Our results provide evidence that A1AT has an inhibitory activity directed only against executioner caspases. We demonstrate that this activity requires the RCL and can be profoundly inhibited by exposure of the protein to CS both in vitro
and in vivo
. In contrast, the single amino acid mutation in the ZZ form of A1AT did not affect its antiapoptotic activity. One of the main posttranslational modifications of A1AT in patients with COPD is that of oxidation, thought to be due to exposure to CS components (20
). Reactive oxygen and nitrogen species, which are increased in smokers and COPD patients, may target and modify the A1AT methionine-358 and cysteine-232 residues of the RCL, respectively. These changes could alter the A1AT function against elastase (21
). A similar effect may impair the ability of A1AT to directly interact with target caspases, as demonstrated by isothermal titration calorimetry assays. Interestingly, only the acute exposure to CS, but not the disease state of COPD, led to a decrease in the anti–caspase-3 activity of A1AT. This suggests that the oxidative burden that may exist in this chronic disease in the absence of smoking is not sufficient to alter the ability of A1AT to interact with the caspases in ex vivo
cell-free conditions. It still remains to be determined whether the A1AT from ex-smokers with COPD has impaired ability to access intracellular caspases in vivo
. We now provide additional information regarding the properties of oxidized A1AT, to include a loss of anticaspase activity, in addition to previously known loss of antielastase action and proinflammatory effects of triggering chemokine release and recruitment of inflammatory cells (23
The fact that the RCL-null A1AT construct failed to inhibit caspase-3 or -6 activities confirmed a critical role of this domain in multiple serpin functions, because the RCL domain is essential for the antielastase activity of A1AT. Even more drastically than the oxidation of its residues, the deletion of the RCL significantly alters the structural properties of the A1AT protein. Because the conformational plasticity of the A1AT is highly vulnerable to structural changes in the protein and is required for its function as an antiprotease, it is not surprising that the RCL-null mutant failed to inhibit caspase-3 activity. However, it was somewhat surprising that the single point mutation causing the ZZ phenotype, which would be expected to affect A1AT plasticity by making it prone to polymerization, did not affect the antiapoptotic function of the protein in vitro
and in vivo
. A similar lack of effect of ZZ-A1AT on the antiapoptotic activity of A1AT was recently reported by Greene et al.
). It is possible that unlike the effect on elastase, certain levels of protein polymerization do not inhibit the ability of the RCL to interact with caspases. Alam et. al.
recently demonstrated that the oxidation of ZZ-A1AT precedes and promotes its polymerization (25
). Because the RCL-null A1AT does not form polymers but failed to inhibit caspase-3, it is likely that the oxidation of the RCL, rather than the oxidation-induced polymerization, promotes loss of A1AT anti-apoptotic function. This possibility is supported by our previously reported finding that polymerization of A1AT obtained by heating the protein caused a significant loss of anti–capsase-3 activity, not seen with the recombinant ZZ protein. Of note, although the bacterial expression of ZZ-A1AT may generate an incompletely functional mutant due to lack of glycosylation, the ZZ-A1AT production in vivo
via AAV should not have this limitation.
This report builds on our previous studies that identified a novel protective mechanism of A1AT in the lung, that of inhibition of the apoptotic effector caspase-3 (3
). Here we identified the other executioner caspases -7 and -6, but not the initiator caspases, as unique targets for A1AT. Executioner caspases can be activated by the extrinsic and intrinsic apoptosis pathways, which in turn are triggered by stimuli such as proinflammatory cytokines or stimuli causing mitochondrial stress, respectively (26
). Although there is some functional redundancy among executioner caspases, it has been reported that the effector caspase-6 can activate caspase-3 (28
). This could explain why we observed increases in endogenous caspase-3/7 activity following intratracheal caspase-6 instillation. It is possible that the effect of the MM- or ZZ-A1AT in vivo
may be a consequence of inhibition of either caspase-6 or the downstream caspase-3/7. While MM-and ZZ-A1AT comparably inhibited the casapase-6–induced rise in caspase-3/7 activity, ZZ-A1AT decreased the baseline caspase activity. A similar observation of a potent inhibition of caspase-3 by ZZ-A1AT was recently made in human bronchial epithelial cells (24
), and the difference between the two proteins was attributed to ZZ-A1AT leading to trans-activation of nuclear factor–κB and up-regulation of the cellular inhibitor of apoptosis. Hence, MM- and ZZ-A1AT may inhibit apoptosis via different mechanisms, which could explain the differences we observed in the regulation of baseline caspase-3/7 activity.
Considering the vital role of apoptosis regulation in COPD, we performed a comprehensive analysis of A1AT inhibitory ability on all caspases known to be involved in apoptosis (caspase-2, -3, -6, -7, -8, -9 and-10) and also on the inflammatory caspases (-1, -4 and -5). A1AT inhibited all effector caspases (-3, -6 and -7), but none of the initiator caspases, and had only a mild but non-significant inhibitory effect on caspase-1 of the inflammatory caspases. The marked inhibitory effect on caspase-6 activity in vitro
was recapitulated in vivo
. We cannot rule out that other caspase activation could be inhibited in complex in vivo
models in which A1AT may act on other upstream targets that lead to the activation of such caspases. This, or more favorable in vivo
conditions, may explain the recently reported inhibitory effect of A1AT on caspase-1 activation in a model of myocardial infarction (29
The clinical significance of our work is related to the finding of increased apoptosis of alveolar epithelial and endothelial cells in the lungs of COPD patients who smoke cigarettes and in animal models of emphysema (26
). Our data suggest that the effect of cigarette smoking on the A1AT antiapoptotic function is reversible and may be linked to the turnover of the protein as it is synthesized from the liver or to the restoration of native A1AT by reduction of the oxidized protein. Limitations of our clinical study include the small number of patients and the fact that no PFTs were performed in the control, no-clinical-diagnosis-of-COPD-smoker group, leaving the possibility that individuals in this group may have had asymptomatic COPD. Future studies will need to address the possibility that the A1AT function at a given time may vary depending on the time since CS exposure and to also address the functional impact of normalizing the function of A1AT in lung endothelial cells on the progression of emphysema in vivo
Finally, in addition to lung destruction there is evidence that cardiovascular morbidities are increased in COPD. Smoking cessation has been noted to result in a sharp reduction in the risk of stroke, myocardial infarction, and mortality from cardiovascular diseases in patients with or without COPD (33
). Extrapolation of our work on pulmonary endothelium to include the antiapoptotic effect of A1AT on other vascular beds might help explain some of the rapid beneficial effects on the systemic or coronary circulation that have been noted following smoking cessation.