title: A prominent role of PDIA6 in processing of misfolded proinsulin.
abstract: Despite its critical role in maintaining glucose homeostasis, surprisingly little is known about proinsulin folding in the endoplasmic reticulum. In this study we aimed to understand the chaperones involved in the maturation and degradation of proinsulin. We generated pancreatic beta cell lines expressing FLAG-tagged proinsulin. Several chaperones (including BiP, PDIA6, calnexin, calreticulin, GRP170, Erdj3 and ribophorin II) co-immunoprecipitated with proinsulin suggesting a role for these proteins in folding. To investigate the chaperones responsible for targeting misfolded proinsulin for degradation, we also created a beta cell line expressing FLAG-tagged proinsulin carrying the Akita mutation (Cys96Tyr). All chaperones found to be associated with wild type proinsulin also co-immunoprecipitated with Akita proinsulin. However, one additional protein, namely P58(IPK), specifically precipitated with Akita proinsulin and approximately ten fold more PDIA6, but not other PDI family members, was bound to Akita proinsulin. The latter suggests that PDIA6 may act as a key reductase and target misfolded proinsulin to the ER-degradation pathway. The preferential association of PDIA6 to Akita proinsulin was also confirmed in another beta cell line (βTC-6). Furthermore, for the first time, a physiologically relevant substrate for PDIA6 has been evidenced. Thus, this study has identified several chaperones/foldases that associated with wild type proinsulin and has also provided a comprehensive interactome for Akita misfolded proinsulin.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia.
authors.forename: Dhana G
authors.initials: DG
authors.lastname: Gorasia
authors.affiliation: Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia; Infection and Immunity Program, Biomolecular Discovery Institute and Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia.
authors.forename: Nadine L
authors.initials: NL
authors.lastname: Dudek
authors.affiliation: Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia.
authors.forename: Helena
authors.initials: H
authors.lastname: Safavi-Hemami
authors.affiliation: Infection and Immunity Program, Biomolecular Discovery Institute and Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia.
authors.forename: Rochelle Ayala
authors.initials: RA
authors.lastname: Perez
authors.affiliation: Infection and Immunity Program, Biomolecular Discovery Institute and Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia.
authors.forename: Ralf B
authors.initials: RB
authors.lastname: Schittenhelm
authors.affiliation: Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Philippa M
authors.initials: PM
authors.lastname: Saunders
authors.affiliation: Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia.
authors.forename: Sheena
authors.initials: S
authors.lastname: Wee
authors.affiliation: Department of Pharmacology and Therapeutics, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Jon E
authors.initials: JE
authors.lastname: Mangum
authors.affiliation: Department of Pharmacology and Therapeutics, The University of Melbourne, Melbourne, Victoria, Australia; Department of Paediatrics, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hubbard
authors.affiliation: Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia; Infection and Immunity Program, Biomolecular Discovery Institute and Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia. Electronic address: Anthony.purcell@monash.edu.
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Purcell
mesh_terms: D000818:Animals; D002460:Cell Line; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011384:Proinsulin / Q000737:chemistry*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000502:physiology*; D017510:Protein Folding
chemical_list: D011384:Proinsulin; C526507:Pdia6 protein, mouse; D019704:Protein Disulfide-Isomerases
keywords: Chaperones; Disulphide bonds; ERAD; PDI; Proinsulin; Protein folding