title: GCK-MODY diabetes as a protein misfolding disease: the mutation R275C promotes protein misfolding, self-association and cellular degradation.
abstract: GCK-MODY, dominantly inherited mild hyperglycemia, is associated with more than 600 mutations in the glucokinase gene. Different molecular mechanisms have been shown to explain GCK-MODY. Here, we report a Pakistani family harboring the glucokinase mutation c.823C>T (p.R275C). The recombinant and in cellulo expressed mutant pancreatic enzyme revealed slightly increased enzyme activity (kcat) and normal affinity for α-D-glucose, and resistance to limited proteolysis by trypsin comparable with wild-type. When stably expressed in HEK293 cells and MIN6 β-cells (at different levels), the mutant protein appeared misfolded and unstable with a propensity to form dimers and aggregates. Its degradation rate was increased, involving the lysosomal and proteasomal quality control systems. On mutation, a hydrogen bond between the R275 side-chain and the carbonyl oxygen of D267 is broken, destabilizing the F260-L271 loop structure and the protein. This promotes the formation of dimers/aggregates and suggests that an increased cellular degradation is the molecular mechanism by which R275C causes GCK-MODY.
authors.affiliation: KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway; Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway.
authors.forename: Maria
authors.initials: M
authors.lastname: Negahdar
authors.affiliation: KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway; Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway; Department of Biomedicine, University of Bergen, Bergen, Norway; Section of Islet Cell Biology and Regenerative Medicine, Joslin Diabetes Center, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Ingvild
authors.initials: I
authors.lastname: Aukrust
authors.affiliation: KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway; Department of Biomedicine, University of Bergen, Bergen, Norway; Department of Pediatrics, Haukeland University Hospital, Bergen, Norway.
authors.forename: Janne
authors.initials: J
authors.lastname: Molnes
authors.affiliation: KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway; Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway; Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Marie H
authors.initials: MH
authors.lastname: Solheim
authors.affiliation: KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway; Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway; Department of Pediatrics, Haukeland University Hospital, Bergen, Norway.
authors.forename: Bente B
authors.initials: BB
authors.lastname: Johansson
authors.affiliation: KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway; Institute of Medicine, University of Bergen, Bergen, Norway; Hormone Laboratory, Haukeland University Hospital, Bergen, Norway.
authors.forename: Jørn V
authors.initials: JV
authors.lastname: Sagen
authors.affiliation: Pediatric Department Ullevaal, Oslo University Hospital, Oslo, Norway; Faculty of Medicine, University of Oslo, Oslo, Norway.
authors.forename: Knut
authors.initials: K
authors.lastname: Dahl-Jørgensen
authors.affiliation: Section of Islet Cell Biology and Regenerative Medicine, Joslin Diabetes Center, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Rohit N
authors.initials: RN
authors.lastname: Kulkarni
authors.affiliation: KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway.
authors.forename: Oddmund
authors.initials: O
authors.lastname: Søvik
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Torgeir
authors.initials: T
authors.lastname: Flatmark
authors.affiliation: KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway; Department of Pediatrics, Haukeland University Hospital, Bergen, Norway. Electronic address: pal.njolstad@uib.no.
authors.forename: Pål R
authors.initials: PR
authors.lastname: Njølstad
authors.affiliation: KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway; Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway; Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Lise
authors.initials: L
authors.lastname: Bjørkhaug
mesh_terms: D000328:Adult; D003924:Diabetes Mellitus, Type 2 / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D004795:Enzyme Stability; D005190:Family; D005260:Female; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D010154:Pakistan; D010375:Pedigree; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017510:Protein Folding; D055503:Protein Multimerization; D059748:Proteolysis; D057165:Proteostasis Deficiencies / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D005941:Glucokinase; D046988:Proteasome Endopeptidase Complex; D005947:Glucose
keywords: Aggregation; BCA; Catalytic activity; Cellular protein degradation; DMEM; GCK; GCK-MODY diabetes; GKRP; Glc; HEK; IFG; MIN; MODY; PDB; PFK-2/FBPase; PNS; Protein misfolding; RRL; Self-association; bicinchoninic acid; bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; dulbecco’s modified eagles medium; glucokinase gene; glucokinase regulatory protein; hGK; human embryonic kidney; human glucokinase; impaired fasting glucose; maturity-onset diabetes of the young; mouse insulinoma; post nuclear supernatant; protein data bank; rabbit reticulocyte lysate; α-D-glucose