Department of Biological Sciences and Geology, Queensborough Community College, City University of New York, Bayside, New York, United States of America.
Department of Natural and Applied Sciences, Cheyney University of Pennsylvania, Cheyney, Pennsylvania, United States of America.
PLoS One. 2022 Apr 29;17(4):e0258794. doi: 10.1371/journal.pone.0258794. eCollection 2022.
Aminoglycoside antibiotics interfere with the selection of cognate tRNAs during translation, resulting in the synthesis of aberrant proteins that are the ultimate cause of cell death. However, the toxic potential of aberrant proteins and how they avoid degradation by the cell's protein quality control (QC) machinery are not understood. Here we report that levels of the heat shock (HS) transcription factor σ32 increased sharply following exposure of Escherichia coli to the aminoglycoside kanamycin (Kan), suggesting that at least some of the aberrant proteins synthesized in these cells were recognized as substrates by DnaK, a molecular chaperone that regulates the HS response, the major protein QC pathway in bacteria. To further investigate aberrant protein toxic potential and interaction with cell QC factors, we studied an acutely toxic 48-residue polypeptide (ARF48) that is encoded by an alternate reading frame in a plant cDNA. As occurred in cells exposed to Kan, σ32 levels were strongly elevated following ARF48 expression, suggesting that ARF48 was recognized as a substrate by DnaK. Paradoxically, an internal 10-residue region that was tightly bound by DnaK in vitro also was required for the ARF48 toxic effect. Despite the increased levels of σ32, levels of several HS proteins were unchanged following ARF48 expression, suggesting that the HS response had been aborted. Nucleoids were condensed and cell permeability increased rapidly following ARF48 expression, together suggesting that ARF48 disrupts DNA-membrane interactions that could be required for efficient gene expression. Our results are consistent with earlier studies showing that aberrant proteins induced by aminoglycoside antibiotics disrupt cell membrane integrity. Insights into the mechanism for this effect could be gained by further study of the ARF48 model system.
氨基糖苷类抗生素在翻译过程中干扰同源 tRNA 的选择，导致异常蛋白质的合成，这是细胞死亡的最终原因。然而，异常蛋白质的毒性潜力以及它们如何避免被细胞的蛋白质质量控制（QC）机制降解尚不清楚。在这里，我们报告说，在暴露于氨基糖苷类抗生素卡那霉素（Kan）后，大肠杆菌中热休克（HS）转录因子σ32 的水平急剧上升，这表明至少部分在这些细胞中合成的异常蛋白质被分子伴侣 DnaK 识别为底物，DnaK 调节 HS 反应，是细菌中主要的蛋白质 QC 途径。为了进一步研究异常蛋白质的毒性潜力及其与细胞 QC 因子的相互作用，我们研究了一种编码植物 cDNA 中一个替代阅读框的 48 个残基的多肽（ARF48）。与 Kan 暴露的细胞一样，σ32 水平在 ARF48 表达后强烈升高，这表明 ARF48 被 DnaK 识别为底物。矛盾的是，DnaK 在体外紧密结合的一个内部 10 个残基区域也是 ARF48 毒性作用所必需的。尽管 σ32 水平增加，但 ARF48 表达后几种 HS 蛋白的水平不变，这表明 HS 反应已经中止。ARF48 表达后核体迅速凝聚，细胞通透性增加，这表明 ARF48 破坏了 DNA-膜相互作用，这可能是有效基因表达所必需的。我们的结果与早期研究一致，表明氨基糖苷类抗生素诱导的异常蛋白质破坏细胞膜完整性。通过对 ARF48 模型系统的进一步研究，可以深入了解这种效应的机制。
