Glutathionylation of the Bacterial Hsp70 Chaperone DnaK Provides a Link between Oxidative Stress and the Heat Shock Response.
Glutathionylation of the Bacterial Hsp70 Chaperone DnaK Provides a Link between Oxidative Stress and the Heat Shock Response.
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细菌 Hsp70 伴侣 DnaK 的谷胱甘肽化提供了氧化应激和热休克反应之间的联系。
DOI：
10.1074/jbc.m115.673608
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发表时间：
2016-03-25
期刊：
影响因子：
--
通讯作者：
Perrett S
中科院分区：
文献类型：
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作者：
Zhang H;Yang J;Wu S;Gong W;Chen C;Perrett S
DnaK 是主要的细菌 Hsp70，参与 DNA 复制、蛋白质折叠和应激反应。 DnaK 与 Hsp40 共伴侣 DnaJ 和核苷酸交换因子 GrpE 合作。在非应激条件下，DnaK 与热休克转录因子 σ32 结合并促进其降解。由于细胞 ATP 和硫醇修饰（例如谷胱甘肽化）的消耗，氧化应激导致 DnaK 暂时失活，直到恢复正常的细胞 ATP 水平和还原环境。然而，DnaK 谷胱甘肽化的生物学意义仍不清楚，谷胱甘肽化调节 DnaK 活性的机制也不清楚。我们研究了大肠杆菌 DnaK 进行 S-谷胱甘肽化的条件。我们在遭受氧化应激的大肠杆菌细胞裂解物中观察到 DnaK 的谷胱甘肽化。我们还使用 apo 状态的纯化 DnaK 获得了均匀的谷胱甘肽化 DnaK。我们发现 DnaK 的谷胱甘肽化可逆地改变二级结构和三级构象，导致核苷酸和肽结合能力降低。 DnaK 的分子伴侣活性通过谷胱甘肽化可逆性下调，并伴随结构变化。我们发现，当 DnaK 被谷胱甘肽化时，DnaK 与 DnaJ、GrpE 或 σ32 的相互作用变得较弱，而去谷胱甘肽化后，相互作用又恢复。这项研究证实，氧化条件下谷胱甘肽化下调 DnaK 的功能，这种下调可能促进 σ32 从与 DnaK 的相互作用中释放，从而触发热休克反应。这种机制提供了细菌中氧化应激和热休克反应之间的联系。
DnaK is the major bacterial Hsp70, participating in DNA replication, protein folding, and the stress response. DnaK cooperates with the Hsp40 co-chaperone DnaJ and the nucleotide exchange factor GrpE. Under non-stress conditions, DnaK binds to the heat shock transcription factor σ32 and facilitates its degradation. Oxidative stress results in temporary inactivation of DnaK due to depletion of cellular ATP and thiol modifications such as glutathionylation until normal cellular ATP levels and a reducing environment are restored. However, the biological significance of DnaK glutathionylation remains unknown, and the mechanisms by which glutathionylation may regulate the activity of DnaK are also unclear. We investigated the conditions under which Escherichia coli DnaK undergoes S-glutathionylation. We observed glutathionylation of DnaK in lysates of E. coli cells that had been subjected to oxidative stress. We also obtained homogeneously glutathionylated DnaK using purified DnaK in the apo state. We found that glutathionylation of DnaK reversibly changes the secondary structure and tertiary conformation, leading to reduced nucleotide and peptide binding ability. The chaperone activity of DnaK was reversibly down-regulated by glutathionylation, accompanying the structural changes. We found that interaction of DnaK with DnaJ, GrpE, or σ32 becomes weaker when DnaK is glutathionylated, and the interaction is restored upon deglutathionylation. This study confirms that glutathionylation down-regulates the functions of DnaK under oxidizing conditions, and this down-regulation may facilitate release of σ32 from its interaction with DnaK, thus triggering the heat shock response. Such a mechanism provides a link between oxidative stress and the heat shock response in bacteria.