Self-assembled monolayer-assisted silicon nanowire biosensor for detection of protein-DNA interactions in nuclear extracts from breast cancer cell

The large number of estrogen receptor (ER) binding sites of various sequence patterns requires a sensitive detection to differentiate between subtle differences in ER-DNA binding affinities. A self-assembled monolayer (SAM)-assisted silicon nanowire (SiNW) biosensor for specific and highly sensitive detection of protein-DNA interactions, remarkably in nuclear extracts prepared from breast cancer cells, is presented. As a typical model, estrogen receptor element (ERE, dsDNA) and estrogen receptor alpha (ERα, protein) binding was adopted in the work. The SiNW surface was coated with a vinyl-terminated SAM, and the termination of the surface was changed to carboxylic acid via oxidation. DNA modified with amine group was subsequently immobilized on the SiNW surface. Protein-DNA binding was finally investigated by the functionalized SiNW biosensor. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were employed to characterize the stepwise functionalization of the SAM and DNA on bare silicon surface, and to visualize protein-DNA binding on the SiNW surface, respectively. We observed that ERα had high sequence specificity to the SiNW biosensor which was functionalized with three different EREs including wild-type, mutant and scrambled DNA sequences. We also demonstrate that the specific DNA-functionalized SiNW biosensor was capable of detecting ERα as low as 10 fM. Impressively, the developed SiNW biosensor was able to detect ERα-DNA interactions in nuclear extracts from breast cancer cells. The SAM-assisted SiNW biosensor, as a label-free and highly sensitive tool, shows a potential in studying protein-DNA interactions.     

The DNA binding domain of estrogen receptor alpha is required for high-affinity nuclear interaction induced by estradiol

Estrogen receptor alpha (ER) is a member of the nuclear hormone receptor family, which upon binding estrogen shows increased apparent affinity for nuclear components (tight nuclear binding). The nuclear components that mediate this tight nuclear binding have been proposed to include both ER-DNA interactions and ER-protein interactions. In this paper, we demonstrate that tight nuclear binding of ER upon estrogen occupation requires ER-DNA interactions. Hormone-bound ER can be extracted from the nucleus in low-salt buffer using various polyanions, which mimic the phosphate backbone of DNA. The importance of specific ER-DNA interactions in mediating tight nuclear binding is also supported by the 380-fold lower concentration of the ERE oligonucleotide necessary to extract estrogen-occupied ER from the nucleus compared to the polyanions. We also demonstrate that estrogen-induced tight nuclear binding requires both the nuclear localization domain and the DNA binding domain of ER. Finally, enzymatic degradation of nuclear DNA allows us to recover 45% of tight nuclear-bound ER. We further demonstrate that ER-AIB1 interaction is not required for estrogen-induced tight nuclear binding. Taken together, we propose a model in which tight nuclear binding of the estrogen-occupied ER is predominantly mediated by ER-DNA interactions. The effects of estrogen binding on altering DNA binding in whole cells are proposed to occur through estrogen-induced changes in ER-chaperone protein interactions, which alter the DNA accessibility of ER but do not directly change the affinity of the ER for DNA, which is similar for both unoccupied and occupied ER.     

Selective mutations in estrogen receptor alpha D-domain alters nuclear translocation and non-estrogen response element gene regulatory mechanisms

The three main mechanisms of ERα action are: 1) nuclear, genomic, direct DNA binding, 2) nuclear, genomic, "tethered"-mediated, protein-protein interactions, and 3) non-nuclear, non-genomic, rapid action responses. Reports suggest the D-domain or hinge region of ERα plays an important role in mechanisms 1 and 2 above. Studies demonstrating the functionality of the ERα hinge region have resected the full D-domain; therefore, site directed mutations were made to attribute precise sequence functionality to this domain. This study focuses on the characterization and properties of three novel site directed ERα- D-domain mutants. The Hinge 1 (H1) ERα mutant has disrupted nuclear localization, can no longer perform tethered mediated responses and has lost interaction with c-Jun, but retains estrogen response element (ERE)-mediated functions as demonstrated by confocal microscopy, reporter assays, endogenous gene expression and co-immunoprecipitation. The H2 ERα mutant is non-nuclear, but translocates to the nucleus with estradiol (E2) treatment and maintains ERE-mediated functionality. The H2+NES ERα mutant does not maintain nuclear translocation with hormone binding, no longer activates ERE-target genes, functions in ERE- or tethered-mediated luciferase assays, but does retain the non-genomic, non-nuclear, rapid action response. These studies reveal the sequence(s) in the ERα hinge region that are involved in tethered-mediated actions as well as nuclear localization and attribute important functionality to this region of the receptor. In addition, the properties of these ERα mutants will allow future studies to further dissect and characterize the three main ERα mechanisms of action and determine the mechanistic role each action has in estrogen hormone regulation.     

PES1与雌激素受体存在相互作用

雌激素(E2)和雌激素受体(ER)在E2诱发的肿瘤中起着极其重要的作用.ER共调节因子通过与ER相互作用调节其生物学功能.PES1主要表达于E2的重要靶器官如乳腺、卵巢等组织中,并在乳腺癌细胞中高表达.用PCR技术构建HA标签的PES1全长以及1～322aa、312 ～588aa和414～588aa三个不同功能区片段的重组质粒.将不同的重组质粒与FLAG-ERα和或FLAGC-ERβ共转染293T细胞后进行免疫共沉淀,以验证PES1与ER是否有相互作用以及相互作用的区域.用含雌激素受体作用元件的荧光素酶报告基因( ERE-LUC)检测PES1对ERα和ERβ转录激活活性的影响.结果表明,PES1与ERα和ERβ均相互作用,且PES1的1～ 322aa区域与ERα和ERβ相结合.PES1能特异地、E2非依赖性抑制ERβ的转录激活活性.实验结果显示,PES1是一个新的ER共调节因子,需要进一步研究其在ERβ信号通路及其在E2诱发的肿瘤的作用.     

Evaluation of two- and three-dimensional streptavidin binding platforms for surface plasmon resonance spectroscopy studies of DNA hybridization and protein-DNA binding

Surface plasmon resonance (SPR) spectroscopy has been used to study DNA assembly, DNA hybridization, and protein-DNA interactions on two streptavidin (SA) sensor chips. On one chip, SA molecules are immobilized on a biotin-exposed surface, forming an ordered two-dimensional (2D) SA monolayer. The other chip, BIAcore's SA chip, contains SA molecules immobilized within a three-dimensional (3D) carboxylated dextran matrix. Compared to the 2D chip, the 3D SA matrix allows for a slower immobilization rate of biotinylated DNA due to diffusion limitation in the dextran matrix, but with twice the amount of the immobilized DNA due to the greater number of reactive sites, which in turn enables a higher sensitivity for DNA hybridization detection. Interestingly, having a greater DNA probe dispersion in the 3D matrix does not induce a higher DNA hybridization efficiency. In a study of protein binding to immobilized DNA (estrogen receptor to estrogen response elements), aiming at assessing the DNA sequence dependent protein binding behavior, the 2D and 3D chips produce different binding characteristics. On the 2D chip, the protein binding exhibits a better selectivity to the specific sequences, regardless of binding stringency (e.g. salt concentration), whereas on the 3D chip, the liquid handling system needs to be optimized in order to minimize transport limitations and to detect small affinity differences. Through this study we demonstrate that the physicochemical structure of SPR chips affects the apparent binding behaviors of biomolecules. When interpreting SPR binding curves and selecting a sensor chip, these effects should be taken into account.     

Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.     

Ritonavir binds to and downregulates estrogen receptors: Molecular mechanism of promoting early atherosclerosis

Estrogenic actions are closely related to cardiovascular disease. Ritonavir (RTV), a human immunodeficiency virus (HIV) protease inhibitor, induces atherosclerosis in an estrogen-related manner. However, how RTV induce pathological phenotypes through estrogen pathway remains unclear. In this study, we found that RTV increases thickness of coronary artery walls of Sprague Dawley rats and plasma free fatty acids (FFA) levels. In addition, RTV could induce foam cell formation, downregulate both estrogen receptor α (ERα) and ERβ expression, upregulate G protein-coupled estrogen receptor (GPER) expression, and all of them could be partially blocked by 17β-estradiol (E2), suggesting RTV acts as an antagonist for E2. Computational modeling shows a similar interaction with ERα between RTV and 2-aryl indoles, which are highly subtype-selective ligands for ERα. We also found that RTV directly bound to ERα and selectively inhibited the nuclear localization of ERα, and residue Leu536 in the hydrophobic core of ligand binding domain (LBD) was essential for the interaction with RTV. In addition, RTV did not change the secondary structure of ERα-LBD like E2, which explained how ERα lost the capacity of nuclear translocation under the treatment of RTV. All of the evidences suggest that ritonavir acts as an antagonist for 17β-estradiol in regulating α subtype estrogen receptor function and early events of atherosclerosis.     

Unexpected equivalent potency of a constrained chromene enantiomeric pair rationalized by co-crystal structures in complex with estrogen receptor alpha

Despite tremendous progress made in the understanding of the ERα signaling pathway and the approval of many therapeutic agents, ER+?breast cancer continues to be a leading cause of cancer death in women. We set out to discover compounds with a dual mechanism of action in which they not only compete with estradiol for binding with ERα, but also can induce the degradation of the ERα protein itself. We were attracted to the constrained chromenes containing a tetracyclic benzopyranobenzoxepine scaffold, which were reported as potent selective estrogen receptor modulators (SERMs). Incorporation of a fluoromethyl azetidine side chain yielded highly potent and efficacious selective estrogen receptor degraders (SERDs), such as 16aa and surprisingly, also its enantiomeric pair 16ab. Co-crystal structures of the enantiomeric pair 16aa and 16ab in complex with ERα revealed default (mimics the A-D rings of endogenous ligand estradiol) and core-flipped binding modes, rationalizing the equivalent potency observed for these enantiomers in the ERα degradation and MCF-7 anti-proliferation assays.     

Estrogen receptor alpha interacts with 17β-hydroxysteroid dehydrogenase type 10 in mitochondria

Estrogen receptor alpha (ERα) is present in the nucleus, the cytosol and in mitochondria. The rat ERα ligand binding domain was employed as bait in a bacterial two-hybrid screening of a human heart cDNA library to detect novel protein-protein interaction partners of ERα in the heart. 17β-Hydroxysteroid dehydrogenase type 10 (17β-HSD10), which converts potent (17β-estradiol) to less potent estrogens (estrone), co-localized with 17β-HSD10 in the mitochondria of rat cardiac myocytes. GST pull-down experiments confirmed the interaction of ERα and 17β-HSD10. These findings suggest that the ERα estrogen receptor might be involved in regulating intracellular estrogen levels by modulating 17β-HSD10 activity.     

Binding of the estrogen receptor to DNA. The role of waters.

Molecular dynamics simulations are carried out to investigate the binding of the estrogen receptor, a member of the nuclear hormone receptor family, to specific and non-specific DNA. Two systems have been simulated, each based on the crystallographic structure of a complex of a dimer of the estrogen receptor DNA binding domain with DNA. One structure includes the dimer and a consensus segment of DNA, ds(CCAGGTCACAGTGACCTGG); the other structure includes the dimer and a nonconsensus segment of DNA, ds(CCAGAACACAGTGACCTGG). The simulations involve an atomic model of the protein-DNA complex, counterions, and a sphere of explicit water with a radius of 45 A. The molecular dynamics package NAMD was used to obtain 100 ps of dynamics for each system with complete long-range electrostatic interactions. Analysis of the simulations revealed differences in the protein-DNA interactions for consensus and nonconsensus sequences, a bending and unwinding of the DNA, a slight rearrangement of several amino acid side chains, and inclusion of water molecules at the protein-DNA interface region. Our results indicate that binding specificity and stability is conferred by a network of direct and water mediated protein-DNA hydrogen bonds. For the consensus sequence, the network involves three water molecules, residues Glu-25, Lys-28, Lys-32, Arg-33, and bases of the DNA. The binding differs for the nonconsensus DNA sequence in which case the fluctuating network of hydrogen bonds allows water molecules to enter the protein-DNA interface. We conclude that water plays a role in furnishing DNA binding specificity to nuclear hormone receptors.