Zinc Proteomics: Interactions of Zn2+, Cd2+, and Metal-binding Ligands with Zn-Binding Sites in the Proteome

Abstract

Cadmium ion causes toxicity in humans, most prominently in the kidney. This thesis focuses on mechanisms by which Cd2+ harms kidney proximal tubule cells. Previous experiments have shown the time-dependent distribution of Cd2+ and Zn2+ within supernatant of pig kidney, LLC-PK1 cells after exposure to Cd2+ plus the ionophore pyrithione1. The first part of this research describes the trafficking of cadmium within LLC-PK1 proximal tubule cells, including the time dependent distribution, speciation, and quantification of Cd2+ and Zn2+ in cells exposed to Cd2+ and Cd2+ plus pyrithione. The latter treatment introduces Cd2+ into cells at time zero and permits the clean observation of its subsequent trafficking pathway. Cd2+ binds first to the Proteome and exchanges with Zn2+ bound as specific Zn-proteins. Later, Cd2+ shifts into newly synthesized metallothionein. A 1:1 stoichiometry of the Cd-Zn exchange reaction was determined by titrations in cells and isolated cell Proteome. Zn2+ displaced from the Zn-Proteome became bound to other, adventitious sites of binding within the entire Proteome. The results support the hypothesis that non-specifically bound Zn2+ mobilized by Cd2+ activates the transcription factor, MTF-1, leading to the induction of metallothionein synthesis. With these results in hand, the research turned to understanding at the protein level, how Cd2+ interacts with cell constituents. Important questions were what are the identities of the proteins that bind Cd2+? Are these proteins normally Zn-proteins? How can such proteins be detected analytically? Recently, native SDS-PAGE, or NSDS-PAGE, was developed that showed refined separation of proteins even as their native properties and bound metal ions remained intact2. This section of the defense explores the development of a new approach for the identification of zinc- and cadmium-containing proteins in complex mixtures. NSDS-PAGE was used to separate proteins with retention of their zinc and cadmium contents. Then, laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was utilized to detect low amounts of these metals in proteins along the length of the gel electrophoresis lane. A companion technique of fluorescent visualization of Zn-proteins with the zinc sensor, TSQ, was used in tandem with LA-ICP-MS to strengthen the detection of Zn- and Cd-proteins3. Using these methods, the replacement of Zn2+ by Cd2+ in Zn-proteins was observed. In the presence of Zn-metallothionein, the restoration of Zn2+ into these proteins was also seen for the first time as metallothionein successfully competed for Cd2+. The last part of this defense addresses the hypothesis that ligand binding to members of the Zn-proteome can occur with a wide variety of ligands. The ability of ligands to form adduct species with specific and non-specifically bound zinc was surveyed using a competitive titration experiment using the Zn2+ sensor TSQ. Of direct interest to the Cd2+ research, pyrithione can form ternary adducts with both types of cellular zinc. 1. Namdarghanbari, M. et al. J Biol Inorg Chem (2011) 16: 1087 2. Nowakowski, D. et al. Metallomics, 2014, 6, 1068 3. Meeusen, J. et al. Inorganic Chemistry 2011 50 (16), 7563-7573