Johnstone D B, Farr S B
Department of Cellular and Molecular Toxicology, Harvard School of Public Health, Boston, MA 02115.
EMBO J. 1991 Dec;10(12):3897-904. doi: 10.1002/j.1460-2075.1991.tb04959.x.
The dinucleotide AppppA (5',5'''-P1, P4-diadenosine tetraphosphate) is rapidly synthesized in cells exposed to heat stress or oxidative stress. Stress-induced AppppA accumulation has been observed in all cell types studied to date. In order to study the function(s) of AppppA, we created a mutation in the Escherichia coli gene that encodes the sole AppppN hydrolase (apaH). High levels of AppppA have subsequently been shown to affect many cellular processes, including expression of catabolite repressible genes and the ability to survive starvation, oxidative stress and near-UV irradiation. Nevertheless, the precise role of AppppA remains undefined. In order to better understand the mechanism by which AppppA exerts its effects, we attempted to determine which proteins bind to AppppA by synthesizing (alpha'-32P) 8-N3AppppA for use in photocrosslinking experiments with extract derived from cells with different genetic backgrounds and exposed to various stress conditions. We report here that several E. coli proteins bind AppppA, including the heat shock and oxidative stress proteins DnaK, GroEL, E89, C45 and C40. In addition, we show that apaH mutants, which have high basal levels of AppppA, are hypersensitive to killing by heat.
二核苷酸AppppA（5'，5'''-P1，P4-二腺苷四磷酸）在遭受热应激或氧化应激的细胞中会迅速合成。迄今为止，在所有研究过的细胞类型中均观察到应激诱导的AppppA积累。为了研究AppppA的功能，我们在编码唯一的AppppN水解酶（apaH）的大肠杆菌基因中制造了一个突变。随后发现高水平的AppppA会影响许多细胞过程，包括分解代谢阻遏基因的表达以及在饥饿、氧化应激和近紫外线照射下的存活能力。然而，AppppA的确切作用仍不明确。为了更好地理解AppppA发挥作用的机制，我们通过合成（α'-32P）8-N3AppppA来确定哪些蛋白质与AppppA结合，以便用于对来自不同遗传背景且暴露于各种应激条件下的细胞提取物进行光交联实验。我们在此报告，几种大肠杆菌蛋白质可结合AppppA，包括热休克蛋白和氧化应激蛋白DnaK、GroEL、E89、C45和C40。此外，我们还表明，具有高基础水平AppppA的apaH突变体对热杀伤高度敏感。
