Many metal-related conditions impacting human health are due to exposure to harmful non-native metal ions such as lead, cadmium or arsenic. These metals have been associated with increased risk for a variety of cancers including that of the skin, lung, liver, and bladder. The underlying mechanisms of carcinogenesis, and even toxicity, are still unclear. An area that has generated considerable attention recently is the emergence of metal ion xenoestrogens (metalloestrogens) and their relation to cancer of the breast. Metals such as Cd²⁺ and As³⁺ can bind to the hormone binding site of estrogen receptor ERα. Metalloestrogens act as agonists and can lead to cellular proliferation of breast tissue.

             The full-length structure of human ERα has yet to be determined. Amide H/D exchange will be used to localize the binding site for metalloestrogens in the hormone binding domain of full-length ERα. These experiments will also address how the binding of specific metal ions can conformationally mimic estradiol-based activation of ERα. Also, this work will address whether the affinity, association/dissociation kinetics, and/or coordination sphere for a given metal are correlated with the level of estrogenic activity.

Bioinorganic Chemistry of Proteins Involved

In Metal-Related Diseases 

2. Characterizing Metalloestrogen Interactions with Estrogen Receptor-a

Research

             There is increasing evidence that inhibition of DNA repair proteins are important factors in metal-induced carcinogenesis. Many, but not all, of these proteins rely on native metal ions for their structure or function. The prevailing hypothesis is that heavy metals can replace these native metals leading to inactivation. One such protein, Xeroderma pigmentosum group A protein (XPA), is involved in nucleotide excision repair. It is known that genetic mutations in XPA result in the disease Xeroderma Pigmentosa which is characterized by hypersensitivity to sunlight and skin cancer. XPA functions by binding to damaged DNA and serving as marker for assembly of other lesion repair proteins. These protein-protein interactions are thought to be mediated by a zinc-finger domain. Since XPA function is inactivated by nM in vivo concentrations of Cd²⁺, substitution of Zn²⁺ in the zinc finger domain may contribute to faulty protein-protein interactions required for nucleotide excision repair.

             Our goal is to study the equilibrium binding of heavy metals to XPA and correlate metal binding constants and coordination environment with the ability to recognize synthesized DNA templates with appropriate lesions. Also, using H-D exchange mass spectrometry, we are attempting to characterize the effects of heavy metal binding on the structure of XPA as compared to the wild-type structure. An E. coli paralogue of XPA is also available for study.

3.  Heavy Metal Inactivation of DNA Repair Proteins

             Friedreich's ataxia (FRDA) is a debilitating neurodegenerative disease caused by decreased levels of the mitochondrial protein frataxin. FRDA is marked by increased sensitivity to oxidative stress, mitochondrial respiratory chain dysfunction, and mitochondrial iron accumulation. The exact physiological role of frataxin in FRDA is unclear. Although there is no consensus as to the biological function, frataxin may be a chaperone which delivers iron to proteins involved in iron regulation (aconitase), heme biosynthesis (ferrochelatase), and iron-sulfur cluster assembly (ISU). The mechanism of protein interactions and Fe²⁺ transfer is still unknown. Since frataxin associates with three distinct and structurally unrelated proteins in vivo and in vitro, it is important to determine what recognition factor defines the protein interaction. Other characterized metal chaperones structurally resemble their target proteins giving the chaperones "built-in" specificity, but no such correlation is evident for frataxin and its known binding partners. Establishing frataxin's involvement in three separate but overlapping iron metabolism pathways and defining frataxin interactions on a molecular level is essential for new therapeutic approaches to treat FRDA.

             The overall objectives for the proposed research are to understand how frataxin functions in iron regulatory and metabolic pathways and to elucidate the structural characteristics of frataxin in sufficient detail to gain a basic understanding of the biological behavior as it relates to FRDA. The specific aims are to define the regions which are involved in metal-binding and self-association in the absence of a protein partner, to characterize the molecular mechanisms of frataxin recognition for three distinct proteins, and to elucidate the metal coordination and transfer mechanism. Recently, our lab has demonstrated that metal binding sites on frataxin can be localized using amide HD exchange mass spectrometry. This is important since crystal structures have yet to reveal the exact metal binding sites.

1. Investigations into the Fe²⁺-Chaperone Function of Frataxin