Clusterin Binds to A1-42 Oligomers with High Affinity and Interferes with Peptide Aggregation by Inhibiting Primary and Secondary Nucleation
Clusterin Binds to A1-42 Oligomers with High Affinity and Interferes with Peptide Aggregation by Inhibiting Primary and Secondary Nucleation
复制标题
DOI：
10.1074/jbc.m115.689539
复制
发表时间：
2016-03-25
影响因子：
4.8
通讯作者：
Gobbi, Marco
中科院分区：
文献类型：
--
作者：
Beeg, Marten;Stravalaci, Matteo;Gobbi, Marco
淀粉样蛋白(A)的聚集是导致阿尔茨海默病中存在的神经元损伤的基本致病机制，可溶性A低聚物被认为是主要的毒性罪魁祸首。因此，更好的知识和特定的靶向途径，导致这些有害的物种可能导致有价值的治疗策略。我们描述了分子伴侣簇蛋白的一些作用，为其潜在的神经保护作用提供了新的和更详细的证据。使用经典的硫黄素T测定，我们观察到对聚集过程的剂量依赖性抑制。不同条件下的整体时间过程分析表明，聚簇蛋白对伸长率没有影响，但主要干扰成核过程（包括初级和次级），减少可用于纤维进一步生长的细胞核数量。然后，使用最近开发的基于表面等离子体共振的免疫分析，我们获得了集群蛋白与生物相关的a（1-42）低聚物高亲和力（K-D = 1 nm）相互作用的直接证据，这些相互作用被选择性地捕获在传感器芯片上。此外，通过同样的技术，我们观察到亚化学计量浓度的聚簇蛋白阻止了低聚物与抗体4G8的相互作用，这表明伴侣蛋白屏蔽了暴露在低聚物组装上的疏水残基。最后，我们发现，在最近建立的秀丽隐杆线虫体内模型中，用聚簇蛋白预孵育可以拮抗A（1-42）寡聚物的毒性作用。这些数据证实了簇蛋白与暴露在细胞核/ A寡聚物上的生物活性区域的相互作用（1-42），为伴侣蛋白的神经保护作用提供了分子基础。
The aggregation of amyloid protein (A) is a fundamental pathogenic mechanism leading to the neuronal damage present in Alzheimer disease, and soluble A oligomers are thought to be a major toxic culprit. Thus, better knowledge and specific targeting of the pathways that lead to these noxious species may result in valuable therapeutic strategies. We characterized some effects of the molecular chaperone clusterin, providing new and more detailed evidence of its potential neuroprotective effects. Using a classical thioflavin T assay, we observed a dose-dependent inhibition of the aggregation process. The global analysis of time courses under different conditions demonstrated that clusterin has no effect on the elongation rate but mainly interferes with the nucleation processes (both primary and secondary), reducing the number of nuclei available for further fibril growth. Then, using a recently developed immunoassay based on surface plasmon resonance, we obtained direct evidence of a high-affinity (K-D = 1 nm) interaction of clusterin with biologically relevant A(1-42) oligomers, selectively captured on the sensor chip. Moreover, with the same technology, we observed that substoichiometric concentrations of clusterin prevent oligomer interaction with the antibody 4G8, suggesting that the chaperone shields hydrophobic residues exposed on the oligomeric assemblies. Finally, we found that preincubation with clusterin antagonizes the toxic effects of A(1-42) oligomers, as evaluated in a recently developed in vivo model in Caenorhabditis elegans. These data substantiate the interaction of clusterin with biologically active regions exposed on nuclei/oligomers of A(1-42), providing a molecular basis for the neuroprotective effects of the chaperone.