Effect of Cigarette Smoke Exposure and Structural Modifications on the α-1 Antitrypsin Interaction with Caspases

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.

Cytosolic, Autocrine Alpha-1 Proteinase Inhibitor (A1PI) Inhibits Caspase-1 and Blocks IL-1β Dependent Cytokine Release in Monocytes

“…These results are similar to previous studies which invoke A1PI-M interactions with cytosolic executioner caspases 3 and 6 [25]–[26], and recently caspase-1 [27], as critical for staving off cell death in endothelial cells, epithelial cells, and cardiomyocytes. As in previous studies, we observed that an excess of A1PI was required to limit caspase activity in a cell free assay, which is highly reliant on the purity and potency of the enzyme preparation.…”

“…As in previous studies, we observed that an excess of A1PI was required to limit caspase activity in a cell free assay, which is highly reliant on the purity and potency of the enzyme preparation. One previous report showed very limited A1PI inhibition of caspase-1 enzymatic activity in a cell free enzyme assay, compared with caspase-3 and caspase-6 [26]. We note that the preparations of recombinant caspase-1 enzyme and reaction conditions used in our study were quite different and may underlie a sensitivity difference between the two assay systems.…”

α1-Antitrypsin Modulates Lung Endothelial Cell Inflammatory Responses to TNF-α

“…However, the role of A1AT in endothelial cell responses to inflammation remains unclear. We and others have shown that A1AT performs an antiapoptotic function in lung endothelial cells by inhibiting executioner caspases 3, 6, and 7 (4,22,23). This activity requires an active uptake of A1AT by the lung endothelium, which normally does not produce A1AT (24).…”

Intrapleural Administration of an AAVrh.10 Vector Coding for Human α1-Antitrypsin for the Treatment of α1-Antitrypsin Deficiency

“…The most common form of a1AT deficiency is homozygous inheritance of the Z allele (lysine replacing glutamic acid 342 in the normal M allele) leading to intrahepatic polymerization of a1AT that results in reduced secretion, and, in turn, reduced serum a1AT levels (Nukiwa et al, 1986;Curiel et al, 1989;Brantly et al, 1991;Lomas et al, 1992;Carrell and Lomas, 2002;Lomas and Parfrey, 2004). Cigarette smoking markedly accelerates the disease, with diminished lifespan of about 20 years (Brantly et al, 1988a,b;Crystal et al, Sandhaus, 2009;Tuder et al, 2010;Alam et al, 2011;Lockett et al, 2012).…”