Escherichia coli HdeB is an acid stress chaperone
Escherichia coli HdeB is an acid stress chaperone
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DOI：
10.1128/jb.01522-06
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发表时间：
2007-01-01
影响因子：
3.2
通讯作者：
Richarme, Gilbert
中科院分区：
文献类型：
--
作者：
Kern, Renee;Malki, Abderrahim;Richarme, Gilbert
我们克隆、表达并纯化了hdeB基因产物，它属于hdeAB酸胁迫操纵子。我们采用离心-休克法从细菌中提取HdeB，并通过离子交换层析和羟基磷灰石层析将其纯化至均一。通过质谱分析确认其身份。HdeB在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中的分子量为10 kDa，与其预期分子量相符。我们平行纯化了酸应激分子伴侣HdeA，以比较两种分子伴侣。hdeA和hdeB突变体在酸胁迫下都显示出降低的活力，并且只有HdeA/HdeB表达质粒可以将其活力恢复至接近野生型水平，这表明这两种蛋白质都是细菌周质针对酸胁迫的最佳保护所需的。两种突变体的周质提取物在酸性pH下聚集，表明HdeA和HdeB是蛋白质溶解所需的。在pH 2时，通过添加HdeA防止周质提取物的聚集，如先前所报道的，但仅通过HdeB略微减少。然而，在pH 3时，HdeB在防止周质蛋白聚集方面比HdeA更有效。几种模型底物蛋白在酸性pH下的增溶支持以下假设：在体外，HdeA在pH 2下的蛋白增溶中起主要作用，并且两种蛋白质都参与pH 3下的蛋白增溶。与HdeA一样，HdeB在酸性pH下暴露疏水表面，这与其在酸性pH下的伴侣性质的外观一致。HdeB与HdeA一样，在中性pH下从二聚体解离成酸性pH下的单体，但其解离在pH 3下完成，而HdeA的解离在更酸性的pH下完成。我们可以得出结论，大肠杆菌具有两个酸应激分子伴侣，防止周质蛋白质聚集在酸性pH值。
We cloned, expressed, and purified the hdeB gene product, which belongs to the hdeAB acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ion-exchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The hdeA and hdeB mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that Escherichia coli possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.