Analysis of the regulation mechanism of β-secretase function

淀粉样分泌酶，BACE1，是一种膜结合的天冬氨酸蛋白酶，主要参与淀粉样蛋白(ββ-Protein，Aβ)的生成，在阿尔茨海默病患者的大脑中积聚。抑制BACE1被认为是治疗这种疾病的一种潜在有效的方法。我们使用人神经母细胞瘤SH-SY5Y细胞进行蛋白质组学分析，以寻找与BACE1相互作用的蛋白。我们的方法结合了甘油密度梯度分级、免疫沉淀和LC/MS/MS分析。我们鉴定Nogo-B(reeton4B；RTN4B)是BACE1的结合蛋白。用转染的HEK293细胞进行的免疫共沉淀实验证实了BACE1与Nogo-B或其同系物RTN3之间的物理结合。在表达瑞典突变型淀粉样前体蛋白(APP)的HEK293细胞中，过表达Nogo-B或Rtn3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末端片段的细胞的Aβ分泌。这些数据表明，Nogo-B和RTN3可以负向调节APP的BACE1裂解。已知BACE1的胞外区被部分裂解产生可溶性BACE1。免疫沉淀-Western印迹分析表明，高表达BACE1的SH-SY5Y细胞胞外释放少量但明显的BACE1全蛋白和可溶性BACE1。我们的数据进一步表明，全长BACE1的释放通过抑制BACE1的脱落而增强，并且与可溶性BACE1的释放平行发生。这些数据表明，全长BACE1的胞外释放可能是一个重要的生理过程。

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.