Stable Isotope Labeling Reveals Novel Insights Into Ubiquitin-Mediated Protein Aggregation With Age, Calorie Restriction, and Rapamycin Treatment
Stable Isotope Labeling Reveals Novel Insights Into Ubiquitin-Mediated Protein Aggregation With Age, Calorie Restriction, and Rapamycin Treatment
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DOI：
10.1093/gerona/glx047
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发表时间：
2018-05-01
影响因子：
5.1
通讯作者：
Rabinovitch, Peter S.
中科院分区：
文献类型：
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作者：
Basisty, Nathan B.;Liu, Yuxin;Rabinovitch, Peter S.
150 多年前，人们首次在衰老的人体组织中发现了随着年龄增长而积累的蛋白质聚集现象，此后几乎在每个人体组织中都出现了这种现象。泛素修饰是不溶性蛋白质聚集体的典型标志；然而，大多数与年龄相关的内含物的成分仍然相对未知。为了检查体内年龄相关蛋白聚集的情况，我们对热量限制 (CR)、补充雷帕霉素 (RP) 和对照饮食的年轻和年老小鼠进行了基于抗体的泛素化蛋白下拉，并结合代谢标记和质谱分析。我们发现，老年小鼠中许多泛素化蛋白的丰度有所增加，并且相对于未修饰的对应物，预先存在的（未标记的）泛素化蛋白的保留率更高，符合预期的长寿聚集蛋白随着年龄增长而积累的情况。 CR 和 RP 都深刻影响泛素组的组成、半衰期和蛋白质的不溶性，这与它们动员这些与年龄相关的积累的能力一致。最后，共聚焦显微镜证实了两种预测最高的聚集蛋白（角蛋白 8/18 和过氧化氢酶）的聚集，以及 CR 和 RP 对其的减弱。稳定同位素标记是一种强大的工具，可以获得对蛋白质稳态机制（包括蛋白质聚集）的新见解，并且可用于识别衰老和蛋白质聚集疾病的新治疗靶点。
Accumulation of protein aggregates with age was first described in aged human tissue over 150 years ago and has since been described in virtually every human tissue. Ubiquitin modifications are a canonical marker of insoluble protein aggregates; however, the composition of most age-related inclusions remains relatively unknown. To examine the landscape of age-related protein aggregation in vivo, we performed an antibody-based pulldown of ubiquitinated proteins coupled with metabolic labeling and mass spectrometry on young and old mice on calorie restriction (CR), rapamycin (RP)-supplemented, and control diets. We show increased abundance of many ubiquitinated proteins in old mice and greater retention of preexisting (unlabeled) ubiquitinated proteins relative to their unmodified counterparts-fitting the expected profile of age-increased accumulation of long-lived aggregating proteins. Both CR and RP profoundly affected ubiquitinome composition, half-live, and the insolubility of proteins, consistent with their ability to mobilize these age-associated accumulations. Finally, confocal microscopy confirmed the aggregation of two of the top predicted aggregating proteins, keratins 8/18 and catalase, as well as their attenuation by CR and RP. Stable-isotope labeling is a powerful tool to gain novel insights into proteostasis mechanisms, including protein aggregation, and could be used to identify novel therapeutic targets in aging and protein aggregation diseases.