antitrypsin is a protease inhibitor belonging to the serpin family. Serpin polymerisation is at the core of a class of genetic conformational diseases called serpinopathies. These polymers are known to be unbranched, flexible, and heterogeneous in size with a beads-on-a-string appearance viewed by negative stain electron microscopy. Here, we use atomic force microscopy and time-lapse dynamic light scattering to measure polymer size and shape for wild-type (M) and Glu342→Lys (Z) α1-antitrypsin, the most common variant that leads to severe pathological deficiency. Our data for small polymers deposited onto mica and in solution reveal a power law relation between the polymer size, namely the end-to-end distance or the hydrodynamic radius, and the polymer mass, proportional to the contour length. We use the scaling concepts of polymer physics to assess that α1-antitrypsin polymers are random linear chains with a low persistence length.

The development of highly potent and selective small molecule correctors of Z α1-antitrypsin misfolding.

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.

Prognostic value of clinically important deterioration in COPD: IMPACT trial analysis.

INTRODUCTION: Clinically important deterioration (CID) is a multicomponent measure for assessing disease worsening in chronic obstructive pulmonary disease (COPD). This analysis investigated the prognostic value of a CID event on future clinical outcomes and the effect of single-inhaler triple versus dual therapy on reducing CID risk in patients in the IMPACT trial. METHODS: IMPACT was a phase III, double-blind, 52-week, multicentre trial. Patients with symptomatic COPD and at least one moderate/severe exacerbation in the prior year were randomised 2:2:1 to fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) 100/62.5/25 µg, FF/VI 100/25 µg or UMEC/VI 62.5/25 µg. CID at the time-point of interest was defined as a moderate/severe exacerbation, ≥100 mL decrease in trough forced expiratory volume in 1 s or deterioration in health status (increase of ≥4.0 units in St George's Respiratory Questionnaire total score or increase of ≥2.0 units in COPD Assessment Test score) from baseline. A treatment-independent post hoc prognostic analysis compared clinical outcomes up to week 52 in patients with/without a CID by week 28. A prospective analysis evaluated time to first CID with each treatment. RESULTS: Patients with a CID by week 28 had significantly increased exacerbation rates after week 28, smaller improvements in lung function and health status at week 52 (all p<0.001), and increased risk of all-cause mortality after week 28 versus patients who were CID-free. FF/UMEC/VI significantly reduced CID risk versus dual therapies (all p<0.001). CONCLUSIONS: Prevention of short-term disease worsening was associated with better long-term clinical outcomes. FF/UMEC/VI reduced CID risk versus dual therapies; this effect may improve long-term prognosis in this population.

Conversion of the death inhibitor ARC to a killer activates pancreatic ß cell death in diabetes.

Loss of insulin-secreting pancreatic ß cells through apoptosis contributes to the progression of type 2 diabetes, but underlying mechanisms remain elusive. Here, we identify a pathway in which the cell death inhibitor ARC paradoxically becomes a killer during diabetes. While cytoplasmic ARC maintains ß cell viability and pancreatic architecture, a pool of ARC relocates to the nucleus to induce ß cell apoptosis in humans with diabetes and several pathophysiologically distinct mouse models. ß cell death results through the coordinate downregulation of serpins (serine protease inhibitors) not previously known to be synthesized and secreted by ß cells. Loss of the serpin α1-antitrypsin from the extracellular space unleashes elastase, triggering the disruption of ß cell anchorage and subsequent cell death. Administration of α1-antitrypsin to mice with diabetes prevents ß cell death and metabolic abnormalities. These data uncover a pathway for ß cell loss in type 2 diabetes and identify an FDA-approved drug that may impede progression of this syndrome.

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

Severe α1 -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1 -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 α1 -antitrypsin. The lead compound blocks Z α1 -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1 -antitrypsin threefold in 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 polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1 -antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z α1 -antitrypsin deficiency.

The molecular species responsible for α1 -antitrypsin deficiency are suppressed by a small molecule chaperone.

The formation of ordered Z (Glu342Lys) α1 -antitrypsin polymers in hepatocytes is central to liver disease in α1 -antitrypsin deficiency. In vitro experiments have identified an intermediate conformational state (M*) that precedes polymer formation, but this has yet to be identified in vivo. Moreover, the mechanism of polymer formation and their fate in cells have been incompletely characterised. We have used cell models of disease in conjunction with conformation-selective monoclonal antibodies and a small molecule inhibitor of polymerisation to define the dynamics of polymer formation, accumulation and secretion. Pulse-chase experiments demonstrate that Z α1 -antitrypsin accumulates as short-chain polymers that partition with soluble cellular components and are partially secreted by cells. These precede the formation of larger, insoluble polymers with a longer half-life (10.9 ± 1.7 h and 20.9 ± 7.4 h for soluble and insoluble polymers, respectively). The M* intermediate (or a by-product thereof) was identified in the cells by a conformation-specific monoclonal antibody. This was completely abrogated by treatment with the small molecule, which also blocked the formation of intracellular polymers. These data allow us to conclude that the M* conformation is central to polymerisation of Z α1 -antitrypsin in vivo; preventing its accumulation represents a tractable approach for pharmacological treatment of this condition; polymers are partially secreted; and polymers exist as two distinct populations in cells whose different dynamics have likely consequences for the aetiology of the disease.