Ravi Mahadeva

α1-Antitrypsin polymerization and the serpinopathies: pathobiology and prospects for therapy

In the last 35 years there have been tremendous advances in our understanding of α1-antitrypsin deficiency. Z α1-antitrypsin polymerizes within the liver to cause cirrhosis. The molecular basis of polymer formation has now been elucidated with biochemical, cellular, and structural studies. The current goals are to determine the cellular response to polymeric α1-antitrypsin and to develop therapeutic strategies to block polymerization in vivo. The recognition of the association between plasma deficiency of α1-antitrypsin and emphysema has led to the proteinase-antiproteinase hypothesis of lung disease. α1-Antitrypsin polymers may have an important role in the progression of emphysema, but this requires further investigation. The evolution of more sophisticated cell and animal models of disease offers a real prospect of dissecting the role of injurious pathways in the pathogenesis of emphysema.

Clinical outcome in relation to care in centres specialising in cystic fibrosis: cross sectional study

Abstract Objectives: To assess the effect on clinical outcome of managing paediatric and adult patients with cystic fibrosis at specialised cystic fibrosis centres. Design: Cross sectional study. Setting: Two adult cystic fibrosis centres in the United Kingdom. Subjects: Patients from an adult cystic fibrosis centre in Manchester were subdivided into those who had received continuous care from paediatric and adult cystic fibrosis centres (group A), and those who had received paediatric care in a centre not specialising in cystic fibrosis followed by adult care in a cystic fibrosis centre (group B). Group C were referrals to the new adult cystic fibrosis centre in Cambridge who had received neither paediatric nor adult centre care for their cystic fibrosis. Main outcome measures: Body mass index (weight (kg)/height (m2)), lung function (forced expiratory volume in one second (FEV1 percentage of predicted)), the Northern chest x ray film score, and age at colonisation with Pseudomonas aeruginosa. Results: A prominent stepwise increase in body mass index was associated with increasing amounts of care at a cystic fibrosis centre; 18.3, 20.2, and 21.3 for groups C, B, and A respectively (P<0.001). Improved nutritional status was correlated with a higher FEV1 and better (lower) chest x ray film scores; r=0.52 and −0.45 respectively (P<0.001 for both). Conclusion: These findings provide the first direct evidence that management of cystic fibrosis in paediatric and adult cystic fibrosis centres results in a better clinical outcome, and strongly supports the provision of these specialist services. Key messages Management of patients with cystic fibrosis in paediatric and adult cystic fibrosis centres results in an improved clinical outcome Improved clinical outcome occurred in cystic fibrosis centres despite earlier and more frequent colonisation with Pseudomonas aeruginosa Nutritional status is an important predictor of lung disease in cystic fibrosis

A KINETIC MECHANISM FOR THE POLYMERIZATION OF ALPHA 1-ANTITRYPSIN

The mutation in the Z deficiency variant of α1-antitrypsin perturbs the structure of the protein to allow a unique intermolecular linkage. These loop-sheet polymers are retained within the endoplasmic reticulum of hepatocytes to form inclusions that are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. The process of polymer formation has been investigated here by intrinsic tryptophan fluorescence, fluorescence polarization, circular dichroic spectra and extrinsic fluorescence with 8-anilino-1-naphthalenesulfonic acid and tetramethylrhodamine-5-iodoacetamide. These biophysical techniques have demonstrated that α1-antitrypsin polymerization is a two-stage process and have allowed the calculation of rates for both of these steps. The initial fast phase is unimolecular and likely to represent temperature-induced protein unfolding, while the slow phase is bimolecular and associated with loop-sheet interaction and polymer formation. The naturally occurring Z, S, and I variants and recombinant site-directed reactive loop and shutter domain mutants of α1-antitrypsin were used to demonstrate the close association between protein stability and rate of α1-antitrypsin polymerization. Taken together, these data allow us to propose a kinetic mechanism for α1-antitrypsin polymer formation that involves the generation of an unstable intermediate, which can form polymers or generate latent protein.

Heteropolymerization of S, I, and Z α1-antitrypsin and liver cirrhosis

The association between Z alpha1-antitrypsin deficiency and juvenile cirrhosis is well-recognized, and there is now convincing evidence that the hepatic inclusions are the result of entangled polymers of mutant Z alpha1-antitrypsin. Four percent of the northern European Caucasian population are heterozygotes for the Z variant, but even more common is S alpha1-antitrypsin, which is found in up to 28% of southern Europeans. The S variant is known to have an increased susceptibility to polymerization, although this is marginal compared with the more conformationally unstable Z variant. There has been speculation that the two may interact to produce cirrhosis, but this has never been demonstrated experimentally. This hypothesis was raised again by the observation reported here of a mixed heterozygote for Z alpha1-antitrypsin and another conformationally unstable variant (I alpha1-antitrypsin; 39Arg-->Cys) identified in a 34-year-old man with cirrhosis related to alpha1-antitrypsin deficiency. The conformational stability of the I variant has been characterized, and we have used fluorescence resonance energy transfer to demonstrate the formation of heteropolymers between S and Z alpha1-antitrypsin. Taken together, these results indicate that not only may mixed variants form heteropolymers, but that this can causally lead to the development of cirrhosis.

Polymers of Z α1-Antitrypsin Co-Localize with Neutrophils in Emphysematous Alveoli and Are Chemotactic in Vivo

The molecular mechanisms that cause emphysema are complex but most theories suggest that an excess of proteinases is a crucial requirement. This paradigm is exemplified by severe deficiency of the key anti-elastase within the lung: alpha(1)-antitrypsin. The Z mutant of alpha(1)-antitrypsin has a point mutation Glu342Lys in the hinge region of the molecule that renders it prone to intermolecular linkage and loop-sheet polymerization. Polymers of Z alpha(1)-antitrypsin aggregate within the liver leading to juvenile liver cirrhosis and the resultant plasma deficiency predisposes to premature emphysema. We show here that polymeric alpha(1)-anti-trypsin co-localizes with neutrophils in the alveoli of individuals with Z alpha(1)-antitrypsin-related emphysema. The significance of this finding is underscored by the excess of neutrophils in these individuals and the demonstration that polymers cause an influx of neutrophils when instilled into murine lungs. Polymers exert their effect directly on neutrophils rather than via inflammatory cytokines. These data provide an explanation for the accelerated tissue destruction that is characteristic of Z alpha(1)-antitrypsin-related emphysema. The transition of native Z alpha(1)-antitrypsin to polymers inactivates its anti-proteinase function, and also converts it to a proinflammatory stimulus. These findings may also explain the progression of emphysema in some individuals despite alpha(1)-antitrypsin replacement therapy.

Alpha 1 -antitrypsin deficiency, cirrhosis and emphysema

Emphysema is a chronic progressive lung disease characterised by abnormal permanent enlargement of airspaces as a result of destruction of alveolar walls.1 Most patients develop emphysema as a consequence of smoking but 1–2% of patients with emphysema develop the condition as a result of a genetic deficiency of the plasma proteinase inhibitor α1-antitrypsin. The two common deficiency variants of α1-antitrypsin, S and Z, result from point mutations in the α1-antitrypsin gene2-4 and are named on the basis of their slower electrophoretic mobility on isoelectric focusing analysis compared with the normal M allele.5 S α1-antitrypsin (264Glu→Val) is found in up to 28% of Southern Europeans and, although it results in plasma α1-antitrypsin levels that are 60% of the M allele, it is not associated with any pulmonary sequelae. The Z variant (342Glu→Lys) results in a more severe deficiency that is characterised, in the homozygote, by plasma α1-antitrypsin levels of 10% of the normal M allele and by levels of 60% in the MZ heterozygote (50% from the M allele and 10% from the Z allele). The Z mutation results in the accumulation of α1-antitrypsin in the rough endoplasmic reticulum of the liver (fig 1A) and predisposes the homozygote to juvenile hepatitis, cirrhosis,6 and hepatocellular carcinoma.7 Z α1-antitrypsin inclusions are associated with abnormal liver function tests in over 90% of Z homozygotes in the first year of life but only 10–15% of these develop the prolonged cholestatic jaundice that can progress to cirrhosis and the requirement for hepatic transplantation.6 8 Figure 1 (A) Electron micrograph of a hepatocyte from the liver of a child with Z α1-antitrypsin deficiency. The arrow shows the accumulation of α1-antitrypsin …

Anti-inflammatory and immunomodulatory properties of α1-antitrypsin without inhibition of elastase

The rationale of α1-antitrypsin (AAT) augmentation therapy to treat progressive emphysema in AAT-deficient patients is based on inhibition of neutrophil elastase; however, the benefit of this treatment remains unclear. Here we show that clinical grade AAT (with elastase inhibitory activity) and a recombinant form of AAT (rAAT) without anti-elastase activity reduces lung inflammatory responses to LPS in elastase-deficient mice. WT and elastase-deficient mice treated with either native AAT or rAAT exhibited significant reductions in infiltrating neutrophils (23% and 68%), lavage fluid levels of TNF-α (70% and 80%), and the neutrophil chemokine KC (CXCL1) (64% and 90%), respectively. Lung parenchyma TNF-α, DNA damage-inducible transcript 3 and X-box binding protein-1 mRNA levels were reduced in both mouse strains treated with AAT; significantly lower levels of these genes, as well as IL-1β gene expression, were observed in lungs of AAT-deficient patients treated with AAT therapy compared with untreated patients. In vitro, LPS-induced cytokines from WT and elastase-deficient mouse neutrophils, as well as neutrophils of healthy humans, were similarly reduced by AAT or rAAT; human neutrophils adhering to endothelial cells were decreased by 60–80% (P < 0.001) with either AAT or rAAT. In mouse pancreatic islet macrophages, LPS-induced surface expression of MHC II, Toll-like receptor-2 and -4 were markedly lower (80%, P < 0.001) when exposed to either AAT or rAAT. Consistently, in vivo and in vitro, rAAT reduced inflammatory responses at concentrations 40- to 100-fold lower than native plasma-derived AAT. These data provide evidence that the anti-inflammatory and immunomodulatory properties of AAT can be independent of elastase inhibition.

Mutant neuroserpin (S49P) that causes familial encephalopathy with neuroserpin inclusion bodies is a poor proteinase inhibitor and readily forms polymers in vitro

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by intraneuronal inclusions of mutant neuroserpin. We report here the expression, purification, and characterization of wild-type neuroserpin and neuroserpin containing the S49P mutation that causes FENIB. Wild-type neuroserpin formed SDS-stable complexes with tPA with an association rate constant and K i of 1.2 × 104 m −1 s−1 and 5.8 nm, respectively. In contrast, S49P neuroserpin formed unstable complexes with an association rate constant and K i of 0.3 × 104 m −1 s−1 and 533.3 nm, respectively. An assessment by circular dichroism showed that S49P neuroserpin had a lower melting temperature than wild-type protein (49.9 and 56.6 °C, respectively) and more readily formed loop-sheet polymers under physiological conditions. Neither the wild-type nor S49P neuroserpin accepted the P7-P2 α1-anti-trypsin or P14-P3 antithrombin-reactive loop peptides that have been shown to block polymer formation in other members of the serpin superfamily. Taken together, these data demonstrate that S49P neuroserpin is a poor proteinase inhibitor and readily forms loop-sheet polymers. These findings provide strong support for the role of neuroserpin polymerization in the formation of the intraneuronal inclusions that are characteristic of FENIB.

Oxidation of Z α1-antitrypsin by cigarette smoke induces polymerization: a novel mechanism of early-onset emphysema.

The acceleration of chronic obstructive pulmonary disease (COPD) by cigarette smoke (CS) in individuals with severe genetic deficiency of α(1)-antitrypsin (Z-AT [Glu342Lys]) exemplifies the critical importance of gene-environmental interactions to the development of COPD. We investigated the molecular basis for the interaction between Z-AT and CS. Female mice (8-10 wk old) transgenic for normal (M-AT) or Z-AT on CBA background were exposed to four 1R3F cigarettes daily for 5 days. Age and sex matched littermates not exposed to CS were used as controls. Bronchoalveolar lavage fluid and lung homogenates were assessed for inflammatory cells, neutrophil elastase, and AT conformers. Z-AT was purified from plasma, exposed to CS extract, and assessed for the development and temporal relationship between AT conformers. Mice transgenic for Z-AT developed a significant increase in pulmonary polymers after acute CS exposure (P = 0.001). There were also increased neutrophils in CS-Z lungs versus controls (P < 0.001), which were tightly correlated with polymer concentrations (r(2) = 0.93). Oxidation of human plasma Z-AT by CS or N-chlorosuccinimide greatly accelerated polymerization (P = 0.004), which could be abrogated by antioxidants (P = 0.359 versus Z control). Our data show that CS accelerates polymerization of Z-AT by oxidative modification, which, in so doing, further reduces pulmonary defense and increases neutrophil influx into the lungs. These novel findings provide a molecular explanation for the striking observation of premature emphysema in ZZ homozygotes who smoke. Further work is required to assess whether antioxidant therapy may be beneficial in Z-AT-related COPD.

α1-Antitrypsin deficiency, liver disease and emphysema

alpha(1)-Antitrypsin is a member of the serine proteinase inhibitor (serpin) superfamily and a potent inhibitor of neutrophil elastase. The most important deficiency variant of alpha(1)-antitrypsin arises from the Z mutation (Glu342Lys). This mutation perturbs the proteins tertiary structure to promote a precise, sequential intermolecular linkage that results in polymer formation. These polymers accumulate within the endoplasmic reticulum of the hepatocyte forming inclusion bodies that are associated with neonatal hepatitis, juvenile cirrhosis and adult hepatocellular carcinoma. The resultant secretory defect leads to plasma deficiency of alpha(1)-antitrypsin. This exposes lung tissue to uncontrolled proteolytic attack from neutrophil elastase, culminating in alveolar destruction. Thus, the Z alpha(1)-antitrypsin homozygote is predisposed to developing early onset basal, panacinar emphysema. In this review, we summarise the current understanding of the pathobiology of alpha(1)-antitrypsin deficiency and the associated liver cirrhosis and emphysema. We show how this knowledge has led to the development of novel therapeutic approaches to treat this condition.