Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains

Light chain amyloidosis (AL) is caused by the aberrant overproduction of immunoglobulin light chains (LCs). The resulting abnormally high LC concentrations in blood lead to deposit formation in the heart and other target organs. Organ damage is caused not only by the accumulation of bulky amyloid deposits, but extensive clinical data indicate that circulating soluble LCs also exert cardiotoxic effects. The nematode C. elegans has been validated to recapitulate LC soluble toxicity in vivo, and in such a model a role for copper ions in increasing LC soluble toxicity has been reported. Here, we applied microscale thermophoresis, isothermal calorimetry and thermal melting to demonstrate the specific binding of Cu2+ to the variable domain of amyloidogenic H7 with a sub-micromolar affinity. Histidine residues present in the LC sequence are not involved in the binding, and yet their mutation to Ala reduces the soluble toxicity of H7. Copper ions bind to and destabilize the variable domains and induce a limited stabilization in this domain. In summary, the data reported here, elucidate the biochemical bases of the Cu2+-induced toxicity; moreover, they also show that copper binding is just one of the several biochemical traits contributing to LC soluble in vivo toxicity.

Effect of Cu(II) and Conserved Copper Binding Sites on Multicopper Oxidase CopA and Characterization of the BioMnOx

The microbial manganese removal process is believed to be the catalytic oxidation of Mn(II) by manganese oxidase. In this study, the multicopper oxidase CopA was purified and found to have high manganese oxidation activity in vitro and Cu(II) can significantly enhance its manganese oxidation activity. The gene site-directed mutagenesis was used to mutate four conserved copper binding sites of CopA and then obtain four mutant strains. The manganese removal efficiency of the four strains was determined to find that H120 is the catalytic active site of the CopA. Protein modification analysis of CopA obtained under different conditions by mass spectrometry revealed that the loss of Cu(Ⅱ) and the mutation of the conserved copper binding site H120 resulted in the loss of modification of ethoxyformyl and quinone, the number of modifications was reduced and the position of modification was changed, eventually causing a decrease in protein activity. It reveals that Cu(II) and H120 play an indispensable role in the manganese oxidation of the multicopper oxidase CopA. The Mn valence state of BioMnOx was analyzed by XPS, finding that both the strain-mediated product and the CopA-mediated product were composed of MnO2 and Mn3O4 and the average valence of Mn is 3.2.

Intestinal Mucin Is a Chaperone of Multivalent Copper

Mucus protects the body by many mechanisms, but a role in managing toxic transition metals was not previously known. Here we report that secreted mucins, the major mucus glycoproteins coating the respiratory and intestinal epithelia, are specific copper-binding proteins. Most remarkably, the intestinal mucin, MUC2, has two juxtaposed copper binding sites, one that accommodates Cu2+ and the other Cu1+, which can be formed in situ by reduction with vitamin C. Copper is an essential trace metal because it is a cofactor for a variety of enzymes catalyzing electron transfer reactions, but copper damages macromolecules when unregulated. We observed that MUC2 protects against copper toxicity while permitting nutritional uptake into cells. These findings introduce mucins, produced in massive quantities to guard extensive mucosal surfaces, as extracellular copper chaperones and potentially important players in physiological copper homeostasis.

Ionophore Ability of Carnosine and Its Trehalose Conjugate Assists Copper Signal in Triggering Brain-Derived Neurotrophic Factor and Vascular Endothelial Growth Factor Activation In Vitro

l-carnosine (β-alanyl-l-histidine) (Car hereafter) is a natural dipeptide widely distributed in mammalian tissues and reaching high concentrations (0.7–2.0 mM) in the brain. The molecular features of the dipeptide underlie the antioxidant, anti-aggregating and metal chelating ability showed in a large number of physiological effects, while the biological mechanisms involved in the protective role found against several diseases cannot be explained on the basis of the above-mentioned properties alone, requiring further research efforts. It has been reported that l-carnosine increases the secretion and expression of various neurotrophic factors and affects copper homeostasis in nervous cells inducing Cu cellular uptake in keeping with the key metal-sensing system. Having in mind this l-carnosine ability, here we report the copper-binding and ionophore ability of l-carnosine to activate tyrosine kinase cascade pathways in PC12 cells and stimulate the expression of BDNF. Furthermore, the study was extended to verify the ability of the dipeptide to favor copper signaling inducing the expression of VEGF. Being aware that the potential protective action of l-carnosine is drastically hampered by its hydrolysis, we also report on the behavior of a conjugate of l-carnosine with trehalose that blocks the carnosinase degradative activity. Overall, our findings describe a copper tuning effect on the ability of l-carnosine and, particularly its conjugate, to activate tyrosine kinase cascade pathways.

Adaptive changes in the fungal cell wall mediate copper homeostasis

Copper homeostasis mechanisms are essential for microbial adaption to changing copper levels within the host during infection. In the opportunistic fungal pathogen Cryptococcus neoformans (Cn), the Cn Cbi1/Bim1 protein is a newly identified copper binding and release protein that is highly induced during copper limitation. Recent studies demonstrated that Cbi1 functions in copper uptake through the Ctr1 copper transporter during copper limitation. However, the mechanism of Cbi1 action is unknown. The fungal cell wall is a dynamic structure primarily composed of carbohydrate polymers, such as chitin and chitosan, polymers known to strongly bind copper ions. We demonstrated that Cbi1 depletion affects cell wall integrity and architecture, connecting copper homeostasis with adaptive changes within the fungal cell wall. The cbi1 ? mutant strain possesses an aberrant cell wall gene transcriptional signature as well as defects in chitin and chitosan deposition. These changes are reflected in altered macrophage activation and changes in the expression of specific virulence-associated phenotypes. Furthermore, using Cn strains defective in chitosan biosynthesis, we demonstrated that cell wall chitosan modulates the ability of the fungal cell to withstand copper stress. In conclusion, our data suggest a dual role for the fungal cell wall, in particular the inner chitin / chitosan layer, in protection against toxic levels of copper and providing a source of metal ion availability during copper starvation. Given the previously described role for Cbi1 in copper uptake, we propose that this copper-binding protein is involved in shuttling copper from the cell wall to the copper transporter Ctr1 for regulated microbial copper uptake.

Improvement of laccase activity by silencing PacC in Ganoderma lucidum

Ganoderma lucidum is a representative white-rot fungus that has great potential to degrade lignocellulose biomass. Laccase is recognized as a class of the most important lignin-degrading enzymes in G. lucidum. However, the comprehensive regulatory mechanisms of laccase are still lacking. Based on the genome sequence of G. lucidum, 15 laccase genes were identified and their encoding proteins were analyzed in this study. All of the laccase proteins are predicted to be multicopper oxidases with conserved copper-binding domains. Most laccase proteins were secreted enzymes in addition to Lac14 in which the signal peptide could not be predicted. The activity of all laccases showed the highest level at pH 3.0 or pH 7.0, with total laccase activity of approximately 200 U/mg protein. Silencing PacC resulted in a 5.2 fold increase in laccase activity compared with WT. Five laccase genes (lac1, lac6, lac9, lac10 and lac14) showed an increased transcription levels (approximately 1.5-5.6 fold) in the PacC-silenced strains versus that in WT, while other laccase genes were downregulated or unchanged. The extracellular pH value was about 3.1, which was more acidic in the PacC-silenced strains than in the WT (pH 3.5). Moreover, maintaining the fermentation pH resulted in a downregulation of laccase activity which is induced by silencing PacC Our findings indicate that in addition to its function in acidification of environmental pH, PacC plays an important role in regulating laccase activity in fungi.

Integrative Omics Analyses Reveal the Effects of Copper Ions on Salvianolic Acid Biosynthesis

Salvianolic acids, a group of secondary metabolites produced by Salvia miltiorrhiza, are widely used for treating cerebrovascular diseases. Copper is recognized as a necessary microelement and plays an essential role in plant growth. At present, the effect of copper on the biosynthesis of SalAs is unknown. Here, an integrated metabolomic and transcriptomic approach, coupled with biochemical analyses, was employed to dissect the mechanisms by which copper ions induced the biosynthesis of SalAs. In this study, we identified that a low concentration (5 μM) of copper ions could promote growth of S. miltiorrhiza and the biosynthesis of SalAs. Results of the metabolomics analysis showed that 160 metabolites (90 increased and 70 decreased) were significantly changed in S. miltiorrhiza treated with low concentration of copper ions. The differential metabolites were mainly involved in amino acid metabolism, the pentose phosphate pathway, and carbon fixation in photosynthetic organisms. The contents of chlorophyll a, chlorophyll b, and total chlorophyll were significantly increased in leaves of low concentration of copper-treated S. miltiorrhiza plants. Importantly, core SalA biosynthetic genes (laccases and rosmarinic acid synthase), SalA biosynthesis-related transcription factors (MYBs and zinc finger CCCH domain-containing protein 33), and chloroplast proteins-encoding genes (blue copper protein and chlorophyll-binding protein) were upregulated in the treated samples as indicated by a comprehensive transcriptomic analysis. Bioinformatics and enzyme activity analyses showed that laccase 20 contained copper-binding motifs, and its activity in low concentration of copper ions-treated S. miltiorrhiza was much higher than that in the control. Our results demonstrate that enhancement of copper ions of the accumulation of SalAs might be through regulating laccase 20, MYBs, and zinc finger transcription factors, and photosynthetic genes.