Brca1

Protein

" Within each finger there can be different numbers of cysteine and histidine residues tetrahedrally coordinated to zinc. Individual zinc finger proteins possess different numbers of zinc finger motifs sequentially repeated within a binding domain that targets nucleic acid (RNA or DNA, single or double stranded) or protein.

" Within each finger there can be different numbers of cysteine and histidine residues tetrahedrally coordinated to zinc. Individual zinc finger proteins possess different numbers of zinc finger motifs sequentially repeated within a binding domain that targets nucleic acid (RNA or DNA, single or double stranded) or protein.

plays a purely structural role. Less than 20 years ago, the first of many zinc-based motifs termed "zinc fingers" was identified in a eukaryotic transcription factor (TFIIIA), where it was determined to be essential for the DNA-binding ability of the factor. Since then, more than 10 different classes of zinc finger motifs have been discovered and characterized, many for their ability to bind nucleic acids (DNA, RNA, and heteroduplexes of DNA-RNA) in a sequence-specific manner, with others thought to specifically mediate protein-protein interactions (e.g., dimeriza-tion box in C-terminal zinc finger of steroid receptors). Unlike the typical mode of Zn2+ coordination within the catalytic center of enzymes, tetrahedral coordination of Zn2+ within zinc fingers characteristically involves two to four potentially redox-reactive sulfhydryl (cysteine) groups.1-3"5

It is estimated that zinc finger proteins constitute up to 1% of all human gene products, each of these proteins containing from 2 to 37 repeats of cysteine (± histidine)-containing zinc finger motifs.1'3 Table I summarizes the known zinc finger motif classes and illustrates each with a representative protein or protein family. X-ray crystallography of many DNA-bound zinc finger domains has shown stabilized folded and helical structures within a single domain that binds to specific

3 A. Klug and J. W. R. Schwabe, FASEB J. 9, 597 (1995).

4 K.-D. Kroncke and V. Kolb-Bachofen, Methods Enzymol. 269, 279 (1996).

5 X. Wu, N. H. Bishopric, D. J. Discher, B. J. Murphy, and K. A. Webster, Mol. Cell. Biol. 16, 1035 (1996).

bases within the major groove of a duplexed DNA target site (DNA response element). This apparent structural commonality belies the fact that most zinc finger proteins are still biochemically and structurally poorly characterized. In fact, a vast but not well-characterized subset of zinc finger proteins, including many implicated in human tumorigenesis, appears to act by binding directly to macromolecules other than nucleic acids (e.g., the CyS3HisCys4-type LIM domain proteins, Cys3HisCys4 RING finger proteins such as BRCA1 and BRCA2, and the Cys3HisCys3His cysteine-rich subdomain of protein kinase C).13,6'7 A better characterized subset of zinc finger proteins includes transcription factors implicated in both mammalian aging and tumorigenesis, and comprises diverse superfamilies whose major groove DNA binding results in either direct gene upregulation or its repression (e.g., the Cys4-type zinc finger steroid receptor superfamily or the Cys2His2-type zinc finger Sp/Kruppel-like superfamily).8'9 However, these same DNA-binding transcription factors can also regulate gene expression without directly binding DNA; instead, they use one of their zinc fingers to mediate protein-protein interactions (e.g., dimerization) with other DNA-bound transcription factors.610 Despite the redox insensitivity of the coordinated Zn2+ in these transcription factors, a large number of them are known to be redox regulated by intracellular levels of reactive oxygen species (ROS), which can impair their DNA-binding activity and alter their proteinprotein interactions with the oxidation of coordinating cysteines and ejection of Zn2+ that maintains the critical zinc finger structure.6,11,12 Apart from hydrogen peroxide and shorter lived superoxide and hydroxyl radicals, other ROS produced intracellularly under both normal and diseased conditions include the free radical product of nitric oxide synthase, NO, which is also known to structurally impair the DNA-binding and gene-regulating function of zinc finger transcription factors.13

Redox Sensitivity of Zinc Finger Transcription Factors Such as Estrogen Receptor

It is well established that intracellular oxidative stress and ROS are produced by growth factors as well as by many other exogenous agents and during normal physiologic responses (e.g., hypoxia-reperfusion injury, inflammation).14 If

6 L. Zheng, S. Li, T. G. Boyer, and W.-H. Lee, Oncogene 19, 6159 (2000).

7 S. Kuroda, C. Tokunaga, Y. Kiyohara, O. Higuchi, H. Konishi, K. Mizuno, G. N. Gill, and U. Kikkawa, J. Biol. Chem. 271, 31029 (1996).

10 J. Liden, F. Delaunay, I. Rafter, J.-A. Gustafsson, and S. Okret, J. Biol. Chem. 272, 21467 (1997).

11 Y. Sun and L. W. Oberley, Free Radic. Biol. Med. 21, 335 (1996).

12 R. G. Allen and M. Tresini, Free Radic. Biol. Med. 28,463 (2000).

13 K.-D. Kroncke and C. Carlberg, FASEB J. 13, 166 (2000).

14 B. S. Berlett and E. R. Stadtman, J. Biol. Chem. 272, 20313 (1997).

intracellular glutathione (GSH), antioxidant, and/or superoxide dismutase (SOD)-detoxifying systems of the cell are deficient or overwhelmed, increases in intracellular ROS produce various cellular responses ranging from cell proliferation to apoptosis or senescence, and contribute to either transient stress injury or more permanent manifestations such as aging and malignancy.14"16 Of physiologic interest, natural aging is known to be associated with in vivo accumulation of oxyradi-cal tissue damage, resulting in selective loss of Spl and glucocorticoid receptor DNA-binding activities but without significant decline in the tissue content of these zinc finger transcription factors.11 In preclinical models of breast tumori-genesis, growth-associated periodic fluctuations in blood flow through the tumor microvasculature have been found to be sufficient to induce hypoxia-reperfusion injury, with the resulting redox-sensitive gene induction thought to be associated with enhanced tumor aggressiveness and therapeutic resistance.17

A critical member of the nuclear and steroid receptor superfamily essential for normal reproductive gland function and whose transcriptional overexpression contributes to the development of most human breast cancers is the zinc finger transcription factor estrogen receptor (ER, a isoform).8 ER activity is regulated allosterically by ligand binding with either an agonist (estrogen) or antagonist (antiestrogen). On binding to an agonistic ligand, homodimerization and DNA binding by the activated ER complex occurs, producing trans-activation of ER-inducible genes. As with many other well-described transcription factors, a number of intracellular proteins and conditions (e.g., ER phosphorylation) are thought to modulate the binding of ligand-activated steroid receptors to cognate response elements (e.g., the estrogen response element, ERE) within the inducible gene promoter. Dimerization of ER, in particular, is necessary for DNA binding; and at least two dimerization domains have been mapped within the full-length receptor, one in the DNA-binding domain near the second zinc finger and the other in the more C-terminal ligand-binding domain of ER. Interference with either of the two Cys4-type zinc finger structures located in the ER DNA-binding domain diminishes ER dimerization, DNA binding, and frani-activation of ERE-containing target gene promoters.

Structural changes in the zinc finger DNA-binding domain can certainly be induced by amino acid alterations, or by loss or substitutions of the two tetrahedrally coordinated Zn2+ cations either by chelation (e.g., treatment with o-phenanthro-line) or replacement with another coordinating metal ion. In this latter regard, transition metal ions of environmental or physiologic concern include Cu(II), Cd(II), Ni(II), and Cr(II); and of these, Cu(II) is of special interest because of its significant increase in patients with malignant breast disease, its known ability to inhibit

15 T. Finkel and N. J. Holbrook, Nature (London) 408, 239 (2000).

17 H. Kimura, R. D. Braun, E. T. Ong, R. Hsu, T. W. Secomb, D. Papahadjopoulos, and K. Hong, Cancer Res. 56, 5522 (1996).

ER function both in vitro and in vivo, and the ease with which it replaces Zn2+ in the ER DNA-binding domain (forming four bicoordinated Cu2+ complexes of higher affinity than the Zn2+ complexes), leading to an alteration in ER tertiary structure.18-20 Although the concentration of Zn2+ exceeds that of Cu2+ in most normal cells, the intracellular and intranuclear levels of Zn2+ and Cu2+ in different tissues, including estrogen target organs, support the possibility that Cu2+ may occupy the ER DNA-binding domain (DBD) in some human breast tumors.21

As a Cys4-type zinc finger protein, ER is also expected to be redox sensitive. Interestingly, the trans-activating and DNA-binding capacities of ER have previously been shown to be modulated by the redox effector protein, thioredoxin.22 Moreover, thiol-specific oxidation of ER appears to account at least partially for the fact that otherwise intact (67 kDa) and immunoreactive ER present in about one-third of all ER-overexpressing primary breast tumors appears to be completely unable to bind DNA when assayed in vitro in either the presence or absence of exogenous zinc.23-25 This ER DNA-binding dysfunction is partially reversible under strong thiol-reducing conditions (excess dithiothreitol [DTT]), indicating that mild cysteine oxidation has occurred with tumor growth in some of these samples. However, free radical intermediates formed under stronger oxidant stress can also alkylate cysteine and noncysteine residues in and outside of the ER DNA-binding domain, which would then result in nonreversible ER dysfunction, as found in the majority of breast tumor samples exhibiting loss of ER DNA-binding capacity.

Thus, we have proposed that enhanced intratumor ROS production accompanying aggressive tumor cell proliferation and invasion, in association with inadequate tumor neovascularization and a fluctuating supply of oxygen, potentially produces a subset of ER-overexpressing breast tumors that are not only more aggressive in their clinical behavior but also unresponsive to endocrine therapy (e.g., with the antiestrogen tamoxifen), because of the structural damage induced in their ER zinc finger DNA-binding domains. Clinical studies are pending to prove that this subset of ER-overexpressing breast tumors contain such oxidatively damaged and dysfunctional ER. In the interim, however, mass spectrometric analysis of oxidant-stressed recombinant ER protein and ER-overexpressing human breast cancer cell lines (e.g., MCF-7) is providing important new mechanistic

18 J. H. Fishman and J. Fishman, Biochem. Biophys. Res. Commun. 152, 783 (1988).

19 P. F. Predki and B. Sarkar, J. Biol. Chem. 267, 5842 (1992).

20 T. W. Hutchens, M. H. Allen, C. M. Li, and T.-T. Yip, FEBS Lett. 309, 170 (1992).

21 J. H. Freedman, R. J. Weiner, and J. Peisach, J. Biol. Chem. 261, 11840 (1986).

22 S.-I. Hayashi, K. Haijiro-Nakanishi, Y. Makino, H. Eguchi, J. Yodoi, and H. Tanaka, Nucleic Acids Res. 25,4035 (1997).

23 G. K. Scott, P. Kushner, J. L. Vigne, and C. C. Benz, J. Clin. Invest. 88, 700 (1991).

24 P. A. Montgomery, G. K. Scott, M. C. Luce, M. Kaufmann, and C. C. Benz, Breast Cancer Res. Treat. 26, 181 (1993).

25 X. Liang, B. Lu, G. K. Scott, C.-H. Chang, M. A. Baldwin, and C. C. Benz, Mol. Cell. Endocrinol. 146, 151 (1998).

insights into the structural changes induced by oxidant stress of zinc finger transcription factors.26

Monitoring Protein Oxidation by Electrospray Ionization Mass Spectrometry

Most oxidation processes in proteins involve a change in molecular mass, such as the addition of 16 Da for conversion of a methionine residue to methionine sulfoxide or 32 Da for formation of the sulfone. Therefore mass spectrometry (MS) is well suited to monitor protein oxidation. Oxidation of cysteine by disulfide formation may be regarded as a special case as the mass difference between two reduced cysteine residues and the disulfide-linked product cystine is only 2 Da, and only for smaller proteins can such a difference be measured unambiguously. If necessary, chemical alkylation of free thiols may be used to give substantially larger mass differences between the reduced and oxidized states. This provides the additional advantage that, once alkylated, the thiol groups are resistant to further oxidation. Electrospray ionization (ESI)27 is particularly advantageous as it ionizes directly from solution, is fully compatible with the analysis of involatile biomolecules including peptides and proteins, and can be interfaced with high-performance liquid chromatography (HPLC). Thus, not only may specific cysteine residues that are more or less susceptible to oxidation be identified by proteolytic digestion of a partially oxidized protein and analysis of individual peptides by HPLC-MS, cysteines modified by addition of the oxidizing moiety may also be revealed. ESI-MS carried out under nondenaturing conditions is finding increasing application for monitoring the biophysical properties of proteins and the formation of noncova-lent complexes, allowing additional information about the structural consequences of protein oxidation to be derived.28'29 MS also gives an unambiguous measure of stoichiometry for complex formation, for example, it confirmed that the DBD of vitamin D binds to DNA as a dimer, and that this assembly requires a total of four Zn2+ ions.30

Several MS studies of the DBDs of ER and other nuclear hormone receptors have concentrated on their metal-binding properties, as these DBDs possess Cys4-liganded zinc fingers that are essential for normal activity. Thus ESI-MS showed that Cu2+ can replace Zn2+ in the ER-DBD, possibly with higher affinity and with the higher stoichiometry of two Cu2+ ions per zinc finger,20 and the glucocorticoid receptor DNA-binding domain (GR-DBD) can bind two Cd2+ ions as

26 R. M. Whittal, C. C. Benz, G. Scott, J. Semyonov, A. L. Burlingame, and M. A. Baldwin, Biochemistry 39, 8406 (2000).

27 J. B. Fenn, M. Mann, C. K. Meng, S. F. Wong, and C. M. Whitehouse, Science 246,64 (1989).

29 A. M. Last and C. V. Robinson, Curr. Opin. Chem. Biol. 3, 564 (1999).

30 T. D. Veenstra, L. M. Benson, T. A. Craig, A. J. Tomlinson, R. Kumar, and S. Naylor, Nat. Biotechnol. 16, 262 (1998).

an alternative to Zn2+.31 The study by Veenstra el al., referred to above, showed that either zinc or cadmium allowed the vitamin D receptor DBD to bind to DNA.30 More recently, we demonstrated that impaired DNA binding of ER resulting from zinc finger oxidation could be monitored by ESI-MS, and that the susceptibility of the second zinc finger to oxidation was enhanced relative to the first.26 Other zinc finger proteins studied by mass spectrometry include the HIV type 1 nucleopcapsid protein P7 (NcP7).32 Unlike the two Cys4 zinc fingers of ER, NcP7 contains two Cys3His zinc finger motifs. Nevertheless, there are marked similarities between the two proteins, in that the C-terminal zinc finger demonstrated a higher susceptibility toward oxidation in both cases. When NcP7 was oxidized by 2,2/-dithiopyridine, oxidation intermediates were observed in which either one or two cysteines were linked to thiopyridine, with the corresponding loss of one or two Zn2+ ions. By contrast, the more reactive disulfiram gave only a single adduct with the loss of a single Zn2+.32 Similarly, we have observed an intermediate formed by the addition of S-nitrosylglutathione to cysteine in ER-DBD when this material is used as an oxidizing agent.32a

Other established analytical methods can provide valuable complementary information. These include electrophoretic mobility shift assays (EMSAs), metal ion titrations, and various spectroscopic techniques including absorption spectroscopy, circular dichroism, and nuclear magnetic resonance. Absorption spectroscopy established that for peptide analogs of a simian NcP, Zn2+ is complexed in a zinc finger with tetrahedral geometry; Ni2+ also gives a native-like tetrahedral complex, whereas replacement of Zn2+ by Co2+ or Cd2+ results in square planar structures that are more sensitive to oxidation.33

Equipment for Electrospray Ionization-Mass Spectrometry and High-Performance Liquid Chromatography-Mass Spectrometry

ESI-MS can be effective with several different types of mass spectrometer. Initial studies of the purity and of the oxidation and reduction of ER-DBD employed a quadrupole mass spectrometer of relatively modest performance. In other studies we used an orthogonal acceleration time-of-flight mass spectrometer (oa-ToFMS) of higher resolving power and higher sensitivity.26 Magnetic sector instruments have also been employed, but for ESI-MS these are mostly being superseded by quadrupoles, ion traps, and oa-TOFs or Qoa-TOFs. The purity and oxidation state

31 H. E. Witkowska, C. H. L. Shackleton, K. Dahlman-Wright, J. Y. Kim, and J.-A. Gustaffson,./. Am. Chem. Soc. 117,3320(1995).

32 Y. Hathout, D. Fabris, M. S. Han, R. C. Sowder II, L. E. Henderson, and C. Fenselau, Drug Metab. Dispos. 24, 1395 (1996).

32a J. E. Meza, C. C. Benz, G. K. Scott, and M. A. Baldwin, in preparation (2002).

33 X. Chen, M. Chu, and D. P. Giedroc, J. Biol. Inorg. Chem. 5, 93 (2000).

of a peptide or protein can be determined by ESI from acidified solution, infusing from a syringe pump a 5-10 /iM solution of the protein or peptide in H20-methanol (1:1, v/v) acidified with 1% (v/v) acetic acid, directly into the ESI source. Alternatively, an ESI-MS instrument is readily interfaced with the HPLC, perhaps using a 1-mm i.d. reversed-phase (RP) column with 300-A pore size and 5-/xm particle size. This allows samples to be desalted by injection onto the HPLC before being transferred to the mass spectrometer. Typically solvent A is water with 0.06% (v/v) trifluoroacetic acid (TFA) to control pH, and solvent B is 80% (v/v) acetonitrile with 0.052% (v/v) TFA at a flow rate of 50 /xl/min. Mass spectrometric sensitivity may be adversely affected by TFA, in which case this may be replaced with formic acid, and acetonitrile can be replaced by a 5 :2 mixture of ethanol and n-propanol.34 The column effluent should pass through a UV flow cell and then, depending on the optimum flow rate for the ESI source, may be split such that ~10% passes into the ESI source of the mass spectrometer and the balance can be collected in fractions for later studies. Alternatively, a capillary column operating at lower flow rate will allow the entire effluent to be transferred without splitting. Mass spectra should be recorded and accumulated continuously throughout the running of each sample. The UV chromatogram recorded at 215 nm can then be compared with the total ion current (TIC) trace obtained from the mass spectrometer. ESI spectra corresponding to peaks in the UV and TIC traces can be selected manually for averaging and deconvolution, using software provided by the mass spectrometer manufacturer.

The conventional RP-HPLC conditions of low pH are denaturing and do not support the formation or maintenance of noncovalent complexes. To study a metal-loaded holoprotein such as a zinc finger protein at neutral pH, it must be introduced via a syringe pump. The buffer should contain 5-10 ¡iM protein in 2.5 mM ammonium acetate solution (pH 7.4)-10% (v/v) methanol, and varying concentrations of Zn2+ diluted from zinc(II) sulfate stock solution. A reducing agent such as 100 /xM DTT may be included to inhibit aerobic oxidation. The methanol is needed to enhance the spray formation. Ammonium acetate is favored because it is volatile and will not contaminate the ESI source with salt deposits; ammonium formate or bicarbonate can also be used at either lower or higher pH respectively. Each solution should be infused to establish a steady state, and then the spectra can be recorded and averaged over a period of a few minutes.

Interpretation of Mass Spectra

Under normal ESI-MS conditions, peptides and proteins become protonated at the basic residues, that is, arginine, lysine, and histidine and the amino terminus,

34 K. F. Medzihradszky, D. A. Maltby, S. C. Hall, C. A. Settineri, and A. L. Burlingame, J. Am. Soc.

giving a distribution of multiply charged species. This is observed as a series of peaks, separated according to their mass/charge (m/z) ratios. This series can be mathematically deconvoluted to give a single profile for the molecular weight. Because of heavy isotopes such as 13C, 15N, and lsO, each molecule gives a cluster of peaks with individual components separated by 1 Da. These are easily separated for small peptides, the "monoisotopic" molecular weight being defined by the mass of the first peak in the cluster. For larger peptides and proteins these may not be resolved, in which case an "average" mass is derived from the overall profile of the cluster.35 Quantitative data can be derived from the raw mass spectra by addition of the signal intensities from all of the individual charge states, or from peak intensities in the deconvoluted spectra. For recombinant ER-DBD of Mr ~11.2 kDa, the isotopic components at adjacent masses could not be resolved by either mass spectrometer, and therefore the measured mass of each peak, representing an average of the distribution of all isotopes present, was compared with the value calculated from average atomic masses. For synthetic peptides and peptides originating from tryptic digestion, the isotopic clusters were resolved by the oa-TOF mass spectrometer to give separate peaks, giving the monoisotopic molecular mass.

Recombinant Estrogen Receptor DNA-Binding Domain

Using well-established techniques, the 84-residue DNA-binding domain of the estrogen receptor (ER-DBD) with an N-terminal histidine tag was expressed in Escherichia coli as a protein of 99 amino acids (Fig. 1), and purified by immobilized metal ion affinity chromatography (IMAC). Activity was confirmed by a positive EMS A response for binding to the cognate ERE. Some samples were subjected to chemical treatments such as oxidation, reduction, or alkylation before separation and analysis by HPLC-MS. Other experiments were conducted by direct infusion of samples into the ESI mass spectrometer without HPLC separation. The UV chromatogram and total ion current trace for a typical HPLC separation showed a single chromatographic peak, but ESI-MS revealed that this contained two coelut-ing components. The measured Mr of the lower mass component (11,232.7 Da) was close to that calculated for fully reduced ER-DBD (11,232.9 Da), whereas the mass of the second component was higher by ~14 Da. Digestion with trypsin and HPLC-MS separation identified the adduct as an N-terminal methylation, presumably occurring during protein expression.

To confirm the presence of nine cysteine residues, ER-DBD was reduced with 2 mM DTT and reacted with 6 mM iodoacetic acid (IAA) for 1 hr at 4° to

35 S. A. Carr, A. L. Burlingame, and M. A. Baldwin, in "Mass Spectrometry in Biology and Medicine" (A. L. Burlingame, S. A. Carr, and M. A. Baldwin, eds.), p. 553. Humana Press, Totowa, NJ, 2000.

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