Reactivity of zinc finger cysteines: Chemical modifications within labile zinc fingers in estrogen receptor

Abstract

Estrogen receptor (ER, alpha isoform) is a 67 kDa zinc finger transcription factor that plays a fundamental role in both normal reproductive gland development and breast carcinogenesis, and also represents a critical molecular target for breast cancer therapy. We are investigating the structural consequences of chemical exposures thought to modify essential zinc finger cysteine residues in human ER. The current study employs mass spectrometry to probe ER zinc finger structural changes induced by a redox-reactive vitamin K3 analog, menadione; a commonly used cysteine alkylator, iodoacetic acid; and a thiol alkylating fluorophore, monobromobimane. Although they are slower to react, the sterically bulkier reagents, monobromobimane and menadione, effectively alkylate the most susceptible ER zinc finger cysteine sulfhydryl groups. Menadione arylation results first in Michael addition of the hydroquinone followed by rapid oxidation to the corresponding quinone, evidenced by a 2 Da mass loss per cysteine residue. Mass spectrometric analysis performed under MALDI conditions reveals both hydroquinone and quinone forms of arylated menadione, whereas only the quinone product is detectable under ESI conditions. Tandem mass spectrometry of a synthetic peptide encompassing the C-terminal half of the structurally more labile second zinc finger of ER (ZnF2B) demonstrates that the two nucleophilic thiols in ZnF2B (Cys-237, Cys-240) are not chemically equivalent in their reactivity to bromobimane or menadione, consistent with their unequal positioning near basic amino acids that affect thiol pKa, thereby rendering Cys-240 more reactive than Cys-237. These findings demonstrate important differential susceptibility of ER zinc finger cysteine residues to thiol reactions. (J Am Soc Mass Spectrom

Systematic Mapping of Posttranslational Modifications in Human Estrogen Receptor-α with Emphasis on Novel Phosphorylation Sites

“…Cys 530 was observed only in Glu-C digests, and Cys 381 was not observed at all. Our studies on the oxidation of cysteine residues in the DBD have been described elsewhere (8,10,11).…”

“…The LC-MS/MS spectrum of the MH 2 2ϩ is illustrated in Fig. 4A: b 1 and b 2 ions confirm N-terminal acetylation, whereas y 2 and y 3 exclude the possibility of the acetyl group being located on Lys 8 . Ac-2 TMTLHTKASGMALLHQIQGNE was also identified with a Mascot score of 72 (Glu-C digest; spectrum not shown).…”

“…Two of the most common and important PTMs that modulate the activity of ER␣ are redox-based modifications of cysteine residues (7)(8)(9)(10)(11)(12) and phosphorylation of serine, threonine, and tyrosine residues (13). Biochemical techniques used previously to map ER␣ phosphorylation sites utilized radioactive labeling with 32 P (14 -16), Edman degradation, deletion and/or point mutations, and Western blots (17)(18)(19).…”

Modulation of nuclear receptor function by cellular redox poise

“…ER in living cells was also subject to oxidative damage induced by both H 2 O 2 and menadione as demonstrated by a loss of DNA binding activity and the ability of ER to activate transcription of a reporter construct. Mass spectrometry studies revealed that ZF2 is the primary target of oxidative damage via disulfide formation and subsequent loss of Zn 2+ (Figure 3, B) [105, 106]. Being more flexible than ZF1, the “open” structure of ZF2 could allow easier access of ROS to catalyze disulfide bond formation.…”

Novel Pathways Associated with Quinone-Induced Stress in Breast Cancer Cells

“…Some reactants (e.g., diamide) produce thiol-reversible loss of ER DNA-binding; others (e.g., iodoacetamide, bromobimane, K3) produce irreversible structural and functional changes in ER. Understanding the various types of structural changes produced in ER by either environmental or endogenous exposures to redox-active and arylating quinones is now possible using optimized immunoaffinity enrichment procedures and modern mass spectrometry (MS) techniques (Whittal et al 2000;Baldwin and Benz 2002;Meza et al 2003;Atsriku et al 2005). Figure 5 shows the functional domains of ER, highlighting the zinc finger structures and Figure 5 Thiol arylation of ER assessed by mass spectrometry.…”