Bibekbrata Gooptu

Mechanisms of emphysema in α1-antitrypsin deficiency: molecular and cellular insights

The severe, early onset emphysema that occurs in patients with circulating deficiency of α1-antitrypsin (α1-AT) attests to the importance of this protease inhibitor in maintaining lung parenchymal integrity. It has led to the powerful concept of protease:antiprotease balance being crucial to alveolar homeostasis. Pathogenic mutations cause α1-AT to self-associate into polymer chains that accumulate intracellularly rather than proceeding along the secretory pathway. Polymerisation of α1-AT abolishes antiprotease activity and confers toxic gain-of-function effects. Since α1-AT is predominantly synthesised in the liver, where it does not play a major homeostatic role, the directly toxic effects of polymerisation are clearest here. However, data from molecular, cellular, animal and ex vivo studies indicate that intrapulmonary polymerisation of α1-AT and inflammatory positive feedback loops may augment the destructive effects of decreased antiprotease levels in the lung. This review integrates the findings from these different approaches and highlights how multiple pathways may converge to give the severe, panacinar emphysema phenotype seen in α1-AT deficiency.

The molecular and cellular pathology of α1-antitrypsin deficiency

Since its discovery 50 years ago, α₁-antitrypsin deficiency has represented a case study in molecular medicine, with careful clinical characterisation guiding genetic, biochemical, biophysical, structural, cellular, and in vivo studies. Here we highlight the milestones in understanding the disease mechanisms and show how they have spurred the development of novel therapeutic strategies. α₁-Antitrypsin deficiency is an archetypal conformational disease. Its pathogenesis demonstrates the interplay between protein folding and quality control mechanisms, with aberrant conformational changes causing liver and lung disease through combined loss- and toxic gain-of-function effects. Moreover, α₁-antitrypsin exemplifies the ability of diverse proteins to self-associate into a range of morphologically distinct polymers, suggesting a mechanism for protein and cell evolution.

Crystallographic and Cellular Characterisation of Two Mechanisms Stabilising the Native Fold of α1-Antitrypsin: Implications for Disease and Drug Design

The common Z mutant (Glu342Lys) of α1-antitrypsin results in the formation of polymers that are retained within hepatocytes. This causes liver disease whilst the plasma deficiency of an important proteinase inhibitor predisposes to emphysema. The Thr114Phe and Gly117Phe mutations border a surface cavity identified as a target for rational drug design. These mutations preserve inhibitory activity but reduce the polymerisation of wild-type native α1-antitrypsin in vitro and increase secretion in a Xenopus oocyte model of disease. To understand these effects, we have crystallised both mutants and solved their structures. The 2.2 Å structure of Thr114Phe α1-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation. The Gly117Phe mutation operates differently, repacking aromatic side chains in the helix F–β-sheet A interface to induce a half-turn downward shift of the adjacent F helix. We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease. Both mutations increase the secretion of Z α1-antitrypsin in the native conformation, but the double mutants remain more polymerogenic than the wild-type (M) protein. Taken together, these data support different mechanisms by which the Thr114Phe and Gly117Phe mutations stabilise the native fold of α1-antitrypsin and increase secretion of monomeric protein in cell models of disease.

Update on alpha-1 antitrypsin deficiency: New therapies

α1-Antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin within the endoplasmic reticulum of hepatocytes. The retention of mutant protein causes hepatic damage and cirrhosis whilst the lack of an important circulating protease inhibitor predisposes the individuals with severe α1-antitrypsin deficiency to early onset emphysema. Our work over the past 25years has led to new paradigms for the liver and lung disease associated with α1-antitrypsin deficiency. We review here the molecular pathology of the cirrhosis and emphysema associated with α1-antitrypsin deficiency and show how an understanding of this condition provided the paradigm for a wider group of disorders that we have termed the serpinopathies. The detailed understanding of the pathobiology of α1-antitrypsin deficiency has identified important disease mechanisms to target. As a result, several novel parallel and complementary therapeutic approaches are in development with some now in clinical trials. We provide an overview of these new therapies for the liver and lung disease associated with α1-antitrypsin deficiency.

In silico assessment of potential druggable pockets on the surface of α1-antitrypsin conformers.

The search for druggable pockets on the surface of a protein is often performed on a single conformer, treated as a rigid body. Transient druggable pockets may be missed in this approach. Here, we describe a methodology for systematic in silico analysis of surface clefts across multiple conformers of the metastable protein α1-antitrypsin (A1AT). Pathological mutations disturb the conformational landscape of A1AT, triggering polymerisation that leads to emphysema and hepatic cirrhosis. Computational screens for small molecule inhibitors of polymerisation have generally focused on one major druggable site visible in all crystal structures of native A1AT. In an alternative approach, we scan all surface clefts observed in crystal structures of A1AT and in 100 computationally produced conformers, mimicking the native solution ensemble. We assess the persistence, variability and druggability of these pockets. Finally, we employ molecular docking using publicly available libraries of small molecules to explore scaffold preferences for each site. Our approach identifies a number of novel target sites for drug design. In particular one transient site shows favourable characteristics for druggability due to high enclosure and hydrophobicity. Hits against this and other druggable sites achieve docking scores corresponding to a Kd in the µM–nM range, comparing favourably with a recently identified promising lead. Preliminary ThermoFluor studies support the docking predictions. In conclusion, our strategy shows considerable promise compared with the conventional single pocket/single conformer approach to in silico screening. Our best-scoring ligands warrant further experimental investigation.

Interactions between N-linked glycosylation and polymerisation of neuroserpin within the endoplasmic reticulum

The neuronal serpin neuroserpin undergoes polymerisation as a consequence of point mutations that alter its conformational stability, leading to a neurodegenerative dementia called familial encephalopathy with neuroserpin inclusion bodies (FENIB). Neuroserpin is a glycoprotein with predicted glycosylation sites at asparagines 157, 321 and 401. We used site‐directed mutagenesis, transient transfection, western blot, metabolic labelling and ELISA to probe the relationship between glycosylation, folding, polymerisation and degradation of neuroserpin in validated cell models of health and disease. Our data show that glycosylation at N157 and N321 plays an important role in maintaining the monomeric state of neuroserpin, and we propose this is the result of steric hindrance or effects on local conformational dynamics that can contribute to polymerisation. Asparagine residue 401 is not glycosylated in wild type neuroserpin and in several polymerogenic variants that cause FENIB, but partial glycosylation was observed in the G392E mutant of neuroserpin that causes severe, early‐onset dementia. Our findings indicate that N401 glycosylation reports lability of the C‐terminal end of neuroserpin in its native state. This C‐terminal lability is not required for neuroserpin polymerisation in the endoplasmic reticulum, but the additional glycan facilitates degradation of the mutant protein during proteasomal impairment. In summary, our results indicate how normal and variant‐specific N‐linked glycosylation events relate to intracellular folding, misfolding, degradation and polymerisation of neuroserpin.

Tacrolimus in idiopathic inflammatory myopathy-associated interstitial lung disease: defining roles and responders

This editorial refers to The efficacy of tacrolimus in patients with interstitial lung diseases complicated with polymyositis or dermatomyositis, by Takashi Kurita et al., doi: 10.1093/rheumatology/keu166, pages 39–44..

Spontaneous pneumothorax can be associated with TGFBR2 mutation.

Primary pneumothorax affects 0.01% of the population. 10% of cases have a family history of pneumothorax but in the majority, a definitive genetic diagnosis is not made. We report a 26-year-old, white British woman who presented with left apical pneumothorax (figure 1a). Previously, she had migraines, multiple stress fractures in her right foot, myopia, easy bruising, lumbar scoliosis and spontaneous dislocation of the right patella. She had no previous history of pneumothoraces or any other respiratory problems, and had never smoked. TGFBR2 mutations that cause Loeys-Dietz syndrome can present as pneumothorax http://ow.ly/SlMkS

S131 Deficiency mutations of α1-antitrypsin differentially affect folding, function and polymerisation

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.