Identification of Human Proteins That Modify Misfolding and Proteotoxicity of Pathogenic Ataxin-1

Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.

携带致病性长聚谷氨酰胺（polyglutamine, polyQ）序列的蛋白质，其自发错折叠并自组装形成不溶性蛋白质聚集体的倾向显著增强。本研究鉴定出21种可在细胞模型系统中影响polyQ诱导的共济失调蛋白1（ataxin-1）错折叠与蛋白毒性的人类蛋白质。通过对这些修饰因子的蛋白质序列进行分析，我们发现共济失调蛋白1毒性增强剂中普遍存在卷曲螺旋（coiled-coil, CC）结构域，而抑制性修饰因子中则不含此类结构域。这提示卷曲螺旋结构域可促进哺乳动物细胞内修饰因子的聚集与毒性增强效应。我们发现，经计算预测拥有N端卷曲螺旋结构域的共济失调蛋白1互作蛋白MED15，在细胞实验中可增强共济失调蛋白1的自发聚集；而缺失该结构域的截短蛋白MED15ΔCC则未表现出此类效应。重组蛋白实验验证了上述结果，并证实MED15的N端卷曲螺旋结构域（MED15CC）本身即可在体外促进共济失调蛋白1的自发聚集。此外，我们观察到携带MED15CC结构域的嵌合Pum1蛋白可在细胞模型系统中促进共济失调蛋白1的聚集；与之形成鲜明对比的是，不含卷曲螺旋结构域的野生型Pum1并不会刺激共济失调蛋白1的聚合。上述结果表明，携带卷曲螺旋结构域的蛋白质在体外与体内均为polyQ介导的蛋白质错折叠与聚集的强效增强剂。