title: Preventing and reversing the cellular consequences of Z alpha-1 antitrypsin accumulation by targeting s4A.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Medicine, University of Cambridge, Level 5, Box 157, Addenbrookes Hospital, Hills Road, Cambridge CB2 0QQ, UK.
authors.forename: Sam
authors.initials: S
authors.lastname: Alam
authors.forename: Jicun
authors.initials: J
authors.lastname: Wang
authors.forename: Sabina
authors.initials: S
authors.lastname: Janciauskiene
authors.forename: Ravi
authors.initials: R
authors.lastname: Mahadeva
mesh_terms: D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004721:Endoplasmic Reticulum / Q000378:metabolism; D014644:Genetic Variation / Q000502:physiology; D057809:HEK293 Cells; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D008107:Liver Diseases / Q000188:drug therapy* / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D058105:Polymerization / Q000187:drug effects; D011656:Pulmonary Emphysema / Q000188:drug therapy / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection / Q000379:methods; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin