The cellular metallome includes thousands of Zn-proteins and hundreds of Fe- and Cu-proteins. When toxic metals interact with cells, it is hypothesized that they bind to sensitive sites and, thereby, elicit their deleterious effects by altering protein function. Binding may occur through exchange with metals present in pre-existent metalloproteins or to other sites that also display substantial affinity for metals. Much remains to be learned about the native metalloproteome and little is known about the binding distribution of toxic metals within the proteome. Without such information, it is not possible to understand at a chemical level how xenobiotic agents exert their cellular impact. Research in these areas has been severely hindered by the lack of methods that provide high resolution separation of the proteome with the retention of bound metals to native proteins. This proposal will test the applicability of a new method of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) called native (N)SDS-PAGE that utilizes milder conditions to achieve both the excellent separation of SDS-PAGE and the retention of metals bound to proteins in their native state. Once dispersed on the gel, metalloproteins will be detected using laser ablation inductively coupled plasma mass spectrometry and then identified by mass spectrometry. For this test, the hypothesis will be examined that Cd2+ binds to proteome through metal exchange reactions with Zn-proteins, resulting in the formation of Cd- proteins with displacement of Zn2+. Further, the contraction of the proteomic distribution of Cd-proteins in the presence of the major Cd-binding protein, metallothionein (MT), will be investigated. The secondary hypothesis will be explored that Cd-proteins and Zn-MT undergo metal exchange to produce Cd-MT and Zn- proteins. In this context, the overall objective of the project is to determine whether NSDS-PAGE in conjunction with LA-ICP-MS can be used as the central methodology to support mechanistic studies that focus on the reaction of exogenous metals with cells and their distribution among cell biomolecules. The following specific aims address this objective:  Test and finalize the procedures for conducting NSDS-PAGE followed by LA-ICP-MS.  Characterize the Cd-proteome formed during in vitro reaction of Cd2+ with the isolated proteome of pig kidney LLC-PK1 cells.  Determine how the in vitro Cd-proteome reacts with apo- and Zn-metallothionein.  Compare in vitro and in vivo reactivity of the proteome with Cd2+ in the absence and presence of metallothionein. Success will open the nascent field of metalloproteomics to a wide range of experimentation into the mechanism of action of toxic metals.
Pollutant metals cause serious toxicity in human populations. Little is known about the molecular mechanisms of toxicity because technology has been lacking to measure binding of toxic metals among intracellular proteins in relation to their pathological effects. This proposal aims to develop a robust method to determine the distribution of toxic metals among such binding sites as a basis for understanding their chemical mechanisms of action and proposing means to intervene in their pathways of toxicity.