Abstract
α-1 Antitrypsin (A1AT) is a serpin with a major protective effect against cigarette smoke-induced emphysema development, and patients with mutations of the A1AT gene display a markedly increased risk for developing emphysema. We reported that A1AT protects lung endothelial cells from apoptosis and inhibits caspase-3 activity. It is not clear if cigarette smoking or A1AT mutations alter the caspase-3 inhibitory activity of A1AT and if this serpin alters the function of other caspases. We tested the hypothesis that the caspase-3 inhibitory activity of A1AT is impaired by cigarette smoking and that the A1AT RCL, the key antiprotease domain of the serpin, is required for its interaction with the caspase. We examined the caspase-3 inhibitory activity of human A1AT purified from plasma of actively smoking and nonsmoking individuals, either affected or unaffected with chronic obstructive pulmonary disease. We also tested the caspase inhibitory activity of two mutant forms of A1AT, the recombinant human piZZ and the RCL-deleted (RCL-null) A1AT forms. A1AT purified from the blood of active smokers exhibited marked attenuation in its caspase-3 inhibitory activity, independent of disease status. In vitro exposure of the normal (MM) form of A1AT to cigarette smoke extract reduced its ability to interact with caspase-3, measured by isothermal titration calorimetry, as did the deletion of the RCL, but not the ZZ point mutation. In cell-free assays A1AT was capable of inhibiting all executioner caspases, -3, -7 and especially -6, but not the initiator or inflammatory caspases. The inhibitory effect of A1AT against caspase-6 was tested in vivo, where overexpression of both human MM and ZZ-A1AT via adeno-associated virus transduction significantly protected against apoptosis and against airspace damage induced by intratracheal instillation of caspase-6 in mice. These data indicate a specific inhibitory effect of A1AT on executioner caspases, which is profoundly attenuated by active exposure to cigarette smoking and is dependent on the protein RCL, but is not affected by the PiZZ mutation.
Highlights
INTRODUCTION α1 antitrypsin (A1AT) deficiency is the most common genetic cause of emphysema, which is the main cause of morbidity in individuals who have A1AT deficiency and who smoke cigarettes
In our previous work we demonstrated that A1AT is capable of direct binding to caspase-3, inhibiting caspase-3–mediated apoptosis [3]
On the basis of our previous results in cell-free models, which showed that incubation of A1AT with cigarette smoke (CS) extract altered its ability to inhibit the activity of caspase-3, we hypothesized that soluble components of CS that are absorbed in the blood of chronic obstructive pulmonary disease (COPD) patients who are smokers may affect the anti–caspase-3 function of plasma A1AT
Summary
INTRODUCTION α1 antitrypsin (A1AT) deficiency is the most common genetic cause of emphysema, which is the main cause of morbidity in individuals who have A1AT deficiency and who smoke cigarettes. Along with lung matrix destruction, oxidative stress and inflammation, cell death of structural components of the parenchyma is one of the key mechanisms involved in emphysema pathogenesis [2]. A1AT is a serine protease inhibitor synthesized by liver hepatocytes and secreted into the circulation, where it neutralizes neutrophil elastase. This effect accounts for inhibition of elastin degra-. We report our investigation of whether A1AT inhibits the activity of other caspases and whether clinically relevant mutations of A1AT impair its anticaspase activity. We assessed whether in vivo exposure to CS is sufficient to impair the circulating A1AT anticaspase activity
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