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GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation.

ABSTRACT: 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 ?-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.

SUBMITTER: Negahdar M

PROVIDER: S-EPMC3444650 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation.

Negahdar Maria M Aukrust Ingvild I Johansson Bente B BB Molnes Janne J Molven Anders A Matschinsky Franz M FM Søvik Oddmund O Kulkarni Rohit N RN Flatmark Torgeir T Njølstad Pål Rasmus PR Bjørkhaug Lise L

Biochimica et biophysica acta 20120720 11

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-L ...[more]

PMID: 22820548

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Project description:OBJECTIVE:Inactivating heterozygous mutations in the GCK gene are a common cause of MODY and result in mild fasting hyperglycaemia, which does not require treatment. We aimed to identify the frequency, clinical and molecular features of GCK mutations in a Turkish paediatric cohort. DESIGN AND PATIENTS:Fifty-four unrelated probands were selected based on the following criteria: age of diagnosis ?17 years, family history of diabetes in at least two generations, anti-GAD/ICA negative, BMI<95.p and follow-up with diet, oral antidiabetic drug or low-dose insulin treatment (?0·5U/kg/d). A MODY probability score (www.diabetesgenes.org) was calculated and 21 patients with a score ?75%, HbA1c levels ?7·5% (58·5 mmol/mol) and fasting blood glucose (FBG) levels 99-145 mg/dl (5·5-8·0 mmol/l) were selected for Sanger sequencing of the GCK gene. Targeted next-generation sequencing for all known monogenic diabetes genes was undertaken for any patient without a GCK gene mutation. RESULTS:GCK gene mutations (pathogenic or likely pathogenic variants) and a novel intronic variant of uncertain significance (c.208 + 3A>T) were identified in 13/54 probands (24%). Twelve of these patients had a MODY probability score ?75%. FBG level and 2-h glucose level in OGTT were 123 ± 14 mg/dl (6·8 ± 0·7 mmol/l) (107-157 mg/dl) and 181 ± 30 mg/dl (10·1 ± 1·6 mmol/l) (136-247 mg/dl), respectively. Average of glucose increment in OGTT was 58 ± 27 mg/dl (3·2 ± 1·5 mmol/l) (19-120 mg/dl), and mean HbA1c level was 6·5 ± 0·5% (47·5 ± 5·5 mmol/mol) (5·9-7·6%). Five novel missense mutations were identified (p.F123S, p.L58P, p.G246A, p.F419C, and p.S151C). Two patients treated with low-dose insulin before the molecular analysis were able to stop treatment. CONCLUSIONS:Approximately 1 in 4 MODY cases in this Turkish paediatric cohort have a GCK mutation. Selection of patients for GCK gene analysis using the MODY probability score was an effective way of identifying most (11/12) patients with a GCK mutation.

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GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation.

Publications

GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation.

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