Cooperative action of Hsp70, Hsp90, and DnaJ proteins in protein renaturation.
Cooperative action of Hsp70, Hsp90, and DnaJ proteins in protein renaturation.
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DOI：
10.1021/bi961825h
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发表时间：
1996-11
期刊：
影响因子：
2.9
通讯作者：
Robert J. Schumacher;W J Hansen;Brian C. Freeman;Emad S. Alnemri;Gerald Litwack;D. Toft
中科院分区：
文献类型：
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作者：
Robert J. Schumacher;W J Hansen;Brian C. Freeman;Emad S. Alnemri;Gerald Litwack;D. Toft
真核细胞质中修复受损蛋白所需的蛋白质在很大程度上仍未被表征。热变性萤火虫荧光素酶的再生容易发生在兔网状细胞裂解物通过atp依赖的过程。早期的研究表明，这种伴随活性可以部分地通过hsp70和hsp90的纯化制剂来重建。通过阐明hsp70和hsp90的重要性，我们扩展了该系统的描述，并测试了其他增强复性的因素。利用突变体hsp70蛋白，我们已经证明hsp70是荧光素酶再生所必需的。我们还发现，通过普通方法纯化的hsp70和hsp90制剂被低水平的DnaJ蛋白污染，而DnaJ蛋白是恢复活性所必需的。当使用不含DnaJ蛋白的hsp70和hsp90制剂时，必须在体系中添加DnaJ蛋白才能获得恢复活性。酵母DnaJ蛋白YDJ-1被发现在这方面非常有效。虽然只有hsp70和DnaJ蛋白可以发生明显的再生，但在网状细胞裂解物的复杂环境和纯化系统中，hsp90也有助于再生过程。然而，使用高纯度的hsp90和格尔达霉素（一种hsp90功能的特异性抑制剂），我们已经确定hsp90不是再生系统的必要组成部分。热休克蛋白90对再生的贡献仅被格尔达霉素部分阻断，这表明该蛋白可能以不止一种方式影响活性。这项研究表明，hsp70、hsp90和DnaJ蛋白协同作用，使真核细胞质中受损的蛋白再生，并提供了一个框架，通过该框架可以识别额外的成分，并可以研究个体伴侣蛋白的贡献。
The proteins required for the repair of damaged proteins in the eukaryotic cytoplasm remain largely uncharacterized. The renaturation of thermally denatured firefly luciferase readily occurs in rabbit reticulocyte lysate by an ATP-dependent process. Earlier studies had shown that this chaperoning activity could be reconstituted, in part, using purified preparations of hsp70 and hsp90. We have extended the description of this system by clarifying the importance of hsp70 and hsp90 and have tested for additional factors that enhance renaturation. Using mutant hsp70 proteins, we have shown that hsp70 is required for luciferase renaturation. We have also found that hsp70 and hsp90 preparations purified by common procedures were contaminated with low levels of DnaJ proteins that are essential for the renaturing activity. When hsp70 and hsp90 preparations free of DnaJ proteins are used, the system must be supplemented with a DnaJ protein to obtain renaturation activity. The yeast DnaJ protein, YDJ-1, was found to be very effective for this purpose. Although significant renaturation can occur with only hsp70 and DnaJ proteins, hsp90 also contributes to the renaturation process, both in the complex environment of reticulocyte lysate and in a purified system. However, using highly purified hsp90 and geldanamycin, a specific inhibitor of hsp90 function, we have determined that hsp90 is not an essential component of the renaturation system. The contribution of hsp90 to renaturation is only partially blocked by geldanamycin, suggesting that this protein may influence activity in more than one way. This study indicates that hsp70, hsp90, and DnaJ proteins function cooperatively to renature damaged proteins in the eukaryotic cytoplasm and provides a framework by which additional components can be identified and individual chaperone contributions can be investigated.