Yang A J, Knauer M, Burdick D A, Glabe C
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717, USA.
J Biol Chem. 1995 Jun 16;270(24):14786-92. doi: 10.1074/jbc.270.24.14786.
We have analyzed the effect of internalized amyloid beta-protein (A beta) 1-42 aggregates on the metabolism of the amyloid precursor protein (APP) in stably transfected 293 cells. The amount of potentially amyloidogenic fragments of APP immunoprecipitated by anti-carboxyl-terminal APP and anti-A beta antibodies is dramatically enhanced by the treatment of the cells with A beta 1-42, which is resistant to degradation, but not A beta 1-28, which does not accumulate in cells. This accumulation of amyloidogenic carboxyl-terminal fragments is specific, since there is relatively little effect of A beta 1-42 on the amount of the nonamyloidogenic alpha-secretase carboxyl-terminal fragment. The amyloidogenic fragments accumulate in the same nonionic detergent-insoluble fraction of the cell that contains the internalized A beta 1-42. Western analysis indicates that a subset of the amyloidogenic fragments react with antibodies that recognize a conformation of A beta that is specifically associated with aggregated forms of A beta, suggesting that the adoption of this aggregation-related conformation may be an early event which precedes the final processing that produces A beta. Pulse-chase analysis of the [35S]Met-labeled 16-kDa amyloidogenic fragment indicates that it is relatively stable in A beta 1-42-treated cells, with a half-life of approximately 50 h. This fragment is degraded with a half-life of 30 min in control cells treated with A beta 1-28. In contrast, the turnover of the nonamyloidogenic alpha-secretase product is not significantly altered by the presence of A beta 1-42. The continuous uptake of A beta 1-42 from the medium is not required for the stimulation of amyloidogenic fragment accumulation, suggesting that the presence of intracellular A beta 1-42 aggregates establishes a new pathway for APP catabolism in cells which leads to the long term stability of the fragments. If these amyloidogenic fragments of APP ultimately give rise to A beta, then the production of A beta may be an autocatalytic, "runaway" process in cells containing A beta 1-42 nuclei. It is conceivable that the accumulation of insoluble APP and amyloidogenic fragments of APP in response to A beta 1-42 aggregates may mimic the pathophysiology of dystrophic neurites, where the accumulation of intracellular APP and APP fragments has been documented by immunohistochemistry.
我们分析了内化的淀粉样β蛋白（Aβ）1-42聚集体对稳定转染的293细胞中淀粉样前体蛋白（APP）代谢的影响。用抗羧基末端APP和抗Aβ抗体免疫沉淀的APP潜在淀粉样生成片段的量，在用抗降解的Aβ1-42处理细胞时显著增加，但用在细胞中不积累的Aβ1-28处理则不然。淀粉样生成羧基末端片段的这种积累是特异性的，因为Aβ1-42对非淀粉样生成α-分泌酶羧基末端片段的量影响相对较小。淀粉样生成片段积累在含有内化Aβ1-42的细胞相同的非离子去污剂不溶性组分中。蛋白质免疫印迹分析表明，一部分淀粉样生成片段与识别Aβ构象的抗体反应，该构象与Aβ聚集形式特异性相关，这表明采用这种与聚集相关的构象可能是产生Aβ的最终加工之前的早期事件。对[35S]甲硫氨酸标记的16 kDa淀粉样生成片段的脉冲追踪分析表明，它在Aβ1-42处理的细胞中相对稳定，半衰期约为50小时。在用Aβ1-28处理的对照细胞中，该片段以30分钟的半衰期降解。相反，非淀粉样生成α-分泌酶产物的周转不受Aβ1-42存在的显著影响。刺激淀粉样生成片段积累不需要从培养基中持续摄取Aβ1-42，这表明细胞内Aβ1-42聚集体的存在为细胞中的APP分解代谢建立了一条新途径，导致片段的长期稳定性。如果APP的这些淀粉样生成片段最终产生Aβ，那么在含有Aβ1-42核的细胞中，Aβ的产生可能是一个自催化的“失控”过程。可以想象，响应Aβ1-42聚集体，不溶性APP和APP淀粉样生成片段的积累可能模拟营养不良性神经突的病理生理学，其中细胞内APP和APP片段的积累已通过免疫组织化学记录。
