GCK-MODY diabetes as a protein misfolding disease: The mutation R275C promotes protein misfolding,self-association and cellular degradation |
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| Authors: | Maria Negahdar,Ingvild Aukrust,Janne Molnes,Marie H. Solheim,Bente B. Johansson,Jø rn V. Sagen,Knut Dahl-Jø rgensen,Rohit N. Kulkarni,Oddmund Sø vik,Torgeir Flatmark,På l R. Njø lstad,Lise Bjø rkhaug |
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| Affiliation: | 1. KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway;2. Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway;3. Department of Biomedicine, University of Bergen, Bergen, Norway;4. Section of Islet Cell Biology and Regenerative Medicine, Joslin Diabetes Center, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA;5. Department of Pediatrics, Haukeland University Hospital, Bergen, Norway;6. Institute of Medicine, University of Bergen, Bergen, Norway;g Hormone Laboratory, Haukeland University Hospital, Bergen, Norway;h Pediatric Department Ullevaal, Oslo University Hospital, Oslo, Norway;i Faculty of Medicine, University of Oslo, Oslo, Norway |
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| 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. |
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| Keywords: | BCA, bicinchoninic acid DMEM, dulbecco&rsquo s modified eagles medium GCK, glucokinase gene Glc, α-D-glucose GKRP, glucokinase regulatory protein HEK, human embryonic kidney hGK, human glucokinase IFG, impaired fasting glucose MIN, mouse insulinoma MODY, maturity-onset diabetes of the young PFK-2/FBPase, bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase PNS, post nuclear supernatant PDB, protein data bank RRL, rabbit reticulocyte lysate |
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