Department of Chemistry, Duke University, 124 Science Drive, Durham, North Carolina 27708, United States.
ACS Chem Biol. 2021 Jan 15;16(1):214-224. doi: 10.1021/acschembio.0c00900. Epub 2020 Dec 11.
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 . The protein folding stability analysis employed a one-pot protocol for ulse roteolysis (PP) in combination with a emi-ryptic peptide nrichment strategy for roteolysis rocedures (STEPP) to generate stability profiles for proteins in cell lysates derived from 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.
金属载体能够诱导细胞内金属超积累，从而赋予其强大的抗菌活性；然而，这些结果背后的靶点和机制尚不清楚。本研究首次利用蛋白质折叠稳定性的全蛋白质组测量与蛋白质表达水平分析相结合的方法来鉴定铜的蛋白质靶点，从而为离子载体诱导的铜毒性提供了新的见解。蛋白质折叠稳定性分析采用了一种一锅法脉冲酶解（PP）与半隐蔽肽富集策略相结合的酶解程序（STEPP），用于生成来自铜暴露的细胞裂解物中蛋白质的稳定性谱，其中铜与两种离子载体（抗菌剂吡硫翁及其β-内酰胺酶激活前药 PcephPT）一起使用。作为这项工作的一部分，上述细胞裂解物也使用传统的定量底部向上蛋白质组学方法进行蛋白质表达水平分析。这里生成的蛋白质折叠稳定性和表达水平谱使我们能够区分离子载体与铜的作用，并揭示了参与代谢、翻译和细胞氧化还原稳态过程的蛋白质在铜驱动下的稳定性变化。在这项分析中鉴定出的 159 种差异稳定化蛋白质的数量明显多于具有差异表达水平的 53 种蛋白质（约 3 倍）。这些结果说明了蛋白质稳定性测量可以为药物作用模式研究中的金属依赖性过程提供独特的信息。
