title: Aberrant disulphide bonding contributes to the ER retention of alpha1-antitrypsin deficiency variants.
abstract: Mutations in alpha1-antitrypsin (AAT) can cause the protein to polymerise and be retained in the endoplasmic reticulum (ER) of hepatocytes. The ensuing systemic AAT deficiency leads to pulmonary emphysema, while intracellular polymers are toxic and cause chronic liver disease. The severity of this process varies considerably between individuals, suggesting the involvement of mechanistic co-factors and potential for therapeutically beneficial interventions. We show in Hepa1.6 cells that the mildly polymerogenic I (Arg39Cys) AAT mutant forms aberrant inter- and intra-molecular disulphide bonds involving the acquired Cys39 and the only cysteine residue in the wild-type (M) sequence (Cys232). Substitution of Cys39 to serine partially restores secretion, showing that disulphide bonding contributes to the intracellular retention of I AAT. Covalent homodimers mediated by inter-Cys232 bonding alone are also observed in cells expressing the common Z and other polymerising AAT variants where conformational behaviour is abnormal, but not in those expressing M AAT. Prevention of such disulphide linkage through the introduction of the Cys232Ser mutation or by treatment of cells with reducing agents increases Z AAT secretion. Our results reveal that disulphide interactions enhance intracellular accumulation of AAT mutants and implicate the oxidative ER state as a pathogenic co-factor. Redox modulation, e.g. by anti-oxidant strategies, may therefore be beneficial in AAT deficiency-associated liver disease.
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Ronzoni
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
authors.forename: Romina
authors.initials: R
authors.lastname: Berardelli
authors.affiliation: Department of Pathology, Spedali Civili, Brescia, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Medicina
authors.affiliation: Università Vita-Salute San Raffaele, Milan, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Sitia
authors.affiliation: Institute of Structural and Molecular Biology/Crystallography, Birkbeck College, University of London, London, UK and Division of Asthma, Allergy and Lung Biology, King's College, London, UK.
authors.forename: Bibek
authors.initials: B
authors.lastname: Gooptu
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy, annamaria.fra@unibs.it.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Fra
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004220:Disulfides / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005838:Genotype; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D009154:Mutation; D058105:Polymerization; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000378:metabolism; D019896:alpha 1-Antitrypsin Deficiency / Q000235:genetics* / Q000378:metabolism
chemical_list: D004220:Disulfides; D000515:alpha 1-Antitrypsin