Alanine substitutions in the GXXXG motif alter C99 cleavage by γ-secretase but not its dimerization
Alanine substitutions in the GXXXG motif alter C99 cleavage by γ-secretase but not its dimerization
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DOI：
10.1111/jnc.13942
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发表时间：
2017-03-01
影响因子：
4.7
通讯作者：
Funamoto, Satoru
中科院分区：
文献类型：
--
作者：
Higashide, Hidekazu;Ishihara, Seiko;Funamoto, Satoru
淀粉样β蛋白(Aβ)是老年斑的主要成分，老年斑是阿尔茨海默病的神经病理特征之一。淀粉样前体蛋白(APP)在β-和γ-分泌酶的作用下发生淀粉样变性，导致ALI的产生。APP在胞外膜旁和跨膜区含有GXXXG基序的串联三重复序列。据报道，GXXXG基序与蛋白质之间的相互作用有关，但APP中的GXXXG基序是否参与底物二聚以及二聚化是否影响伽马分泌依赖的切割仍存在争议。因此，GXXXG基序、底物二聚化和依赖于伽马分泌的切割位点之间的关系尚不清楚。在这里，我们应用蓝色天然聚丙烯酰胺凝胶电泳法检测了APP羧基末端片段(C99)GXXXG基序中的丙氨酸取代对其二聚化和Aβ产生的影响。令人惊讶的是，基序中的丙氨酸取代未能改变洗涤剂可溶状态下的C99二聚。基于细胞和溶解的伽马分泌酶分析表明，增加基序中的丙氨酸取代倾向于减少长的Aβ物种，如Aβ42和Aβ43，同时增加短的Aβ物种。我们的数据表明，GXXXG基序对于Aβ的产生是至关重要的，但对C99二聚体来说并不是。
The amyloid beta (A beta) protein is a major component of senile plaques, one of the neuropathological hallmarks of Alzheimer's disease. Amyloidogenic processing of amyloid precursor protein (APP) by beta- and gamma-secretases leads to production of Ali. APP contains tandem triple repeats of the GXXXG motif in its extracellular juxtamembrane and transmembrane regions. It is reported that the GXXXG motif is related to protein -protein interactions, but it remains controversial whether the GXXXG motif in APP is involved in substrate dimerization and whether dimerization affects gamma-secretasedependent cleavage. Therefore, the relationship between the GXXXG motifs, substrate dimerization, and gamma-secretasedependent cleavage sites remains unclear. Here, we applied blue native poly acrylamide gel electrophoresis to examine the effect of alanine substitutions within the GXXXG motifs of APP carboxyl terminal fragment (C99) on its dimerization and A beta production. Surprisingly, alanine substitutions in the motif failed to alter C99 dimerization in detergent soluble state. Cell based and solubilized gamma-secretase assays demonstrated that increasing alanine substitutions in the motif tended to decrease long A beta species such as A beta 42 and A beta 43 and to increase in short A beta species concomitantly. Our data suggest that the GXXXG motif is crucial for A beta production, but not for C99 dimerization.