Cysteine reactivity distinguishes redox sensing by the heat-inducible and constitutive forms of heat shock protein 70.
Cysteine reactivity distinguishes redox sensing by the heat-inducible and constitutive forms of heat shock protein 70.
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DOI：
10.1016/j.chembiol.2012.07.026
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发表时间：
2012-11-21
影响因子：
--
通讯作者：
Gestwicki JE
中科院分区：
文献类型：
--
作者：
Miyata Y;Rauch JN;Jinwal UK;Thompson AD;Srinivasan S;Dickey CA;Gestwicki JE
热休克蛋白 70 (Hsp70) 分子伴侣家族在应激条件下维持蛋白质稳态方面具有重要功能。几种 Hsp70 亚型，尤其是 Hsp72 (HSPA1A)，在应激反应中显着上调；然而，尚不清楚这些家庭成员是否具有专门适应这些情况的生化特性。氧化还原活性化合物亚甲蓝 (MB) 已被证明可以在体外抑制 Hsp72 的 ATP 酶活性，并在细胞和动物模型中促进 Hsp72 底物 tau 的降解。在此，我们报道 MB 不可逆地使 Hsp72 失活，但不能使几乎相同的、组成型表达的同种型热休克同源 70 (Hsc70；HSPA8) 失活。质谱分析结果表明，MB 氧化了 Hsc70 中不保守的 Cys306。分子模型表明，Cys306 的氧化使 Cys267 暴露于修饰，并且这两个事件都会导致响应 MB 的 ATP 结合丧失。与该模型一致，将 Cys267 和 Cys306 突变为丝氨酸使得 Hsp72 在体外对 MB 具有很大的抗性，并且 C306S 突变体的过度表达在细胞模型中阻止了 MB 介导的 tau 丢失。此外，将 Cys267 和 Cys306 突变为假氧化模拟物天冬氨酸，镜像 MB 处理：C267D 和 C306D 突变体在体外降低了 ATPase 活性，并且 C267/306D 双突变体的过度表达显着降低了细胞中的 tau 水平。总之，这些结果表明，Hsp72（而非 Hsc70）中特定半胱氨酸残基的氧化还原感应可能是分子伴侣对氧化应激反应的重要组成部分。
The heat shock protein 70 (Hsp70) family of molecular chaperones has important functions in maintaining proteostasis under stress conditions. Several Hsp70 isoforms, especially Hsp72 (HSPA1A), are dramatically upregulated in response to stress; however, it is unclear whether these family members have biochemical properties that are specifically adapted to these scenarios. The redox-active compound, methylene blue (MB), has been shown to inhibit the ATPase activity of Hsp72 in vitro and it promotes degradation of the Hsp72 substrate, tau, in cellular and animal models. Here, we report that MB irreversibly inactivates Hsp72 but not the nearly identical, constitutively expressed isoform, heat shock cognate 70 (Hsc70; HSPA8). Mass spectrometry results show that MB oxidizes Cys306, which is not conserved in Hsc70. Molecular models suggested that oxidation of Cys306 exposes Cys267 to modification and that both events contribute to loss of ATP binding in response to MB. Consistent with this model, mutating Cys267 and Cys306 to serine made Hsp72 largely resistant to MB in vitro and over-expression of the C306S mutant blocked MB-mediated loss of tau in a cellular model. Further, mutating Cys267 and Cys306 to the pseudo-oxidation mimic, aspartic acid, mirrored MB treatment: the C267D and C306D mutants had reduced ATPase activity in vitro and over-expression of the C267/306D double mutant significantly reduced tau levels in cells. Together, these results suggest that redox sensing by specific cysteine residues in Hsp72, but not Hsc70, may be an important component of the chaperone response to oxidative stress.
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