Preventing and reversing the cellular consequences of Z alpha-1 antitrypsin accumulation by targeting s4A

Abstract

Background & aims: The Z variant (Glu342Lys) of α(1)-antitrypsin (AT) polymerizes and accumulates in the hepatocyte endoplasmic reticulum (ER) predisposing to neonatal hepatitis and liver cirrhosis. The resultant secretory defect leaves the lungs vulnerable to elastolysis and early-onset emphysema. Our aim in this study was to evaluate the effect of targeting strand 4a (s4A) as a strategy to inhibit polymerization and restore plasma secretion.

Methods: HEK293 cells and HepG2 cells were transfected with Z-AT (Z-AT cells) or control M-AT (M-AT cells). The effect of Ac-TTAI-NH(2) (4M), Ac-FLEAIG-NH(2) (6M), and Ac-SEAAASTAVVIA-NH(2) (12M) on preventing and reversing intracellular Z-AT polymers and secretion of AT was evaluated by pulse-chase/immunoprecipitation, ELISA, and immunoblot with a polymer-specific antibody (ATZII). The ER overload response was assessed by RT-PCR for PERK, calnexin, and RGS16, and ELISA for NF-κB, IL-6, and IL-8.

Results: All peptides prevented the intracellular accumulation of Z-AT (4M>6M>12M) in comparison with control peptides, with detection of the AT-Inhibitor complex in inclusion bodies. In so doing, 4M also significantly increased the concentration of secreted Z-AT and the elastase inhibitory activity. Furthermore, the 4M peptide was able to reverse the intracellular aggregation of Z-AT. The ER accumulation of Z-AT was shown to induce PERK-dependent NF-κB, IL-6, IL-8, and RGS16 and calnexin; all of which could be abrogated effectively by 4M. 4M had no effect on apoptosis or cell viability.

Conclusions: These findings are the first evidence that targeting s4A can prevent the cellular accumulation and deleterious effects of Z-AT and restore its plasma concentrations. As such, this is a major step towards treatment of patients with Z-AT-related disease.