Fredriksson Asa, Ballesteros Manuel, Dukan Sam, Nyström Thomas
Department of Cell and Molecular Biology, Microbiology, Medicinaregatan 9C, 413 90 Göteborg, Sweden.
J Bacteriol. 2005 Jun;187(12):4207-13. doi: 10.1128/JB.187.12.4207-4213.2005.
Protein carbonylation is an irreversible oxidative modification that increases during organism aging and bacterial growth arrest. We analyzed whether the heat shock regulon has a role in defending Escherichia coli cells against this deleterious modification upon entry into stationary phase. Providing the cell with ectopically elevated levels of the heat shock transcription factor, sigma32, effectively reduced stasis-induced carbonylation. Separate overproduction of the major chaperone systems, DnaK/DnaJ and GroEL/GroES, established that the former of these is more important in counteracting protein carbonylation. Deletion of the heat shock proteases Lon and HslVU enhanced carbonylation whereas a clpP deletion alone had no effect. However, ClpP appears to have a role in reducing protein carbonyls in cells lacking Lon and HslVU. Proteomic immunodetection of carbonylated proteins in the wild-type, lon, and hslVU strains demonstrated that the same spectrum of proteins displayed a higher load of carbonyl groups in the lon and hslVU mutants. These proteins included the beta-subunit of RNA polymerase, elongation factors Tu and G, the E1 subunit of the pyruvate dehydrogenase complex, isocitrate dehydrogenase, 6-phosphogluconate dehydrogenase, and serine hydroxymethyltranferase.
蛋白质羰基化是一种不可逆的氧化修饰，在生物体衰老和细菌生长停滞期间会增加。我们分析了热休克调节子在大肠杆菌进入稳定期时抵御这种有害修饰过程中是否发挥作用。向细胞中异位提高热休克转录因子sigma32的水平，可有效减少停滞诱导的羰基化。分别过量表达主要伴侣系统DnaK/DnaJ和GroEL/GroES，结果表明前者在对抗蛋白质羰基化方面更为重要。缺失热休克蛋白酶Lon和HslVU会增强羰基化，而单独缺失clpP则没有影响。然而，ClpP似乎在缺乏Lon和HslVU的细胞中具有减少蛋白质羰基的作用。对野生型、lon和hslVU菌株中羰基化蛋白质的蛋白质组免疫检测表明，相同的蛋白质谱在lon和hslVU突变体中显示出更高的羰基基团负载。这些蛋白质包括RNA聚合酶的β亚基、延伸因子Tu和G、丙酮酸脱氢酶复合体的E1亚基、异柠檬酸脱氢酶、6-磷酸葡萄糖酸脱氢酶和丝氨酸羟甲基转移酶。
