Selective degradation of unfolded proteins by the self-compartmentalizing HtrA protease, a periplasmic heat shock protein in Escherichia coli
Selective degradation of unfolded proteins by the self-compartmentalizing HtrA protease, a periplasmic heat shock protein in Escherichia coli
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DOI：
10.1006/jmbi.1999.3320
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发表时间：
1999-12-17
影响因子：
5.6
通讯作者：
Chung, CH
中科院分区：
文献类型：
--
作者：
Kim, KI;Park, SC;Chung, CH
HtrA具有约500 kDa的高分子量，是一种周质热休克蛋白，其蛋白水解活性对于大肠杆菌在高温下的存活是必需的。为了确定HtrA的结构组织，我们使用了电子显微镜和化学交联分析。HtrA的平均图像与端对取向揭示了一个六元，环形结构与中心腔，其侧面视图显示了两层结构。因此，HtrA表现为由两个堆叠的六聚体环组成的十二聚体。HtrA可以降解热未折叠的柠檬酸合酶和苹果酸脱氢酶，但在其天然形式时不能降解。HtrA降解部分未折叠的酪蛋白更迅速地增加孵育温度。然而，它在所有测试温度下以几乎相同的速率水解完全展开的氧化胰岛素B链。HtrA也能迅速降解用二硫苏糖醇处理胰岛素产生的还原胰岛素B链，但不能降解A链或完整胰岛素。此外，HtrA降解完全未折叠的α-乳白蛋白，其中所有四个二硫键被还原，但不是天然的α-乳白蛋白及其未折叠的中间体含有两个或三个二硫键。这些结果表明，蛋白质底物的解折叠，例如通过暴露于高温或二硫键的还原，对于它们进入双环状HtrA的内室是必不可少的，在该内室中可能发生肽键的裂解。因此，具有自区室化结构的HtrA可能在消除大肠杆菌周质中的未折叠蛋白中起重要作用。(C)北京：科学出版社.
HtrA, which has a high molecular mass of about 500 kDa, is a periplasmic heat shock protein whose proteolytic activity is essential for the survival of Escherichia coli at high temperatures. To determine the structural organization of HtrA, we have used electron microscopy and chemical cross-linking analysis. The averaged image of HtrA with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity, and its side-on view showed a two-layered structure. Thus, HtrA behaves as a dodecamer consisting of two stacks of hexameric ring. HtrA can degrade thermally unfolded citrate synthase and malate dehydrogenase but cannot when in their native form. HtrA degraded partially unfolded casein more rapidly upon increasing the incubation temperature. However, it hydrolyzed oxidized insulin B-chain, which is fully unfolded, at nearly the same rate at all of the temperatures tested. HtrA also rapidly degraded reduced insulin B-chain generated by treatment of insulin with dithiothreitol but not A-chain or intact insulin. Moreover, HtrA degraded fully unfolded alpha-lactalbumin, of which all four disulfide bonds were reduced, but not the native alpha-lactalbumin and its unfolded intermediates containing two or three disulfide bonds. These results indicate that unfolding of the protein substrates, such as by exposure to high temperatures or reduction of disulfide bonds, is essential for their access into the inner chamber of the double ring-shaped HtrA, where cleavage of peptide bonds may occur. Thus, HtrA with a self-compartmentalizing structure may play an important role in elimination of unfolded proteins in the periplasm of Escherichia coli. (C) 1999 Academic Press.