A Screen for Enhancers of Clearance Identifies Huntingtin as a Heat Shock Protein 90 (Hsp90) Client Protein
A Screen for Enhancers of Clearance Identifies Huntingtin as a Heat Shock Protein 90 (Hsp90) Client Protein
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DOI：
10.1074/jbc.m111.294801
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发表时间：
2012-01-06
影响因子：
4.8
通讯作者：
Kaupmann, Klemens
中科院分区：
文献类型：
--
作者：
Baldo, Barbara;Weiss, Andreas;Kaupmann, Klemens
降低突变亨廷顿蛋白 (mHtt) 细胞水平的机制为治疗亨廷顿病 (HD) 提供了有前景的策略。为了鉴定增强 mHtt 降解的化合物，我们使用表达 573 个氨基酸 mHtt 片段的海马 HN10 细胞系进行了高通量筛选。几个命中结构被鉴定为热休克蛋白 90 (Hsp90) 抑制剂。通过时间分辨福斯特共振能量转移测定和蛋白质印迹测量，用这些化合物处理细胞可降低 mHtt 水平，但没有明显的毒性作用。为了表征 mHtt 降解的机制，我们使用了有效的选择性 Hsp90 抑制剂 NVP-AUY922。在 HdhQ150 胚胎干 (ES) 细胞和 ES 细胞衍生的神经元中，NVP-AUY922 处理显着降低了可溶性全长 mHtt 水平。在 HN10 细胞中，在没有任何可检测到的 Hsp70 诱导的情况下，NVP-AUY922 抑制 Hsp90 增强了 mHtt 清除率。此外，用放线菌酮抑制蛋白质合成或在 HdhQ150 ES 细胞中过度表达显性负性热休克因子 1 (Hsf1) 可减弱 Hsp70 诱导，但不影响 NVP-AUY922 介导的 mHtt 清除。总之，这些数据提供的证据表明，直接抑制 Hsp90 伴侣功能对于 mHtt 降解至关重要，而不是诱导热休克反应和 Hsp70 上调。免疫共沉淀实验揭示了突变型和野生型 Htt 与 Hsp90 伴侣的物理相互作用。 Hsp90 抑制破坏了 Htt 通过泛素-蛋白酶体系统的相互作用并诱导清除。我们的数据表明，Htt 是一种 Hsp90 客户蛋白，并且 Hsp90 抑制可能提供一种减少 HD 中 mHtt 的方法。
Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Forster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1(Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.