Department of Histology and Embryology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People's Republic of China.
Department of Histology and Embryology, Xinxiang Medical University, Xinxiang, 453003, People's Republic of China.
Mol Neurobiol. 2018 Oct;55(10):7652-7668. doi: 10.1007/s12035-018-0900-8. Epub 2018 Feb 12.
In neurodegenerative diseases, pathogenic proteins tend to misfold and form aggregates that are difficult to remove and able to induce excessive endoplasmic reticulum (ER) stress, leading to neuronal injury and apoptosis. Homocysteine-induced endoplasmic reticulum protein (Herp), an E3 ubiquitin ligase, is an important early marker of ER stress and is involved in the ubiquitination and degradation of many neurodegenerative proteins. However, in Huntington's disease (HD), a typical polyglutamine disease, whether Herp is also involved in the metabolism and degradation of the pathogenic protein, mutant huntingtin, has not been reported. Therefore, we studied the relationship between Herp and N-terminal fragments of huntingtin (HttN-20Q and HttN-160Q). We found that Herp was able to bind to the overexpressed Htt N-terminal, and this interaction was enhanced by expansion of the polyQ fragment. Confocal microscopy demonstrated that Herp was co-localized with the HttN-160Q aggregates in the cytoplasm and tightly surrounded the aggregates. Overexpression of Herp significantly decreased the amount of soluble and insoluble HttN-160Q, promoted its ubiquitination, and inhibited its cytotoxicity. In contrast, knockdown of Herp resulted in more HttN-160Q protein, less ubiquitination, and stronger cytotoxicity. Inhibition of the autophagy-lysosomal pathway (ALP) had no effect on the function of Herp. However, blocking the ubiquitin-proteasome pathway (UPP) inhibited the reduction in soluble HttN-160Q caused by Herp. Interestingly, blocking the UPP did not weaken the ability of Herp to reduce HttN-160Q aggregates. Deletions of the N-terminal of Herp weakened its ability to inhibit HttN-160Q aggregation but did not result in a significant increase in its soluble form. However, loss of the C-terminal led to a significant increase in soluble HttN-160Q, but Herp still maintained the ability to inhibit aggregate formation. We further found that the expression level of Herp was significantly increased in HD animal and cell models. Our findings suggest that Herp is a newly identified huntingtin-interacting protein that is able to reduce the cytotoxicity of mutant huntingtin by inhibiting its aggregation and promoting its degradation. The N-terminal of Herp serves as the molecular chaperone to inhibit protein aggregation, while its C-terminal functions as an E3 ubiquitin ligase to promote the degradation of misfolded proteins through the UPP. Increased expression of Herp in HD models may be a pro-survival mechanism under stress.
在神经退行性疾病中，致病性蛋白往往会错误折叠并形成难以去除的聚集体，这些聚集体能够诱导内质网（ER）过度应激，导致神经元损伤和凋亡。内质网应激相关蛋白（Herp）是一种 E3 泛素连接酶，是 ER 应激的一个重要早期标志物，参与许多神经退行性蛋白的泛素化和降解。然而，在亨廷顿病（HD）这种典型的多聚谷氨酰胺病中，Herp 是否也参与了致病性蛋白，即突变型亨廷顿蛋白（mutant huntingtin，mHTT）的代谢和降解，尚未有报道。因此，我们研究了 Herp 与亨廷顿蛋白 N 端片段（HttN-20Q 和 HttN-160Q）之间的关系。我们发现 Herp 能够与过表达的 Htt N 端结合，且这种相互作用可被聚谷氨酰胺片段的扩增所增强。共聚焦显微镜显示，Herp 与细胞质中的 HttN-160Q 聚集体共定位，并紧密包围聚集体。过表达 Herp 可显著降低可溶性和不溶性 HttN-160Q 的含量，促进其泛素化，并抑制其细胞毒性。相比之下，敲低 Herp 会导致更多的 HttN-160Q 蛋白、更少的泛素化和更强的细胞毒性。自噬溶酶体途径（ALP）的抑制对 Herp 的功能没有影响。然而，阻断泛素-蛋白酶体途径（UPP）可抑制 Herp 引起的可溶性 HttN-160Q 减少。有趣的是，阻断 UPP 并没有削弱 Herp 降低 HttN-160Q 聚集体的能力。Herp N 端的缺失削弱了其抑制 HttN-160Q 聚集的能力，但并未导致其可溶性形式显著增加。然而，C 端的缺失会导致可溶性 HttN-160Q 显著增加，但 Herp 仍保持抑制聚集体形成的能力。我们进一步发现，在 HD 动物和细胞模型中，Herp 的表达水平显著升高。我们的研究结果表明，Herp 是一种新鉴定的与 huntingtin 相互作用的蛋白，能够通过抑制其聚集和促进其降解来降低突变型 huntingtin 的细胞毒性。Herp 的 N 端作为分子伴侣抑制蛋白聚集，而其 C 端作为 E3 泛素连接酶通过 UPP 促进错误折叠蛋白的降解。在应激条件下，HD 模型中 Herp 的高表达可能是一种生存机制。
