Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones.
Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones.
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Nox5 稳定性和超氧化物产生受 Hsp90 和 CO 伴侣 C 端结合的调节
DOI：
10.1016/j.freeradbiomed.2015.09.019
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发表时间：
2015-12
影响因子：
7.4
通讯作者：
Fulton DJ
中科院分区：
文献类型：
--
作者：
Chen F;Haigh S;Yu Y;Benson T;Wang Y;Li X;Dou H;Bagi Z;Verin AD;Stepp DW;Csanyi G;Chadli A;Weintraub NL;Smith SM;Fulton DJ
热休克蛋白90（Hsp90）是一种分子伴侣，它调控着调节细胞信号传导、增殖和炎症的蛋白质的折叠和稳定性。我们先前已经表明，Hsp90通过调节Nox1 - 3和5（而非Nox4）的活性来控制活性氧物质的产生。本研究的目的是确定Nox5上与Hsp90结合的区域，并确定Hsp90如何调节酶活性。在分离的酶活性测定中，我们发现Hsp90抑制剂选择性地降低超氧化物的产生，但不降低过氧化氢的产生。单独添加Hsp90仅适度增加Nox5酶活性，但与辅助伴侣蛋白Hsp70、HOP、Hsp40和p23联合时，它强烈刺激超氧化物的产生，但不刺激过氧化氢的产生。邻近连接试验表明，Nox5和Hsp90在完整细胞中相互作用。在使用免疫共沉淀方法的细胞裂解物中，Hsp90与Nox5结合但不与Nox4结合，并且结合程度可受钙依赖性刺激的影响。抑制Hsp90会诱导Nox5的全长、无催化活性以及C末端片段（aa398 - 719）的降解。相反，抑制Hsp90不影响N末端片段（aa1 - 550）的表达水平，这表明Hsp90的结合维持了C末端区域的稳定性。在免疫共沉淀试验中，Hsp90仅与Nox5的C末端区域结合。通过缺失分析进一步细化表明，aa490 - 550之间的区域介导Hsp90的结合。反向定位实验表明，Nox5的C末端区域与Hsp90的M结构域（aa310 - 529）结合。除了Hsp90，Nox5还与折叠体的其他成分结合，包括辅助伴侣蛋白Hsp70、HOP、p23和Hsp40。沉默HOP、Hsp40和p23会降低Nox5依赖性超氧化物的产生。相反，Hsp70表达增加会降低Nox5活性，而Hsp70的一种突变体则不会。抑制Hsp90会导致Nox5的高分子量复合物丢失以及单体之间相互作用降低。总之，这些结果表明，Nox5的C末端区域与Hsp90的M结构域结合，并且Hsp90和特定辅助伴侣蛋白的结合促进寡聚化以及超氧化物的高效产生。
Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1-3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398–719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1–550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490–550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310–529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide.