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.

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.