Div. of Radiation Life Science, Dept. of Radiation Life Science and Radiation Medical Science, Research Reactor Institute, Kyoto University, Sennan-gun, Osaka 590-0494, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Nov 1;879(29):3349-52. doi: 10.1016/j.jchromb.2011.08.031. Epub 2011 Aug 30.
The formation and accumulation of D-aspartate residue (D-Asp) in proteins caused by oxidative stress leads to dysfunction and/or denaturation of proteins, and is consequently responsible for aging-related misfolding diseases such as cataracts, prion disease, and Alzheimer's disease. We sought to identify that an unknown protease selectively degrades the noxious D-Asp-containing protein, namely D-aspartyl endopeptidase (DAEP), and finally purified it from the inner mitochondrial membrane of mouse liver. In order to analyze the substrate stereoselectivity of DAEP, we synthesized a peptide corresponding to 55-65 (Thr-Val-Leu-Asp-Ser-Gly-Ile-Ser-Glu-Val-Arg) of human αA-crystallin and its corresponding diastereoisomers in which L-α-Asp was replaced with L-β-, D-α- or D-β-Asp residue at position 58. Following incubation of that peptide with purified DAEP, it was only degraded at D-α-Asp(58), independent of ATP or NAD. This result indicates that DAEP stereoselectively recognizes and degrades its substrate at the internal D-α-Asp residue. DAEP therefore seems to physiologically serve as the quality control system against the noxious D-Asp-containing protein in the long life span of mammals.
氧化应激导致蛋白质中 D-天冬氨酸残基（D-Asp）的形成和积累，导致蛋白质功能障碍和/或变性，从而引发与衰老相关的错误折叠疾病，如白内障、朊病毒病和阿尔茨海默病。我们试图确定一种未知的蛋白酶是否能选择性地降解含有有害 D-Asp 的蛋白质，即 D-天冬氨酰内肽酶（DAEP），并最终从鼠肝的线粒体内膜中纯化出来。为了分析 DAEP 的底物立体选择性，我们合成了一个对应于人αA-晶体蛋白 55-65 位（Thr-Val-Leu-Asp-Ser-Gly-Ile-Ser-Glu-Val-Arg）的肽及其相应的非对映异构体，其中 58 位的 L-α-Asp 被 L-β-、D-α-或 D-β-Asp 取代。将该肽与纯化的 DAEP 孵育后，只有 D-α-Asp（58）被降解，这一过程独立于 ATP 或 NAD。这一结果表明，DAEP 能选择性地识别和降解其内部 D-α-Asp 残基的底物。因此，DAEP 似乎在哺乳动物的长寿命中充当了一种针对含有有害 D-Asp 的蛋白质的质量控制系统。
