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Abstract：
Alzheimer\'s disease (AD) is a progressive neurodegenerative disease characterized by the accumulation and aggregation of extracellular amyloid β (Aβ) peptides and intracellular aggregation of hyper-phosphorylated tau protein. Recent evidence indicates that accumulation and aggregation of intracellular amyloid β peptides may also play a role in disease pathogenesis. This would suggest that intracellular Heat Shock Proteins (HSP) that maintain cellular protein homeostasis might be candidates for disease amelioration. We recently found that DNAJB6, a member of DNAJ family of heat shock proteins, effectively prevented the aggregation of short aggregation-prone peptides containing large poly glutamines (associated with CAG repeat diseases) both in vitro and in cells. Moreover, recent in vitro data showed that DNAJB6 can delay the aggregation of Aβ42 peptides. In this study, we investigated the ability of DNAJB6 to prevent the aggregation of extracellular and intracellular Aβ peptides using transfection of human embryonic kidney 293 (HEK293) cells with Aβ-green fluorescent protein (GFP) fusion construct and performing western blotting and immunofluorescence techniques. We found that DNAJB6 indeed suppresses Aβ-GFP aggregation, but not seeded aggregation initiated by extracellular Aβ peptides. Unexpectedly and unlike what we found for peptide-mediated aggregation, DNAJB6 required interaction with HSP70 to prevent the aggregation of the Aβ-GFP fusion protein and its J-domain was crucial for its anti-aggregation effect. In addition, other DNAJ proteins as well as HSPA1a overexpression also suppressed Aβ-GFP aggregation efficiently. Our findings suggest that Aβ aggregation differs from poly glutamine (Poly Q) peptide induced aggregation in terms of chaperone handling and sheds doubt on the usage of Aβ-GFP fusion construct for studying Aβ peptide aggregation in cells.
摘要：
阿尔茨海默病（AD）是一种进行性神经退行性疾病，其特征是细胞外淀粉样β（Aβ）肽的积累和聚集以及过度磷酸化tau蛋白的细胞内聚集。最近的证据表明，细胞内淀粉样β肽的积累和聚集也可能在疾病的发病机理中起作用。这表明维持细胞蛋白质稳态的细胞内热休克蛋白（HSP）可能是疾病改善的候选者。我们最近发现DNAJB6是热休克蛋白DNAJ家族的一员,在体外和细胞中有效地防止了含有大的聚谷氨酰胺（与CAG重复疾病相关）的短的易聚集肽的聚集。此外，最近的体外数据表明DNAJB6可以延缓Aβ42肽的聚集。在这项研究中,我们使用Aβ-绿色荧光蛋白（GFP）融合构建体转染人胚肾293（HEK293）细胞并进行蛋白质印迹和免疫荧光技术，研究了DNAJB6防止细胞外和细胞内Aβ肽聚集的能力。我们发现DNAJB6确实抑制了Aβ-GFP的聚集，但不是由胞外Aβ肽引发的种子聚集。出乎意料的是，与我们发现的肽介导的聚集不同，DNAJB6需要与HSP70相互作用以防止Aβ-GFP融合蛋白的聚集，其J结构域对于其抗聚集作用至关重要。此外,其他DNAJ蛋白以及HSPA1a过表达也有效抑制Aβ-GFP聚集。我们的发现表明，Aβ聚集在伴侣处理方面与聚谷氨酰胺（PolyQ）肽诱导的聚集不同，并且对使用Aβ-GFP融合构建体来研究细胞中的Aβ肽聚集表示怀疑。
