Superfund Research Program
Toxic Metal Interactions With Cellular Proteins
Project Leader: Dean E. Wilcox
Grant Number: P42ES007373
Funding Period: 2000-2008
This project aims to identify, quantify and understand the molecular events whereby toxic metals disrupt the function of key biological molecules, typically proteins. Since many proteins have well-defined binding sites for one or more essential metals, these are often sites of competition between the essential metal and various toxic metals. Breakthrough insight that helps to explain previous results from Arsenic as an Endocrine Disruptor that arsenic disrupts pathways regulated by the steroid hormones (e.g. glucocorticoid, estrogen, progesterone) has recently been obtained. These hormones bind and activate receptor proteins that then bind to DNA and initiate the transcription of specific genes. The receptors all share a common DNA-binding domain (DBD) that requires two Zn+2 ions bound at sites with four cysteine residues to stabilize the protein structure that is capable of binding to specific DNA sequences. Dr. Wilcox and his laboratory have recently shown, with a combination of isothermal titration calorimetry (ITC) and circular dichroism, (CD) that the methylated form of arsenite, MMAs(III), but not arsenite, is able to compete with Zn+2 ions for these DBD binding sites, and that it stabilizes a protein structure that is different than the active one stabilized by Zn+2. Since arsenite is the form of arsenic that is commonly ingested under low chronic levels of exposure and it is methylated within cells, these results implicate the methylation of arsenite as a mechanism for in situ generation of a species capable of disrupting hormone-regulated pathways. Parallel ITC and CD studies of the cysteine-rich metal detoxification protein metallothionein (MT) have quantified its Zn+2-binding properties to investigate its proposed role in buffering Zn+2 in cells and investigated the competition of arsenite, MMAs(III) and other toxic metals (e.g. Cd+2, Pb+2, Ag+1, Bi+3) with Zn+2 for this protein. Here scientists have found that MMAs(III) does not effectively compete with Zn+2, and the inability of MT to sequester MMAs(III) can be explained by the metal binding thermodynamics that are measured with ITC.