Culvenor Janetta G, Ilaya Nancy T, Ryan Michael T, Canterford Louise, Hoke David E, Williamson Nicholas A, McLean Catriona A, Masters Colin L, Evin Geneviève
Department of Pathology, The University of Melbourne, Australia.
Eur J Biochem. 2004 Jan;271(2):375-85. doi: 10.1046/j.1432-1033.2003.03936.x.
The presenilin proteins are required for intramembrane cleavage of a subset of type 1 membrane proteins including the Alzheimer's disease amyloid precursor protein. Previous studies indicate presenilin proteins form enzymatically active high molecular mass complexes consisting of heterodimers of N- and C-terminal fragments in association with nicastrin, presenilin enhancer-2 and anterior pharynx defective-1 proteins. Using Blue Native gel electrophoresis (BN/PAGE) we have studied endogenous presenilin 1 complex mass, stability and association with nicastrin, presenilin enhancer-2 and anterior pharynx defective-1. Solubilization of mouse or human brain membranes with dodecyl-d-maltoside produced a 360-kDa species reactive with antibodies to presenilin 1. Presenilin 1 complex levels were high in embryonic brain. Complex integrity was sensitive to Triton X-100 and SDS, but stable to reducing agent. Addition of 5 M urea caused complex dissolution and nicastrin to migrate as a subcomplex. Nicastrin and presenilin enhancer-2 were detected in the presenilin 1 complex following BN/PAGE, electroelution and second-dimension analysis. Anterior pharynx defective-1 was detected as an 18-kDa form and 9-kDa C-terminal fragment by standard SDS/PAGE of mouse tissues, and as a predominant 36-kDa band after presenilin 1 complex second-dimension analysis. Membranes from brain cortex of Alzheimer's disease patients, or from cases with presenilin 1 missense mutations, indicated no change in presenilin 1 complex mobility. Higher molecular mass presenilin 1-reactive species were detected in brain containing presenilin 1 exon 9 deletion mutation. This abnormality was confirmed using cells transfected with the same presenilin deletion mutation.
早老素蛋白是包括阿尔茨海默病淀粉样前体蛋白在内的1型膜蛋白亚群进行膜内切割所必需的。先前的研究表明，早老素蛋白形成具有酶活性的高分子量复合物，该复合物由N端和C端片段的异二聚体与尼卡斯特林、早老素增强子2和咽前缺陷1蛋白结合而成。我们使用蓝色原胶电泳（BN/PAGE）研究了内源性早老素1复合物的质量、稳定性以及与尼卡斯特林、早老素增强子2和咽前缺陷1的结合情况。用十二烷基 - d - 麦芽糖苷溶解小鼠或人脑膜产生了一种与早老素1抗体反应的360 kDa物质。早老素1复合物水平在胚胎脑中较高。复合物的完整性对Triton X - 100和SDS敏感，但对还原剂稳定。添加5 M尿素会导致复合物溶解，尼卡斯特林以亚复合物形式迁移。在BN/PAGE、电洗脱和二维分析后，在早老素1复合物中检测到了尼卡斯特林和早老素增强子2。通过小鼠组织的标准SDS/PAGE检测到咽前缺陷1为18 kDa形式和9 kDa C端片段，在早老素1复合物二维分析后检测到其主要为36 kDa条带。来自阿尔茨海默病患者大脑皮层或早老素1错义突变病例的膜显示早老素1复合物迁移率没有变化。在含有早老素1外显子9缺失突变的脑中检测到更高分子量的早老素1反应性物质。使用转染相同早老素缺失突变的细胞证实了这种异常。
