Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: a study of DNAJB6 chaperone.
Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: a study of DNAJB6 chaperone.
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DOI：
10.3389/fnmol.2015.00040
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发表时间：
2015
影响因子：
4.8
通讯作者：
Rashed LA
中科院分区：
文献类型：
--
作者：
Hussein RM;Hashem RM;Rashed LA
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β淀粉样蛋白（a β）肽的积累和聚集以及细胞内超磷酸化tau蛋白的聚集。最近的证据表明，细胞内β淀粉样蛋白肽的积累和聚集也可能在疾病发病机制中发挥作用。这表明维持细胞蛋白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，在体外和细胞中都能有效地阻止含有大聚谷氨酰胺的短聚集倾向肽的聚集（与CAG重复疾病相关）。此外，最近的体外实验表明，DNAJB6可以延缓a - β42肽的聚集。在本研究中，我们利用Aβ-绿色荧光蛋白（GFP）融合构建体转染人胚胎肾293 （HEK293）细胞，并采用western blotting和免疫荧光技术研究了DNAJB6阻止细胞外和细胞内Aβ肽聚集的能力。我们发现DNAJB6确实抑制Aβ- gfp聚集，但不抑制细胞外Aβ肽启动的种子聚集。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相互作用才能阻止Aβ-GFP融合蛋白的聚集，其j结构域对其抗聚集作用至关重要。此外，其他DNAJ蛋白和HSPA1a过表达也能有效抑制a - β- gfp聚集。我们的研究结果表明，Aβ聚集与聚谷氨酰胺（聚Q）肽诱导的聚集在伴侣处理方面存在差异，并对使用Aβ- gfp融合构建物研究细胞中Aβ肽聚集提出了质疑。
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.