GCK-MODY diabetes as a protein misfolding disease: The mutation R275C promotes protein misfolding, self-association and cellular degradation

• Published in: Molecular and cellular endocrinology 2014-01, Vol.382 (1), p.55-65
• Main Authors: Negahdar, Maria, Aukrust, Ingvild, Molnes, Janne, Solheim, Marie H., Johansson, Bente B., Sagen, Jørn V., Dahl-Jørgensen, Knut, Kulkarni, Rohit N., Søvik, Oddmund, Flatmark, Torgeir, Njølstad, Pål R., Bjørkhaug, Lise
• Format: Article
• Language: English
• Subjects: Adult; Aggregates; Aggregation; Catalytic activity; Cellular; Cellular protein degradation; Degradation; Diabetes Mellitus, Type 2 - enzymology; Diabetes Mellitus, Type 2 - genetics; Diabetes Mellitus, Type 2 - pathology; Dimers; Enzyme Stability; Enzymes; Family; Female; GCK-MODY diabetes; Glucokinase - chemistry; Glucokinase - genetics; Glucokinase - metabolism; Glucose - metabolism; HEK293 Cells; Humans; Hyperglycemia; Kinetics; Lysosomes - metabolism; Male; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutation - genetics; Mutations; Pakistan; Pedigree; Phenotype; Proteasome Endopeptidase Complex - metabolism; Protein Folding; Protein misfolding; Protein Multimerization; Proteins; Proteolysis; Proteostasis Deficiencies - enzymology; Proteostasis Deficiencies - genetics; Proteostasis Deficiencies - pathology; Recombinant Proteins - metabolism; Self-association
• ISSN: 0303-7207; 1872-8057
• DOI: 10.1016/j.mce.2013.08.020

•The glucokinase mutation c.823C>T generates a slightly activated protein (p.R275C).•The mutation also causes a misfolded and unstable protein (p.R275C).•The mutant protein promotes the formation of dimers and aggregates.•The functional impact is an increased rate of cellular degradation.•Biochemical effect relates to conformational changes in the F260-L271 loop structure. 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.