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Identification of copper-binding-induced polymeric protein complexes

ABSTRACT: Cuproptosis is characterized by copper ion-induced polymerization of lipoylated proteins. Whether other proteins involved in cuprotosis also undergo polymerization in the presence of copper ions remains to be determined. In this study, we utilized three experimental approaches to identify novel copper-induced polymerized proteins associated with cuprotosis. In the first two approaches, melanoma A375 cells were treated with copper ionophores—either elesclomol (ES) or disulfiram (DSF) in combination with copper—prior to cell lysis. In the third approach, cell lysates were directly incubated with copper ions in vitro. The resulting lysates were then subjected to non-reducing gel electrophoresis to detect proteins that undergo copper-induced polymerization. We expect to identify new target proteins of copper ions and new participating and executing proteins of copper death through this method.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Kejun Xu

LAB HEAD: Kejun Xu

PROVIDER: PXD076672 | Pride | 2026-04-25

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	WLZ-DSF-H.xlsx	Xlsx
	WLZ-ES-H.xlsx	Xlsx
	WLZ_DSF_Slot1-11_1_2946.d.zip	Other
	WLZ_ES_Slot1-13_1_2950.d.zip	Other
	WLZ_Vitro_Slot1-18_1_2960.d.zip	Other

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Project description:Pf-5 is an important biocontrol strain of Pseudomonas fluorsecens, that improves plant growth, mainly via the production of secondary metabolites and growth/colonisation competition. Copper is a broad-spectrum antimicrobial that is effective against bacteria, fungi and even viruses in soluble forms such as copper sulfate and to a lesser extent in solid form, such as copper surfaces. Copper sulfate is commonly sprayed on food crops to increase yield and is becoming more routinely used due to the increasing problem of resistance to standard pesticides. Thus, an obvious question that remains is: what effect is this introduced copper having on these important biocontrol strains? First, the phenotypic effects of copper on carbon utilisation and pH tolerance was tested using Biolog. Interestingly, the ability of Pf-5 to utilise amino acids as a sole carbon source was largely unaltered, but the presence of copper completely eliminated the utilisation of carbohydrates or fatty acids, and diminished the use of carboxylic acids and amines. This could be explained by copper-mediated disruptions of enzymes in metabolic pathways, such as the TCA cycle. The effect on gene expression was examined using reverse transcriptomics, which established molecular bases for the phenotypic results, and confirmed some known mechanisms for copper resistance, efflux and transporter proteins, and highlighted the delicate interplay with cellular control of iron, as well as uncovered the interesting relationship between integrated bacteriophage and copper as a molecular switch. The integrated phage, Prophage 01, was downregulated in the presence of copper and when this phage was activated with Mitomycin C, the copper appeared to stop the phage from going into lytic cycle and protected lysis of the cells. Prophage 01 is integrated between the housekeeping genes mutS and cinA and is conserved throughout many Pseudomonas. However, only Pf-5, out of the 10 strains tested seemed to be protected from cell lysis by copper, indicating that Prophage 01 in Pf-5 has unique features that interact with Cu. Two-condition experiment, where normal culture in rich medium versus cell treated with CuSO4. 3 biological replicates including 3 technical replicates for one of the biological replicates and 2 technical replicates for another of biological replicates. Swap-dye experiments were performed.

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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Identification of copper-binding-induced polymeric protein complexes

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