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Abstract of associated dissertation:
Within a single human cell, there can be more than 10,000 proteins, all of which must be properly synthesized, folded and maintained. About a third of proteins have metal cofactors that are integral to catalytic activity, structure and regulation. Therefore, proper metal levels must be maintained within the cell for all metalloproteins to be functional. Nevertheless, an excess of any biometal can be detrimental to cellular health. The importance of this dynamic regulation of metals is critical for Cu homeostasis. Cu, due to its ability to redox cycle, plays an essential role in many enzymes including those involved in the electron transport chain, and defense mechanisms against reactive oxygen species. Nevertheless, labile Cu must be strictly regulated because even a minimal amount of excess Cu can have detrimental effects on the cell. Cuproptosis, a form of cell death caused by excess Cu, has been shown to cause destabilization of Fe-S cluster proteins, and aggregation of lipoylated proteins. Moreover, Cu-induced protein aggregation has been proposed to be a significant cause of Cu’s toxicity. This work helps address the lack of information about Cu-induced precipitation on a proteomic scale, about the chemical and biophysical properties that influence the susceptibility of a protein to Cu-induced precipitation, and about the mechanistic factors that drive Cu-induced precipitation.
The approach used in this work exposes proteomes in cell lysates to Cu ions in solution environments that are complex but not controlled by the biological pathways that normally regulate metallostasis and proteostasis at the cellular level. Bottom-up proteomics techniques were used to assess individual susceptibilities of individual proteins to precipitation. These experiments provided evidence that proteins sensitive to Cu-induced precipitation had increased cysteine and histidine residues. Electron paramagnetic resonance and inductively coupled plasma mass spectrometry were used to show that Cu precipitates with proteins and is mostly bound in its Cu+ oxidation state. To understand if this was a global phenomenon that occurred across multiple types of cells, bottom-up proteomics methodologies were used on multiple cell lines. It was found that small molecules, such as histidine and histamine, are increased in C. albicans’ lysate and can help proteins resist Cu-induced precipitation. Additionally, these same small molecules helped increase cellular survival of C. albicans grown in the presence of supplemental Cu. To provide insight as to how the redox activity of Cu affects precipitation, bottom-up proteomic methodologies were performed on proteins precipitated by Cu2+, Cu+, and Zn2+. These experiments showed that for both oxidation states of Cu and Zn2+ the same proteins precipitated with the same relative susceptibilities, but the overall concentration of metal differed. Additionally, reversibility assays showed that this protein precipitation was partially reversible for all three metals, but that Cu+ induced precipitation had the lowest reversibility. Lastly, a bottom-up proteomics methodology utilizing iodoacetamide-alkyne was developed for identification of cryptic Cu binding sites on cysteine residues. This methodology provided evidence that there might be sites of tight Cu interaction with cysteines that help potentiate precipitation. Altogether, this work provides critical insights into the effect of Cu ions on protein precipitation.... [Read More]
Total Size
5 files (1.84 MB)
Data Citation
Robison, A. T. R., Sturrock, G. R., Fitzgerald, M. C., & Franz, K. J. (2025). Data from: Analysis of the Effects of Copper Ions on Protein Precipitation [dissertation]. Duke Research Data Repository. https://doi.org/10.7924/r47w6nn4s
Sturrock, G. R., Robison, A. T. R., Dharani, A., Monson, E. E., Franz, K.J., & Fitzgerald, M. C. (2025). Extrinsic and intrinsic factors affect copper-induced protein precipitation across eukaryotic and prokaryotic proteomes. Protein Sci. 34(6):e70158. https://doi.org/10.1002/pro.70158
Robison, A. T. R., Sturrock, G. R., Zaengle-Barone, J. M., Wiebelhaus, N., Dharani, A., Williams, I. G., Fitzgerald, M. C., & Franz, K. J. (2023). Analysis of copper-induced protein precipitation across the E. coli proteome. Metallomics, 15(1) https://doi.org/10.1093/mtomcs/mfac098
Robison, A. T. R. (2025). Analysis of the Effects of Copper Ions on Protein Precipitation [dissertation]. in press. Duke University.
