Toxic metal proteomics: Reaction of the mammalian zinc proteome with Cd2+

Abstract

The hypothesis was tested that Cd2+ undergoes measureable reaction with the Zn-proteome through metal ion exchange chemistry. The Zn-proteome of pig kidney LLCPK1 cells is relatively inert to reaction with competing ligands, including Zinquin acid, EDTA, and apo-metallothionein. Upon reaction of Cd2+ with the Zn-proteome, Cd2+ associates with the proteome and near stoichiometric amounts of Zn2+ become reactive with these chelating agents. The results strongly support the hypothesis that Cd2+ displaces Zn2+ from native proteomic binding sites resulting in the formation of a Cd-proteome. Mobilized Zn2+ becomes adventitiously bound to proteome and available for reaction with added metal binding ligands. Cd-proteome and Zn-metallothionein readily exchange metal ions, raising the possibility that this reaction restores functionality to Cd-proteins. In a parallel experiment, cells were exposed to Cd2+ and pyrithione briefly to generate substantial proteome-bound Cd2+. Upon transition to a Cd2+ free medium, the cells generated new metallothionein protein over time that bound most of the proteomic Cd2+ as well as additional Zn2+.

Impact of Cadmium on Intracellular Zinc Levels in HepG2 Cells: Quantitative Evaluations and Molecular Effects

“…This result clearly indicates that the uptake of cadmium, that uses aspecific transport pathways [ 5 ], occurs to a significantly larger extent to that of zinc, whose concentration is, on the contrary, strictly regulated. This result is consistent with the large increment of the fluorescence intensity of HepG2 cells exposed to 10 μ M Cd as due to cadmium to zinc replacement in proteins and mobilization of the latter metal ion, a mechanism previously proposed [ 7 ] and recently demonstrated in a prokaryotic zinc-finger domain [ 33 ], and proteome extracted from pig kidney LLC-PK1 cells [ 8 ].…”

“…The term “labile” will be used in the following to indicate all of the Zn(II) sensed by Zinquin. The displacement of Zn(II) from the Zn-proteome by Cd(II) and its labilization has been recently demonstrated on the isolated proteome extracted by pig kidney LLC-PK1 cells by using Zinquin as a fluorescent probe [ 8 ]. Here, we are investigating the effect of Cd exposure on the Zn-homeostasis in intact human cells.…”

“…In mammalian cells cadmium toxicity has been closely related to zinc homeostasis. Once inside the cells, cadmium is expected to displace zinc in proteins and enzymes in which zinc has a sulphur-dominated coordination sphere, such as the metallothioneins, the zinc sensor MTF-1, and multiple zinc-finger proteins [ 3 , 7 , 8 ]. However, even though the disturbance of cadmium in zinc homeostasis has already been recognized, its extent has not been a matter of study yet.…”

Developing a Gene Biomarker at the Tipping Point of Adaptive and Adverse Responses in Human Bronchial Epithelial Cells

“…We utilized Zn 2+ exposures to generate oxidative stress in a reproducible manner. This environmentally relevant exposure model may apply to other metal exposures, including cadmium [ 61 ] and mercury [ 62 ], which can displace protein bound Zn, thereby increasing intracellular free Zn 2+ and adaptive and adverse cellular effects [ 63 , 64 ]. Moreover, the enrichment of our biomarker gene set in other publicly available gene expression data of bronchial origin suggests a similar biological response, likely oxidant-mediated, with other environmental exposures and related diseases, including cigarette smoke, bacterial or viral infection, mechanical injury, COPD, and PAHs ( Table 1 ).…”

Proteomic High Affinity Zn2+ Trafficking: Where Does Metallothionein Fit in?

“…Instead, the model presented in Figure 2 portrays trafficking proteins (L 1-m ). Consistent with this emphasis, Zn 2+ added to cells or cell lysates or mobilized from native Zn-proteins by displacement with Cd 2+ associates with the proteome fraction not with glutathione or other low molecular weight molecules [53]. It is within this context that a role for metallothionein in Zn 2+ trafficking must be considered and begins to make sense.…”