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Identification of a Novel Protein Binding to the Endocrine Disruptor Nonylphenol
Abstract
Nonylphenol is an alkyl phenol generated through microbial degradation of non-ionic surfactants in rivers as well as by oxidation and hydrolysis of oxidants in plastic products. Therefore, it is important to assess the endocrine disruption effects of nonylphenol ingested via food and to clarify the molecular mechanism involved. Nonylphenol primarily targets the male reproductive (testes and prostate) system, and may be involved in insufficiency of sperm differentiation and degeneration of the testes and prostate in F1 and F2 mice exposed to nonylphenol at the prenatal stage. In this study, we isolated the human protein NPR1 (Nonylphenol Receptor 1) from a normal human prostate cDNA library as a protein binding to a hormone receptor binding domain of histone acetyltransferase AIB1 in the presence of nonylphenol. Molecular interaction analysis revealed that NPR1 binds nonylphenol with high specificity. Therefore, in addition to the endocrine disruption mechanism occurring through the estrogen receptor, a new mechanism acting via NPR1 was suggested.
... EDs are thought to be toxic primarily to the human reproductive system (genital cancer and decreased sperm count), immune system (asthma and atopic dermatitis), and nervous system (developmental disorders). It has also been reported that the sensitivity to EDs is particularly high in the fetal stage and during childhood [47]. Although only persistent pollutants, such as polychlorinated biphenyl (PCBs), dioxins, and DDT, were originally identified as EDs [48], many other chemicals from different groups, including non-persistent pesticides, phenols, and phthalates, have since been identified as EDs [49][50][51][52][53][54][55]. ...
Neonicotinoid pesticides (NPs) are neurotoxic substances. They are highly effective as insecticides owing to their water solubility, permeability, and long-lasting activity. These molecules are structurally similar to nicotine and act as nicotinic acetylcholine receptor agonists. The administration of NPs to experimental animals reportedly causes neuromuscular and reproductive disorders. Moreover, recently reported problems caused by NPs include damage to land-dwelling creatures (such as mammals and birds), hydrobiology, and ecosystems. This review summarizes the recent reports on NP concentrations detected in river systems in several Japanese regions. These values were lower than the environmental standard values; however, seasonal variations were observed. Furthermore, reports on NP-induced testicular and ovarian toxicity were examined, revealing that the mechanism of injury is mainly driven by oxidative stress. The use of NPs is declining worldwide, except in Japan; therefore, continuous monitoring remains necessary.
Background: Endocrine-disrupting chemicals (EDCs) influence the activity of estrogen receptors (ERs) and alter the function of the endocrine system. However, the diversity of EDC effects and mechanisms of action are poorly understood.
Objectives: We examined the agonistic activity of EDCs through ERα and ERβ. We also investigated the effects of EDCs on ER-mediated target genes.
Methods: HepG2 and HeLa cells were used to determine the agonistic activity of EDCs on ERα and ERβ via the luciferase reporter assay. Ishikawa cells stably expressing ERα were used to determine changes in endogenous ER target gene expression by EDCs.
Results: Twelve EDCs were categorized into three groups on the basis of product class and similarity of chemical structure. As shown by luciferase reporter analysis, the EDCs act as ER agonists in a cell type– and promoter-specific manner. Bisphenol A, bisphenol AF, and 2-2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (group 1) strongly activated ERα estrogen responsive element (ERE)-mediated responses. Daidzein, genistein, kaempferol, and coumestrol (group 2) activated both ERα and ERβ ERE-mediated activities. Endosulfan and kepone (group 3) weakly activated ERα. Only a few EDCs significantly activated the “tethered” mechanism via ERα or ERβ. Results of real-time polymerase chain reaction indicated that bisphenol A and bisphenol AF consistently activated endogenous ER target genes, but the activities of other EDCs on changes of ER target gene expression were compound specific.
Conclusion: Although EDCs with similar chemical structures (in the same group) tended to have comparable ERα and ERβ ERE-mediated activities, similar chemical structure did not correlate with previously reported ligand binding affinities of the EDCs. Using ERα-stable cells, we observed that EDCs differentially induced activity of endogenous ER target genes.
Fos family proteins form stable heterodimers with Jun family proteins, and each heterodimer shows distinctive transactivating
potential for regulating cellular growth, differentiation, and development via AP-1 binding sites. However, the molecular
mechanism underlying dimer specificity and the molecules that facilitate transactivation remain undefined. Here, we show that
BAF60a, a subunit of the SWI·SNF chromatin remodeling complex, is a determinant of the transactivation potential of Fos/Jun
dimers. BAF60a binds to a specific subset of Fos/Jun heterodimers using two different interfaces for c-Fos and c-Jun, respectively.
Only when the functional SWI·SNF complex is present, can c-Fos/c-Jun (high affinity to BAF60a) but not Fra-2/JunD (no affinity
to BAF60a) induce the endogenous AP-1-regulated genes such as collagenase and c-met. These results indicate that a specific subset of Fos/Jun dimers recruits SWI·SNF complex via BAF60a to initiate transcription.
Migration of bisphenol A (BPA) from epoxy-coated can surfaces to infant formula concentrates is reported. Levels of BPA in the undiluted concentrates surveyed in this study range from 0.1 to 13 parts per billion (ppb) as determined by solid phase extraction/high-pressure liquid chromatography with fluorescence detection and confirmation by gas chromatography with mass selective detection. Fourier transform infrared spectroscopy with 30° specular reflectance/transmittance was used to screen formula cans for epoxy coatings. Keywords: Bisphenol A; epoxy; can coating; migration; infant formula
Transcription activation by androgen receptor (AR), which depends on recruitment of coactivators, is required for the initiation and progression of prostate cancer, yet the mechanisms of how hormone-activated AR interacts with coactivators remain unclear. This is because AR, unlike any other nuclear receptor, prefers its own N-terminal FXXLF motif to the canonical LXXLL motifs of coactivators. Through biochemical and crystallographic studies, we identify that steroid receptor coactivator-3 (SRC3) (also named as amplified in breast cancer-1 or AIB1) interacts strongly with AR via synergistic binding of its first and third LXXLL motifs. Mutagenesis and functional studies confirm that SRC3 is a preferred coactivator for hormone-activated AR. Importantly, AR mutations found in prostate cancer patients correlate with their binding potency to SRC3, corroborating with the emerging role of SRC3 as a prostate cancer oncogene. These results provide a molecular mechanism for the selective utilization of SRC3 by hormone-activated AR, and they link the functional relationship between AR and SRC3 to the development and growth of prostate cancer.
Receptors for peptide hormones and neurotransmitters are integral components of the plasma membrane of cells which serve to couple the external milieu to the intracellular regulators of metabolism. These macromolecules are usually high molecular weight glycoproteins, and in many cases appear to have more than one subunit capable of binding the hormone. The interaction of the hormone or neurotransmitter with its receptor is rapid, reversible, and of high affinity and specificity. Many receptors exhibit cooperative properties in hormone binding or biological function. The concentration of receptors on the membrane is a function of continued synthesis and degradation, and may be altered by a variety of factors including the hormone itself. The fluid mosaic nature of the membrane may allow hormone receptors and effectors to exist in free floating states. Further investigations of the hormone-receptor interaction will no doubt yield new insights into both the mechanism of hormone action and membrane structure and function.
Many biological processes rely upon protein-protein interactions. Hence, detailed analysis of these interactions is critical for their understanding. Due to the complexities involved, genetic approaches are often needed. In yeast and phage, genetic characterizations of protein complexes are possible. However, in multicellular organisms, such characterizations are limited by the lack of powerful selection systems. Herein we describe genetic selections that allow single amino acid changes that disrupt protein-protein interactions to be selected from large libraries of randomly generated mutant alleles. The strategy, based on a yeast reverse two-hybrid system, involves a first-step negative selection for mutations that affect interaction, followed by a second-step positive selection for a subset of these mutations that maintain expression of full-length protein (two-step selection). We have selected such mutations in the transcription factor E2F1 that affect its ability to heterodimerize with DP1. The mutations obtained identified a putative helix in the marked box, a region conserved among E2F family members, as an important determinant for interaction. This two-step selection procedure can be used to characterize any interaction domain that can be tested in the two-hybrid system.
The aim of this study was to analyse the multigenerational effects of para-nonylphenol (NP) and resveratrol (RES) on the body weight, organ weight and reproductive fitness of outbred CD-1 mice. The data indicate that in male mice, NP had an effect on the weight of selected reproductive organs and the kidneys in the parental (P) generation males. Effects on selected reproductive organs, the liver and kidneys in the F1-generation males were also seen. In females, effects of NP on body weight and kidney weight were seen in the P generation, but no effects on any measured parameter were seen in the F1 generation. RES had no effect on body weight but did have some effect on selected male and female reproductive organs in the P generation. RES altered the spleen and liver weights of P-generation males and the kidney weight of F1-generation males. Acrosomal integrity (using a monoclonal antibody against intra-acrosomal sperm proteins) was assessed for both generations of NP- and RES-treated mice. A significant reduction in acrosomal integrity was seen in both generations of NP-treated, but not in RES-treated, mice. Fewer offspring were observed in the second litter of the F2 generation of mice treated with NP; no similar effect was seen in RES-treated mice. The litter sex ratio was not different from controls. Unlike RES, NP had a negative effect on spermatogenesis and sperm quality with a resultant impact on in vivo fertility.
1 - CAPLUS AN 2002:943098(Journal)
Analyses of steroid receptors are important for understanding molecular details of transcriptional control, as well as providing
insight as to how an individual transacting factor contributes to cell identity and function. These studies have led to the
identification of a superfamily of regulatory proteins that include receptors for thyroid hormone and the vertebrate morphogen
retinoic acid. Although animals employ complex and often distinct ways to control their physiology and development, the discovery
of receptor-related molecules in a wide range of species suggests that mechanisms underlying morphogenesis and homeostasis
may be more ubiquitous than previously expected.
A trichloroacetic-acid-soluble 14.5-kDa protein (p14.5) has been isolated from human mononuclear phagocytes (MNP) by a combination of trichloroacetic acid extraction, preparative electrophoresis and hydrophobic affinity chromatography; five tryptic peptides were subjected to protein sequencing. The full-length cDNA of the protein was cloned and sequenced from a lambda gt11 human liver library. The cDNA showed a remarkable similarity to a rat protein preferentially expressed in hepatocytes and renal tubular epithelial cells. The encoded protein is 137 amino acids long and similar to members of a new hypothetical family of small proteins with presently unknown function, named YER057c/YJGF. Human recombinant p14.5 inhibits in vitro protein synthesis in a rabbit reticulocyte lysate system. Unlike other inhibitors of protein synthesis, p14.5 is not phosphorylated despite the presence of putative phosphorylation sites. The p14.5 mRNA is weakly expressed in freshly isolated monocytes but is significantly upregulated when these monocytes are subjected to differentiation. This is also reflected by a differentiation-dependent increase in the protein concentration as demonstrated by immunoblots from cytosolic fractions and fluorescence-activated flow cytometry of permeabilized cells. A differentiation-dependent mRNA and protein expression of p14.5 is further suggested by the observation of a low expression in a variety of liver and kidney tumor cells and a high expression in fully differentiated cells as assessed by immunohistochemistry and northern blots. The highest mRNA expression was found in hepatocytes and renal distal tubular epithelial cells and only weak expression was found in other human tissues as evaluated by northern blot analysis. The preferential localization of the immunoreaction product seemed to be cytoplasmatic but, in less differentiated cells, nuclear labeling was occasionally visible. Immunoblotting of subcellular fractions confirmed these data. The high degree of evolutionary conservation of p14.5, the considerable upregulation during cellular differentiation and its potential role as a translational inhibitor may reflect an involvement in basic cellular mechanisms, e.g. a differentiation-dependent regulation of protein synthesis in hepatocytes, renal tubular epithelial cells, smooth muscle cells and MNP.
We report here the identification of a novel cofactor, ACTR, that directly binds nuclear receptors and stimulates their transcriptional activities in a hormone-dependent fashion. ACTR also recruits two other nuclear factors, CBP and P/CAF, and thus plays a central role in creating a multisubunit coactivator complex. In addition, and unexpectedly, we show that purified ACTR is a potent histone acetyltransferase and appears to define a distinct evolutionary branch to this recently described family. Thus, hormonal activation by nuclear receptors involves the mutual recruitment of at least three classes of histone acetyltransferases that may act cooperatively as an enzymatic unit to reverse the effects of histone deacetylase shown to be part of the nuclear receptor corepressor complex.
Co-factor ACTR is frequently overexpressed and/or amplified in multiple types of tumors. The mechanism of its function in prostate cancer (CaP) is still unclear.
The effects of ACTR and androgen receptor (AR) depletion on cell proliferation and gene expression and their functions were analyzed in a panel of androgen-dependent and -independent CaP cells and CWR22 xenograft.
ACTR and AR, but not TIF2, are required for proliferation of androgen-dependent and -independent cells, and for tumor growth. While AR depletion inhibited the expression of cyclin D1, cyclin B, and cdc2, ACTR depletion reduced the expression of cyclin E and cdk2. In response to serum stimulation, AR and ACTR are recruited to the corresponding target gene promoters to activate their expression in androgen-independent manner.
These findings suggest that AR and ACTR may play important roles in androgen ablation resistance by controlling key cell cycle gene expression.
Adverse health effects following prenatal exposures to methylmercury (MeHg) have been apparent from several prospective cohort studies conducted in a fish-eating population. A prospective study in a Faroese birth cohort documented subtle deficits of several functional domains at prenatal MeHg exposure levels previously thought to be safe. Recent additional studies also showed neurobehavioral deficits associated with exposures to polychlorinated biphenyls (PCBs) with concomitant MeHg poisoning. In contrast, a prospective study in the Seychelles did not detect a similar association between MeHg exposure and neurodevelopmental deficits; children of the highest MeHg exposure group showed better scores in some developmental tests than those of the lower exposure groups for both prenatal and postnatal MeHg exposures. This paradoxical difference between both studies is summarized herein. The primary source of human exposure to MeHg is fish. Since a considerable number of pollutants, including polychlorinated biphenyls (PCBs) and pesticides, are also present in fish, and since some organochemical substances including PCBs are also well documented to be neurotoxic to the developing brain from epidemiological studies, the combined effects of these pollutants should be considered in discussing the neurotoxicity of MeHg. In this article, therefore, major prospective cohort studies focusing on the exposures to PCBs were reviewed.
Biolayer interferometry is a novel method for quantifying macromolecules, such as proteins, in solution. The presence of other, non-binding molecules does not interfere with quantification, which allows one to measure the concentration of the molecule of interest in a crude mixture. Here we apply this method to determining the dynamic binding capacity of affinity resins.