Methylation of atypical protein aspartyl residues during the stress response of HeLa cells.
Methylation of atypical protein aspartyl residues during the stress response of HeLa cells.
复制标题
HeLa 细胞应激反应过程中非典型蛋白天冬氨酰残基的甲基化。
DOI：
10.1002/jcp.1041530209
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发表时间：
1992
影响因子：
5.6
通讯作者：
O'Connor,CM
中科院分区：
文献类型：
--
作者：
Ladino,CA;O'Connor,CM
一种蛋白羧基甲基转移酶（PCMT），特异性修饰非典型蛋白L-异天冬氨酰和D-天冬氨酰残基，广泛分布于真核细胞中，但在体内调节其活性的因素尚未确定。有人提出 PCMT 启动结构受损蛋白质的修复。为了测试结构异常细胞蛋白的浓度影响 PCMT 活性的可能性，在暴露于各种压力的 HeLa 细胞中研究了蛋白质羧甲基化反应，这些压力增加了细胞中蛋白质解折叠的程度。在 42°C 孵育期间，蛋白质羧甲基化率增加 70-80%，并在长达 8 小时的时间内保持较高水平。这种持续的增加大于单独对酶的热效应所预测的增加，并且可能反映了蛋白质展开时非典型天冬氨酰位点的暴露以及蛋白质在高温下脱酰胺和异构化速率的增加。与氨基酸类似物 L-氮杂环丁烷-2-羧酸或 L-刀豆氨酸孵育 12 小时后，甲基化率没有增加。对对照细胞和应激细胞的 RNA 制剂进行 Northern 印迹分析，揭示了 HeLa 细胞中 PCMT 的三个主要转录本，长度分别为 1.6、2.6 和 4.5 kb。热休克期间，所有三种转录本的浓度均较对照水平下降约 20%。在与氨基酸类似物一起孵育期间没有观察到 PCMT 转录物浓度的变化。相比之下，在热应激或化学应激后，观察到 hsp70 和泛素转录本的浓度大幅增加。结果表明，PCMT 是细胞的组成部分，其功能在正常条件下以及应激条件下都是必需的，这会加速细胞蛋白质的结构损伤。 © 1992 Wiley-Liss, Inc.
A protein carboxyl methyltransferase (PCMT), which specifically modifies atypical protein L‐isoaspartyl and D‐aspartyl residues, is widely distributed in eucaryotic cells, but the factors that regulate its activity in vivo have not been identified. It has been proposed that the PCMT initiates the repair of structurally damaged proteins. To test the possibility that the concentration of structurally abnormal cellular proteins affects PCMT activity, protein carboxyl methylation reactions were studied in HeLa cells exposed to various stresses that increase the extent of protein unfolding in cells. Protein carboxyl methylation rates increased 70–80% during incubations at 42°C and remained elevated for periods of up to 8 hr. This sustained increase was greater than that predicted from thermal effects on the enzyme alone and may relect the exposure of atypical aspartyl sites as proteins unfold as well as increased rates of protein deamidation and isomerization at elevated temperatures. Methylation rates showed no increases following 12 hr incubations with the amino acid analogs L‐azetidine‐2‐carboxylic acid or L‐canavanine. Northern blot analysis of RNA preparations from control and stressed cells revealed three major transcripts for the PCMT in HeLa cells, which are 1.6, 2.6, and 4.5 kb in length. The concentrations of all three transcripts decreased by ∼ 20% from control levels during heat shock. No changes in PCMT transcript concentrations were observed during incubation with the amino acid analogs. By contrast, large increases in the concentrations of hsp70 and ubiquitin trascripts were observed following either heat or chemical stresses. The results demonstrate that the PCMT is a constitutive component of cells whose function is required under normal conditions as well as during stress conditions, which accelerate structural damage to cellular proteins. © 1992 Wiley‐Liss, Inc.