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Protein stability changes reveal targets of pyrithione-induced Cu toxicity in E. coli

ABSTRACT: The biological activities of metals and ionophores have brought them to the forefront as potent antimicrobial and anticancer agents. However, the biological mechanisms behind these activities are not well understood. This work describes the first utilization of proteome-wide measurements of protein folding stability in combination with protein expression level analyses to identify the protein targets of metals and provides insight into ionophore-induced Cu toxicity in E. coli. Stability and expression level profiles were generated on the proteins in cell lysates derived from E. coli cells exposed to copper in the absence and presence of two ionophores, the antimicrobial agent pyrithione and a β-lactamase-activated prodrug of pyrithione, PcephPT. The differential profiles enabled the effects of Cu to be distinguished from the effects the ionophores. The relatively large number of differentially stabilized proteins identified here were especially informative, and revealed both established and novel mechanisms of action for Cu-induced death.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Escherichia Coli

SUBMITTER: Nancy Corsi

LAB HEAD: Michael C. Fitzgerald

PROVIDER: PXD021198 | Pride | 2020-12-08

REPOSITORIES: Pride

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			Action	DRS
	ProteinExpression_Sample1_Rep1.raw	Raw
	ProteinExpression_Sample1_Rep2.raw	Raw
	ProteinExpression_Sample1_Rep3.raw	Raw
	ProteinExpression_Sample1_prot.xml	Xml
	ProteinExpression_Sample2_Rep1.raw	Raw

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Project description:The transition metal copper (Cu) is an essential element for all living organisms. In plants, Cu plays key roles in electron transport chains of chloroplasts and mitochondria, as well as in cell wall metabolism, ethylene perception, molybdenum cofactor synthesis and oxidative stress protection. Because of its physiological importance, suboptimal concentrations of Cu trigger a re-organization of metabolism to economize on Cu, and pronounced Cu deficiency causes severe growth reduction and defects in male fertility. However, when present in excess, Cu can be highly toxic. Therefore, Cu uptake, utilization and cellular concentrations are strictly regulated. A number of components of the Cu-homeostatic network of Arabidopsis have already been identified. However, the mechanisms that control responses of plant gene expression to Cu-deficiency are only partly understood. In Chlamydomonas reinhardtii, the transcription factor Crr1 is required for activating and/or repressing the expression of a number of genes in response to Cu deficiency. This protein contains a plant-specific DNA-binding domain (SBP domain), ankyrin repeats and a C-terminal cysteine-rich region with similarity to a Drosophila metallothionein (MT). In Arabidopsis, there is a family of 16 proteins with SBP domains named SPL family (SBP-like) of which a subset of proteins have been implicated in flowering time control and floral development. Among all of them, AtSPL7 is the most similar to Crr1 (27 % identity), and AtSPL1 (25 % identity), AtSPL12 (24 % identity) and AtSPL14 (23 % identity) share a common protein architecture. The goal of this work was to obtain a complete account of the response of the Arabidopsis thaliana transcriptome to Cu deficiency, as well as to determine the role of AtSPL7 in the transcriptional response to Cu deficiency. For this purpose, three independent experiments were carried out including Col-0 wild-type and spl7-2 mutant plants grown in Cu-sufficient and Cu-deficient hydroponic media, respectively. Root and shoot transcriptomes were established by RNA-Seq for quantitative comparisons. Sampling of root and shoot tissues of wild-type (WT, Col-0) and spl7-2 mutant plants (the mutant is knock-down for the transcription factor SPL7, which plays a key role in the transcriptional regulation of the copper deficiency responses) cultivated hydroponically in a modified Hoagland's solution under Cu-sufficient (control) and Cu-deficient conditions.

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Protein stability changes reveal targets of pyrithione-induced Cu toxicity in E. coli

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