Institute of Cytology of Russian Academy of Sciences, Tikhoretsky pr., 4, 194064 St Petersburg, Russia.
Hum Mol Genet. 2011 Oct 15;20(20):3953-63. doi: 10.1093/hmg/ddr314. Epub 2011 Jul 20.
The key feature of polyglutamine aggregates accumulating in the course of Huntington disease (HD) is their resistance to protein denaturants, and to date only chaperones are proved to prevent mutant protein aggregation. It was suggested that expanded polyglutamine chains (polyQ) of mutant huntingtin are cross-linked to other proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Here we clarify the roles of GAPDH and molecular chaperone Hsp70 in the formation of sodium dodecyl sulfate (SDS)-insoluble polyQ aggregates. First, the addition of pure GAPDH was found to enhance the aggregation of polyQ in a cell-free model of HD. Secondly, the immunodepletion of GAPDH dose-dependently decreased polyQ aggregation. Finally, siRNA-mediated inhibition of GAPDH protein in SK-N-SH neuroblastoma cells has also reduced the aggregation of cellular polyQ. Regulated over-expression of Hsp70 decreased the amount of GAPDH associated with SDS-insoluble polyQ aggregates. Physical association of Hsp70 and GAPDH in SK-N-SH cells was shown by reciprocal immunoprecipitation and confocal microscopy. Pure Hsp70 dose-dependently inhibited the formation of polyQ aggregates in cell-free model of HD by sequestering both GAPDH and polyQ. We demonstrated that Hsp70 binds to polyQ in adenosine triphosphate-dependent manner, which suggests that Hsp70 exerts a chaperoning activity in the course of this interaction. Binding of Hsp70 to GAPDH was nicotinamide adenine dinucleotide-dependent suggesting another type of association. Based on our findings, we conclude that Hsp70 protects cells in HD by removing/sequestering two intrinsic components of protein aggregates: the polyQ itself and GAPDH. We propose that GAPDH might be an important target for pharmacological treatment of HD and other polyglutamine expansion-related diseases.
亨廷顿病（HD）过程中积累的多聚谷氨酰胺聚集体的主要特征是其对蛋白质变性剂的抗性，迄今为止，只有伴侣蛋白被证明可以防止突变蛋白聚集。有人提出，突变 huntingtin 的扩展多聚谷氨酰胺链（polyQ）与其他蛋白质（如甘油醛-3-磷酸脱氢酶（GAPDH））交联。在这里，我们阐明了 GAPDH 和分子伴侣 Hsp70 在形成十二烷基硫酸钠（SDS）不溶性 polyQ 聚集体中的作用。首先，发现纯 GAPDH 的添加增强了 HD 细胞外模型中 polyQ 的聚集。其次，GAPDH 的免疫耗竭剂量依赖性地降低了 polyQ 聚集。最后，SK-N-SH 神经母细胞瘤细胞中 siRNA 介导的 GAPDH 蛋白抑制也减少了细胞内 polyQ 的聚集。Hsp70 的调节过表达降低了与 SDS 不溶性 polyQ 聚集体相关的 GAPDH 量。通过相互免疫沉淀和共聚焦显微镜显示了 Hsp70 和 GAPDH 在 SK-N-SH 细胞中的物理关联。纯 Hsp70 以依赖于三磷酸腺苷的方式剂量依赖性地抑制 HD 细胞外模型中 polyQ 聚集体的形成，通过隔离 GAPDH 和 polyQ 来实现。我们证明 Hsp70 以三磷酸腺苷依赖的方式与 polyQ 结合，这表明 Hsp70 在这种相互作用中发挥伴侣活性。Hsp70 与 GAPDH 的结合依赖于烟酰胺腺嘌呤二核苷酸，这表明存在另一种类型的关联。基于我们的发现，我们得出结论，Hsp70 通过去除/隔离蛋白聚集体的两个内在成分来保护 HD 中的细胞：聚集体本身和 GAPDH。我们提出 GAPDH 可能是治疗 HD 和其他多聚谷氨酰胺扩展相关疾病的重要靶标。
