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Protein Folding Stability Changes Across the Proteome Reveal Targets of Cu Toxicity in E. coli
ACS Chemical Biology ( IF 4 ) Pub Date : 2020-12-11 , DOI: 10.1021/acschembio.0c00900
Nancy Wiebelhaus 1 , Jacqueline M Zaengle-Barone 1 , Kevin K Hwang 1 , Katherine J Franz 1 , Michael C Fitzgerald 1
Affiliation  

The ability of metal ionophores to induce cellular metal hyperaccumulation endows them with potent antimicrobial activity; however, the targets and mechanisms behind these outcomes are not well understood. This work describes the first utilization of proteome-wide measurements of protein folding stability in combination with protein expression level analysis to identify protein targets of copper, thereby providing new insight into ionophore-induced copper toxicity in E. coli. The protein folding stability analysis employed a one-pot protocol for pulse proteolysis (PP) in combination with a semi-tryptic peptide enrichment strategy for proteolysis procedures (STEPP) to generate stability profiles for proteins in cell lysates derived from E. coli exposed to copper with and without two ionophores, the antimicrobial agent pyrithione and its β-lactamase-activated prodrug, PcephPT. As part of this work, the above cell lysates were also subject to protein expression level analysis using conventional quantitative bottom-up proteomic methods. The protein folding stability and expression level profiles generated here enabled the effects of ionophore vs copper to be distinguished and revealed copper-driven stability changes in proteins involved in processes spanning metabolism, translation, and cell redox homeostasis. The 159 differentially stabilized proteins identified in this analysis were significantly more numerous (∼3×) than the 53 proteins identified with differential expression levels. These results illustrate the unique information that protein stability measurements can provide to decipher metal-dependent processes in drug mode of action studies.

中文翻译:

蛋白质组中蛋白质折叠稳定性的变化揭示了大肠杆菌中铜毒性的靶点

金属离子载体诱导细胞金属超积累的能力赋予它们强大的抗菌活性;然而,这些成果背后的目标和机制尚不清楚。这项工作描述了首次利用蛋白质组范围内的蛋白质折叠稳定性测量结合蛋白质表达水平分析来识别铜的蛋白质靶标,从而为大肠杆菌中离子载体诱导的铜毒性提供新的见解。蛋白质折叠稳定性分析采用一锅法进行脉冲蛋白水解( PP) 结合半胱氨酸富集策略进行蛋白水解用于生成来自大肠杆菌的细胞裂解物中蛋白质的稳定性谱的程序 ( STEPP )暴露于带有和不带有两种离子载体的铜,即抗菌剂吡啶硫酮及其 β-内酰胺酶激活的前药 PcephPT。作为这项工作的一部分,上述细胞裂解物还使用传统的定量自下而上蛋白质组学方法进行蛋白质表达水平分析。此处生成的蛋白质折叠稳定性和表达水平谱能够区分离子载体与铜的影响,并揭示了铜驱动的蛋白质在跨越代谢、翻译和细胞氧化还原稳态的过程中的稳定性变化。在该分析中鉴定的 159 种差异稳定的蛋白质比鉴定的具有差异表达水平的 53 种蛋白质显着更多(~3×)。
更新日期:2021-01-15
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