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

ABSTRACT: 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.

SUBMITTER: Wiebelhaus N

PROVIDER: S-EPMC7984275 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Phytoremediation soil polluted by cadmium has drawn worldwide attention. However, how to improve the efficiency of plant remediation of cadmium contaminated soil remains unknown. Previous studies showed that nitrogen (N) significantly enhances cadmium uptake and accumulation in poplar plants. In order to explore the important role of nitrogen in plants' responses to cadmium stress, this study investigates the poplar proteome and phosphoproteome difference between Cd stress and Cd + N treatment. In total, 6573 proteins were identified, and 5838 of them were quantified. With a fold-change threshold of > 1.3, and a p-value < 0.05, 375 and 108 proteins were up- and down-regulated by Cd stress when compared to the control, respectively. Compared to the Cd stress group, 42 and 89 proteins were up- and down-regulated by Cd + N treatment, respectively. Moreover, 522 and 127 proteins were up- and down-regulated by Cd + N treatment compared to the CK group. In addition, 1471 phosphosites in 721 proteins were identified. Based on a fold-change threshold of > 1.2, and a p-value < 0.05, the Cd stress up-regulated eight proteins containing eight phosphosites, and down-regulated 58 proteins containing 69 phosphosites, whereas N + Cd treatment up-regulated 86 proteins containing 95 phosphosites, and down-regulated 17 proteins containing 17 phosphosites, when compared to Cd stress alone. N + Cd treatment up-regulated 60 proteins containing 74 phosphosites and down-regulated 37 proteins containing 42 phosphosites, when compared to the control. Several putative responses to stress proteins, as well as transcriptional and translational regulation factors, were up-regulated by the addition of exogenous nitrogen following Cd stress. Especially, heat shock protein 70 (HSP70), 14-3-3 protein, peroxidase (POD), zinc finger protein (ZFP), ABC transporter protein, eukaryotic translation initiation factor (elF) and splicing factor 3 B subunit 1-like (SF3BI) were up-regulated by Cd + N treatment at both the proteome and the phosphoproteome levels. Combing the proteomic data and phosphoproteomics data, the mechanism by which exogenous nitrogen can alleviate cadmium toxicity in poplar plants was explained at the molecular level. The results of this study will establish the solid molecular foundation of the phytoremediation method to improve cadmium-contaminated soil.

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

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