Protein Folding Stability Changes Across the Proteome Reveal Targets of Cu Toxicity in E. coli

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.金属离子载体（metal ionophores）诱导细胞内金属过度积累的能力赋予其强效抗菌活性，但其背后的作用靶点与分子机制尚未得到充分阐释。本研究首次将全蛋白质组蛋白质折叠稳定性测量与蛋白质表达水平分析相结合，用于鉴定铜的蛋白质靶点，从而为大肠杆菌（E. coli）中离子载体诱导的铜毒性提供全新研究视角。本研究采用一锅法脉冲蛋白水解（pulse proteolysis, PP）方案，并结合蛋白水解过程半胰蛋白酶肽富集策略（semi-tryptic peptide enrichment strategy for proteolysis procedures, STEPP），对分别暴露于铜（添加与不添加两种离子载体：抗菌剂吡啶硫酮（pyrithione）及其β-内酰胺酶激活前药PcephPT）的大肠杆菌细胞裂解液中的蛋白质生成稳定性谱图。作为本研究的组成部分，上述细胞裂解液还采用常规定量自下而上蛋白质组学方法开展蛋白质表达水平分析。本研究生成的蛋白质折叠稳定性与表达水平谱图，得以区分离子载体与铜各自介导的生物学效应，并揭示了参与代谢、翻译及细胞氧化还原稳态等过程的蛋白质发生铜诱导的稳定性改变。本分析中鉴定得到的159个差异稳定性蛋白质，其数量约为3倍，显著多于鉴定出的53个差异表达蛋白质。上述结果表明，在药物作用模式研究中，蛋白质稳定性测量可提供独特的信息维度，用于解析金属依赖的生物学过程。