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COPPER INDUCES PROTEIN AGGREGATION, A TOXIC PROCESS COMPENSATED BY MOLECULAR CHAPERONES.

ABSTRACT: Copper is a powerful antimicrobial and antiviral agent, which has interest through several biomedical applications. Nevertheless, how copper induces the cellular damage is not fully understood. Two main mechanisms are claimed to be involved, i.e. Cu catalyzed reactive oxygen species production (ROS) and replacements of other essential metal ions, such as Fe in Fe-S clusters by Cu. In the present work, we cumulate strong evidence for the importance of a third mechanism, which proposes that Cu damaging effects are due to its ability to cause massive protein aggregation in a ROS independent mechanism. We further show that Hsp33, a redox-regulated molecular chaperone in Escherichia coli, can prevent Cu-induced protein aggregation, suggesting an important role of the chaperoning system in defense against Cu-toxicity. A closer inspection of the mechanism of how Cu can activate Hsp33 revealed two intriguing features, i) a redox-state specificity of Cu+ vs Cu2+, and ii) an unprecedented mechanism of Hsp33-activation via a non-redox trans-metalation. Indeed, our data suggest that two Cu+ bind to the Zn-Cys4-finger site in Hsp33, which lead to a replacement of one Zn2+ by two Cu+ without Cys oxidation. In contrast, Cu2+ oxidizes the Cys in the Zn-finger, analogue to other oxidative stress effectors of Hsp33. Hence, the present results give several new insights in the antimicrobial mechanism of Cu and how bacteria defend against it.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Escherichia Coli

SUBMITTER: Rosi Fassler

LAB HEAD: Dana Reichmann

PROVIDER: PXD019288 | Pride | 2022-02-10

REPOSITORIES: Pride

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Project description:Copper and iron are essential micronutrients for most living organisms because they participate as cofactors in biological processes including respiration, photosynthesis and oxidative stress protection. In many eukaryotic organisms, including yeast and mammals, copper and iron homeostases are highly interconnected; however such interdependence is not well established in higher plants. Here we propose that COPT2, a high-affinity copper transport protein, functions under copper and iron deficiencies in Arabidopsis thaliana. COPT2 is a plasma membrane protein that functions in copper acquisition and distribution. Characterization of the COPT2 expression pattern indicates a synergic response to copper and iron limitation in roots. We have characterized a knockout of COPT2, copt2-1, that leads to increased resistance to simultaneous copper and iron deficiencies, measured as reduced leaf chlorosis and improved maintenance of the photosynthetic apparatus. We propose that COPT2 expression could play a dual role under Fe deficiency. First, COPT2 participates in the attenuation of copper deficiency responses driven by iron limitation maybe aimed to minimize further iron consume. On the other hand, global expression analyses of copt2-1 mutants versus wild type Arabidopsis plants indicate that low phosphate responses are increased in copt2-1 plants. In this sense, COPT2 function under Fe deficiency counteracts low phosphate responses. These results open up new biotechnological approaches to fight iron deficiency in crops. Four biological replicates of Arabidopsis seedlings were generated for 2 genotypes, Col-0 and copt2-1 mutant; and 3 growth condictions; first one an iron(Fe) and copper(Cu) sufficient medium (+Fe + Cu), second one an Fe deficient and Cu sufficient medium (-Fe+Cu) and third one an Fe and Cu deficient medium (-Fe-Cu). For each growth one comparasion was made, copt2-1 mutant versus Col-0; in each comparasion four biological replicates were made, two replicas were labeled with Cy5 for the mutant sample and Cy3 for the Col-0 sample, while the other two replicas were reversed-labeled.

Project description:Zinc is an essential nutrient because of its role in catalysis and in stabilizing protein structure, but excess zinc can also be deleterious. Four nutritional zinc states have been identified in the alga Chlamydomonas reinhardtii: zinc toxic, zinc replete, zinc deficient and zinc limited. Growth is inhibited in zinc-limited and zinc toxic cells relative to zinc-replete cells, while zinc-deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition responsive changes in gene expression. We identified a subset of genes encoding zinc-handling components, including ZIP family transporters and candidate zinc chaperones. In addition, we noted an impact on two other regulatory pathways, the carbon concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc-deficiency, as a likely consequence of reduced carbonic anhydrase activity, which is validated by mass spectrometry and immunoblot analysis of Cah1, Cah3 and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in air in zinc limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. Surprisingly, we noted also that the Crr1 regulon, which responds to Cu limitation, is also turned on in zinc deficiency, and in fact, Crr1 is required for growth in zinc-limiting conditions. Zinc deficient cells are functionally copper deficient, as evidenced by reduced plastocyanin abundance, even though they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester Cu in a bio-unavailable form, perhaps to prevent mis-metallation of critical zinc sites. Zn-limited wild-type cells were generated by transfer of cells from the first round of growth in medium with no supplemental Zn into TAP medium supplemented or not with 2.5 ÂµM Zn-EDTA The control samples for this study are represented in GSE25622

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COPPER INDUCES PROTEIN AGGREGATION, A TOXIC PROCESS COMPENSATED BY MOLECULAR CHAPERONES.

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