Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress tumor necrosis factor α by phosphorylation
Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress tumor necrosis factor α by phosphorylation
复制标题
DOI：
10.1074/jbc.274.27.18947
复制
发表时间：
1999-07-02
影响因子：
4.8
通讯作者：
Gaestel, M
中科院分区：
文献类型：
--
作者：
Rogalla, T;Ehrnsperger, M;Gaestel, M
来自人（Hsp 27）和小鼠（Hsp 25）的小热休克蛋白（sHsps）形成大的寡聚体，其可以在体外充当分子伴侣，并且当过表达时保护细胞免受热休克和氧化应激。此外，哺乳动物sHsps在响应各种细胞外应激时被MAPKAP激酶2/3在两个或三个丝氨酸残基处快速磷酸化。在这里，我们分析了sHsp磷酸化对其四级结构，伴侣功能和保护氧化应激的影响。我们发现，在体外磷酸化的重组sHsp以及分子模拟的Hsp 27磷酸化导致的寡聚体的大小显着减少。我们证明磷酸化的sHsps和三突变体Hsp 27-S15 D、S78 D、S82 D在体外作为分子伴侣抑制柠檬酸合酶的热变性和促进柠檬酸合酶的重折叠的能力显著降低。同时，分析Hsp 27及其突变体在L929和13.S.1.24细胞中过表达时赋予抗氧化应激的能力。野生型Hsp 27具有抗氧化能力，而三突变体S15 D、S78 D、S82 D不能有效地保护机体免受氧化应激。这些数据表明，sHsps的大寡聚体是必要的伴侣作用和抗氧化应激，而磷酸化下调这些活动的sHsp复合物的解离四聚体。
The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers.