Analysis of the regulation mechanism of β-secretase function

β-分泌酶（β-Secretase，BACE 1）是一种膜结合型β-淀粉基蛋白酶，主要参与β-淀粉样蛋白（Aβ）的生成，A β在阿尔茨海默病患者的大脑中积累。BACE 1的抑制被认为是这种疾病的潜在有效治疗方法。我们进行了蛋白质组学分析，以寻找BACE 1相互作用的蛋白质，使用人类神经母细胞瘤SH-SY 5 Y细胞表达BACE 1与C-末端标签。我们的方法结合了甘油密度梯度分离、免疫沉淀和LC/MS/MS分析。我们鉴定了Nogo-B（reticulon 4 B; RTN 4 B）作为BACE 1的结合蛋白。使用转染的HEK 293细胞的免疫共沉淀实验证实了BACE 1和Nogo-B或其同源物RTN 3之间的物理关联。在表达瑞典突变淀粉样前体蛋白（APP）的HEK 293细胞中，Nogo-B或RTN 3的过表达使Aβ40和Aβ42的分泌减少30-40%。然而，这些RTN蛋白并不影响表达APP C末端片段的细胞中的Aβ分泌。这些数据表明，Nogo-B和RTN 3可以负调节APP的BACE 1切割。已知BACE 1的胞外结构域被部分切割以产生可溶性BACE 1。使用免疫沉淀-蛋白质印迹分析，我们表明，SH-SY 5 Y细胞过度表达BACE 1释放细胞外低，但显着量的BACE 1全蛋白沿着可溶性BACE 1。我们的数据进一步表明全长BACE 1的释放通过抑制BACE 1脱落而增强，并且与可溶性BACE 1释放平行发生。这些数据表明，全长BACE 1的细胞外释放可能是一个生理上重要的过程。

β-Secretase, BACE1 is a membrane-bound aspartyl protease critically involved in generation of amyloid β-protein(Aβ), which accumulates in the brain of Alzheimer's disease patients. Inhibition of BACE1 is considered to be a potentially effective therapeutic approach for this disease. We performed proteomic analyses to search for BACE1-interacting proteins using human neuroblastoma SH-SY5Y cells expressing BACE1 with a C-terminal tag. Our method combined glycerol density gradient fractionation, immunoprecipitaion and LC/MS/MS analysis. We identified Nogo-B (reticulon4B ; RTN4B) as a binding protein of BACE1. Co-immunoprecipitaion experiments using transfected HEK293 cells confirmed the physical association between BACE1 and Nogo-B or its homologue, RTN3. Overexpression of Nogo-B or RTN3 reduced secretion of both Aβ40 and Aβ42 by 30-40% in HEK293 cells expressing Swedish mutant amyloid precursor protein(APP). However, these RTN proteins did not influence Aβ secretion in cells expressing APP C-terminal fragment. These data, indicate that Nogo-B and RTN3 can negatively modulate BACE1 cleavage of APP.The extracellular domain of BACE1 is known to be cleaved partially to generate soluble BACE1. Using immunoprecipitation-Western blot analysis we showed that SH-SY5Y cells overexpressing BACE1 released extracellularly a low but significant amount of BACE1 holoprotein along with soluble BACE1. Our data further indicated that the release of full-length BACE1 was enhanced by inhibition of BACE1 shedding and occurred in parallel with soluble BACE1 release. These data suggest that the extracellular release of full-length BACE1 may be a physiologically significant process.