YajL, Prokaryotic Homolog of Parkinsonism-associated Protein DJ-1, Functions as a Covalent Chaperone for Thiol Proteome
YajL, Prokaryotic Homolog of Parkinsonism-associated Protein DJ-1, Functions as a Covalent Chaperone for Thiol Proteome
复制标题
DOI：
10.1074/jbc.m111.299198
复制
发表时间：
2012-02-17
影响因子：
4.8
通讯作者：
Richarme, Gilbert
中科院分区：
文献类型：
--
作者：
Hai-Tuong Le;Gautier, Valerie;Richarme, Gilbert
YajL是与帕金森病相关蛋白DJ-1最接近的大肠杆菌同源物，DJ-1是一种多功能氧化应激反应蛋白，其生化功能尚不清楚。我们最近报道了蛋白质的聚集在一个yajL突变体中的氧化应激依赖的方式和YajL表现出分子伴侣活性。在这里，我们表明，YajL显示共价分子伴侣和弱蛋白氧化还原酶的活动，依赖于其暴露的半胱氨酸106。它催化还原的RNA酶氧化和乱序RNA酶异构化和胰岛素还原，并在氧化应激时与许多细胞蛋白质形成混合二硫化物。在非还原条件下，用抗YajL抗体对细菌提取物进行免疫印迹，检测混合二硫化物的形成。在YajL亲和柱上从细菌提取物中纯化二硫化物，通过非还原-还原SDS-PAGE分离，并通过质谱鉴定。YajL的共价底物包括核糖体蛋白、氨酰-tRNA合成酶、分子伴侣、过氧化氢酶、过氧化物酶和其他含有催化或FeS簇结合所必需的半胱氨酸的蛋白质，如甘油醛-3-磷酸脱氢酶、醛脱氢酶、乌头酸酶和呼吸链的含FeS簇亚基。此外，我们还发现DJ-1在氧化应激时也与细胞质蛋白形成混合二硫化物。这些结果揭示了氧化应激依赖的伴侣功能的YajL和确定YajL底物参与翻译，应激保护，蛋白质溶解和代谢。他们揭示了半胱氨酸106的关键作用，并表明DJ-1也起着共价伴侣的作用。这些发现与在yajL或DJ-1突变体中观察到的几种缺陷一致，包括翻译缺陷、蛋白质聚集、氧化应激敏感性和代谢缺陷。
YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently reported the aggregation of proteins in a yajL mutant in an oxidative stress-dependent manner and that YajL exhibits chaperone activity. Here, we show that YajL displays covalent chaperone and weak protein oxidoreductase activities that are dependent on its exposed cysteine 106. It catalyzes reduced RNase oxidation and scrambled RNase isomerization and insulin reduction and forms mixed disulfides with many cellular proteins upon oxidative stress. The formation of mixed disulfides was detected by immunoblotting bacterial extracts with anti-YajL antibodies under nonreducing conditions. Disulfides were purified from bacterial extracts on a YajL affinity column, separated by nonreducing-reducing SDS-PAGE, and identified by mass spectrometry. Covalent YajL substrates included ribosomal proteins, aminoacyl-tRNA synthetases, chaperones, catalases, peroxidases, and other proteins containing cysteines essential for catalysis or FeS cluster binding, such as glyceraldehyde-3-phosphate dehydrogenase, aldehyde dehydrogenase, aconitase, and FeS cluster-containing subunits of respiratory chains. In addition, we show that DJ-1 also forms mixed disulfides with cytoplasmic proteins upon oxidative stress. These results shed light on the oxidative stress-dependent chaperone function of YajL and identify YajL substrates involved in translation, stress protection, protein solubilization, and metabolism. They reveal a crucial role for cysteine 106 and suggest that DJ-1 also functions as a covalent chaperone. These findings are consistent with several defects observed in yajL or DJ-1 mutants, including translational defects, protein aggregation, oxidative stress sensitivity, and metabolic deficiencies.