RESUMEN
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 ß-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.
Asunto(s)
Diabetes Mellitus Tipo 2/enzimología , Glucoquinasa/genética , Proteínas Mutantes/metabolismo , Mutación/genética , Pliegue de Proteína , Proteolisis , Deficiencias en la Proteostasis/enzimología , Adulto , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Estabilidad de Enzimas , Familia , Femenino , Glucoquinasa/química , Glucoquinasa/metabolismo , Glucosa/metabolismo , Células HEK293 , Humanos , Cinética , Lisosomas/metabolismo , Masculino , Proteínas Mutantes/química , Pakistán , Linaje , Fenotipo , Complejo de la Endopetidasa Proteasomal/metabolismo , Multimerización de Proteína , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/patología , Proteínas Recombinantes/metabolismoRESUMEN
Glucokinase is the predominant hexokinase expressed in hepatocytes and pancreatic ß-cells, with a pivotal role in regulating glucose-stimulated insulin secretion, illustrated by glucokinase gene mutations causing monogenic diabetes and congenital hyperinsulinemic hypoglycemia. A complex tissue-specific network of mechanisms regulates this enzyme, and a major unanswered question in glucokinase biology is how post-translational modifications control the function of the enzyme. Here, we show that the pancreatic isoform of human glucokinase is SUMOylated in vitro, using recombinant enzymes, and in insulin-secreting model cells. Three N-terminal lysines unique for the pancreatic isoform (Lys-12/Lys-13 and/or Lys-15) may represent one SUMOylation site, with an additional site (Lys-346) common for the pancreatic and the liver isoform. SUMO-1 and E2 overexpression stabilized preferentially the wild-type human pancreatic enzyme in MIN6 ß-cells, and SUMOylation increased the catalytic activity of recombinant human glucokinase in vitro and also of glucokinase in target cells. Small ubiquitin-like modifier conjugation represents a novel form of post-translational modification of the enzyme, and it may have an important regulatory function in pancreatic ß-cells.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Glucoquinasa/química , Páncreas/enzimología , Sumoilación , Animales , Carbohidratos/química , Catálisis , Electroforesis en Gel Bidimensional/métodos , Células Secretoras de Insulina/citología , Cinética , Hígado/enzimología , Espectrometría de Masas/métodos , Ratones , Mutación , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/químicaRESUMEN
GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically 'normal' GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 ß-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations.