Preferential Oxidation of Zinc Finger 2 in Estrogen Receptor DNA-binding Domain Prevents Dimerization and, Hence, DNA Binding

Abstract

For approximately one-third of estrogen receptor (ER)-positive breast cancer patients, extracted tumor ER is unable to bind to its cognate DNA estrogen response element (ERE), an effect that is partly reversible by the thiol-reducing agent dithiothreitol (DTT). Full-length (67 kDa) ER or its 11 kDa recombinant DNA-binding domain (ER-DBD) is also susceptible to loss of structure and function by the action of oxidants such as diamide and hydrogen peroxide; however, prior DNA binding by ER or ER-DBD protects against this oxidant induced loss of function. The ER-DBD contains two (Cys)(4)-liganded zinc finger motifs that cooperate to stabilize a rigid DNA-binding recognition helix and a flexible helix-supported dimerization loop, respectively. Comparisons between synthetic peptide analogues of each zinc finger and recombinant ER-DBD in the presence of zinc by electrophoretic mobility shift assay, circular dichroism, and mass spectrometry confirm that cooperativity between these zinc fingers is required for both ER-DBD structure (alpha-helicity) and function (dimeric DNA binding). Rapid proteolytic digestion of monomeric, non-DNA-bound ER-DBD followed by HPLC-MS analysis of the resulting peptides demonstrates that zinc inhibits thiol oxidation of the DNA-binding finger, but not the finger supporting the flexible dimerization loop, which remains sensitive to internal disulfide formation. These findings indicate that the loss of ER DNA-binding function in extracts from some primary breast tumors and in ER or ER-DBD exposed to thiol-reacting oxidants results from this asymmetric zinc finger susceptibility to disulfide formation that prevents dimerization. Although ER-DBD contains several strategically located methionine residues, they are less susceptible to oxidation than the thiol groups and, thus, afford no protection against cysteine oxidation and consequent loss of ER DNA-binding function.

Effect Modification by Catalase Genotype Suggests a Role for Oxidative Stress in the Association of Hormone Replacement Therapy with Postmenopausal Breast Cancer Risk

“…Although we had only small numbers of subjects in each stratum, we found that the effect of CAT genotype on the association between HRT and breast cancer risk was restricted to ER-positive breast cancer, suggesting that the estrogen response and oxidative stress pathways are somehow intertwined. This result is not unexpected given the fact that binding of ER to DNA is modulated by redox status (36)(37)(38).…”

The Role of Estrogen Receptor β in Prostate Cancer

“…PCa cells can produce increased levels of reactive oxygen species (ROS) and change the redox status of their microenvironment in response to locally produced transforming growth factor β1 (TGFβ1) prooxidant signals as revealed in DU145 PCa cells (54). Interestingly, oxidative stress inactivates ERβ by inhibiting the receptor's dimerization by altering the second zinc-finger motif in the ERβ structure and therefore destabilizing its DNA-binding capacity (55). As a result, ERβ loses its ability to regulate various genes.…”

15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Transcriptional Activity of Estrogen Receptor-α via Covalent Modification of DNA-Binding Domain

“…These two zinc fingers in ERa DBD function cooperatively in ERa dimerization and DNA binding (25,26). In addition, the COOH-terminal zinc finger of ERa is structurally disordered, and the cysteine thiols of this zinc finger have been characterized as particularly susceptible to the attack of electrophilic agents such as 2,2 ¶-dithiobisbenzamide-1 and benzisothiazolone (27)(28)(29).…”