Mechanisms of zinc finger interactions with metal ions and redox molecules

Elucidating interaction mechanisms between small molecules and proteins are central to understanding many aspects of molecular and cell biology including disease processes. The present studies will focus on understanding regulatory mechanisms between proteins and small molecules that include metal ions and redox molecules. These small molecules alter the cell phenotype through normal cell signaling pathways and/or toxicity mechanisms. The following studies will begin to address the following question: what are the molecular mechanisms by which metal ions and redox molecules interact with protein structures and how do these mechanisms ultimately affect cell phenotype? Chemical reactions with protein cysteine (Cys) residues (e.g. in zinc finger proteins) and resultant structural and functional effects are hypothesized to be general mechanisms for the induction and/or alteration of gene expression. Elucidating mechanisms of zinc (Zn2+) binding and release from zinc fingers will lead to a greater understanding of many physiological processes and could help lead to therapies to help protect cells from oxidative stress and inflammation. In the present studies, a number of metal ions and redox molecules including therapeutic gold (I) and selenium compounds are shown to inhibit DNA binding abilities of Cys2His2 zinc finger proteins at micromolar concentrations. These agents are also shown by electrospray ionization mass spectrometry (ESI-MS) to result in Zn2+ release, which is hypothesized to alter Zn2+ homeostasis and thus the cell phenotype. ESI-MS indicates that a thiol-metal ion-thiolate bond exists in Cys2His2 zinc fingers. A selenotrisulfide bond is also observed in Cys2His2 zinc fingers exposed to selenite. Altering the beta-hairpin structure of Cys2His2 zinc fingers effects the formation of selenite-induced selenotrisulfide bonds and effects Zn2+ release thus providing evidence of structural specificity for these reactions. Conformational differences in zinc fingers resulting from interactions with these various ligands are elucidated by circular dichroism (CD). Also in these studies, levels of stress proteins are found to increase in an in vivo animal model exposed a hydrocarbon fuel mixture. This fuel mixture was previously shown to generate oxidative stress and inflammation in cells. Zn2+ release in a number of cellular systems is hypothesized to be associated with oxidative stress and inflammation.