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.

Figure 1

Effect of cigarette smoking and COPD on circulating A1AT’s caspase-3 inhibitory activity. (A) Purity of A1AT eluates obtained by immunopurification from plasma of volunteer subjects, tested via Western blotting (upper panel; representative blot of elutions 1–4 from two subjects) and Coomassie protein staining (lower panel; representative gel of elution 1 from five different subjects). (B) Ex vivo caspase-3 inhibitory activity (% inhibition of caspase-3 activity against a fluorescent substrate; mean + SEM) of A1AT (45.5 μg/mL) purified from the plasma of healthy no-COPD (n = 16) and COPD (n = 13) human volunteers who were either never- or ex-smokers ( , n = 13) or actively smoking cigarettes (■, n = 16).

Figure 2

Interaction of CS-exposed A1AT with capsase-3. Energy (Kcal/mol) generated by the intermolecular interaction between human A1AT (100 μmol/L) added sequentially in 10-μL aliquots (for a total of 29 injections) at 540-s intervals to (A) elastase (positive control); (B) buffer (negative control); (C) caspase-3 (8.9 μmol/L); or between (D) human A1AT preexposed to CS extract (25%) and caspase-3. The energy was measured with an isothermal titration microcalorimeter at 30°C and expressed in relation to the molar ratio of the two reaction components, defined as the ratio of injected reactant to cell solution. Representative of two independent experiments.

Figure 3

Requirement of the RCL for the A1AT inhibition of capsase-3 activity. (A) Western blot of recombinant human MM-A1AT, ZZ-A1AT or RCL-null A1AT produced in E. coli. Representative of n = 2. (B) Kinetic activity of recombinant human caspase-3 (~1 U/μL) against a fluorescently labeled substrate in the presence of purified human recombinant MM-A1AT, ZZ-A1AT or RCL-null A1AT (45 μg/mL) (mean + SEM; n = 3–5); ns, nonsignificant statistical difference.

Figure 4

Caspase-3 inhibitory effect of ZZ-A1AT in vivo. (A) Endogenous lung caspase-3/7 activity measured in a fluorescence activity assay at 3 d following CS exposure in mice. Mice instilled with ZZ-A1AT–expressing AAV, but not those instilled with control AAV, exhibited inhibition of CS-induced caspase-3 activity in the lung (mean + SEM; *P < 0.05; n = 3–5). (B) Zymogram of lung lysates from mice instilled with control vehicle (PBS), control AAV or ZZ-A1AT–expressing AAV exposed to air control or to CS for 3 d. hPC is a positive control (human fibrosarcoma whole cell lysate).

Figure 5

Effect of A1AT on the activity of different classes of caspases. Cell-free kinetic enzymatic activity was measured as follows for different classes of caspases. (A) Executioner caspases: recombinant active caspases -3 (●), -6 (▿) or -7 ( ) were incubated with purified human A1AT (0–500 μg/mL). (B) Initiator caspases (recombinant active caspases -8, -9, -10, -2), executioner caspases (-3, -7, -6) or inflammatory caspases (-1, -4, -5) were incubated with purified human A1AT obtained from pooled plasma (500 μg/mL; n = 3). (C) Executioner caspase-6 incubated with purified A1AT ( ) or recombinant RCL-null A1AT (■) (0–500 μg/mL; n = 3; *P < 0.05 versus caspase-6 activity incubated with vehicle and versus RCL-null A1AT).

Figure 6

Effect of A1AT against executioner caspases in vivo. (A) Representative immunoblots of human MM-A1AT and ZZ-A1AT expression in the lungs of mice instilled with either control Lac-Z-AAV or MM-A1AT or ZZ-A1AT AAV (1 × 10¹⁰; 50 μL), followed, 4 wks later, by active caspase-6 intratracheal instillation (5 U; 50 μL; 48 h). The upper band represents A1AT immunoblots (human recombinant A1AT used as a control in the last lane), and the lower band represents vinculin immunoblots used as a loading control. (B) Endogenous lung caspase-3/7 activity and (C) lung morphometry measurement of surface/volume ratios following active caspase-6 intratracheal instillation in mice (48 h) in the presence of Lac-Z-AAV (vector) empty or overexpressing MM-A1AT, or ZZ-A1AT for 4 wks. Mean + SEM; n = 10.