Negahdar Maria, Aukrust Ingvild, Johansson Bente B, Molnes Janne, Molven Anders, Matschinsky Franz M, Søvik Oddmund, Kulkarni Rohit N, Flatmark Torgeir, Njølstad Pål Rasmus, Bjørkhaug Lise
Department of Clinical Medicine, University of Bergen, N-5020 Bergen, Norway.
Biochim Biophys Acta. 2012 Nov;1822(11):1705-15. doi: 10.1016/j.bbadis.2012.07.005. Epub 2012 Jul 20.
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 β-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.
葡萄糖激酶调节的成年发病型糖尿病（GCK-MODY）是一种常染色体显性遗传的轻度空腹血糖升高疾病，与葡萄糖激酶（GK）编码基因（GCK）中的600多种不同突变有关。当作为重组胰腺蛋白表达时，一些突变会产生具有正常/接近正常催化特性的酶。这些突变导致GCK-MODY的分子机制仍不清楚。在这里，我们旨在探索GK的F260-L270环中两个具有催化“正常”的GCK突变（S263P和G264S）的分子机制。当在HEK293细胞和MIN6β细胞中稳定过表达时，S263P和G264S编码的突变产生了错误折叠的蛋白质，蛋白质质量控制机制使其降解速率增加（S263P>G264S），并且具有自我缔合倾向（G264S>S263P），形成二聚体（抗SDS）和聚集体（部分不溶于Triton X-100），这是通过脉冲追踪实验和亚细胞分级分离确定的。因此，GCK-MODY突变S263P和G264S导致蛋白质错误折叠，从而引起不稳定、细胞二聚化/聚集以及降解速率加快。计算机模拟预测的F260-L270环结构的构象变化被认为通过结构域交换机制介导了两种突变蛋白的二聚化。因此，类似的特性可能代表了其他无法解释的GCK-MODY突变的分子机制，也可能促成了其他先前已表征的GCK-MODY失活突变的疾病机制。
