Search

Supplementary Methods and Figures 1-12 from Different Roles of Estrogen Receptors α and β in the Regulation of E-Cadherin Protein Levels in a Mouse Mammary Epithelial Cell Line

Two estrogen receptors (ERα and ERβ) are found throughout the mammary gland. Evidence indicates that, while ERα transduces proliferation signals, ERβ opposes this effect and is necessary for epithelial differentiation. Using mouse mammary epithelial cells, we have previously shown that activation of ERβ opposes ERα-induced proliferation and increases apoptosis. Furthermore, stable knockdown of ERβ resulted in loss of growth contact inhibition. In this work, we report that loss of ERβ is associated with a decrease of E-cadherin protein levels through different posttranscriptional regulatory mechanisms. Ligand activation of ERα induced E-cadherin extracellular shedding and internalization only in the absence of ERβ, followed by lysosomal degradation. Loss of ERβ also led to an increase of E-cadherin uptake in a ligand-independent manner through mechanisms that required caveolae formation. Proteasome activity was necessary for both mechanisms to operate. Increased E-cadherin internalization correlated with the up-regulation of β-catenin transcriptional activity and impaired morphogenesis on Engelbreth-Holm-Swarm matrix. Taken together, these results emphasize the role of epithelial ERβ in maintaining cell adhesion and a differentiated phenotype and highlight the potential importance of ERβ for the design of specific agonists for use in breast cancer therapy. [Cancer Res 2008;68(21):8695–704]

High-pressure sodium (HPS) lighting is increasingly replaced by LED lighting in lettuce greenhouse cultivation. In contrast to HPS lighting, LEDs do not heat radiation. Therefore, the leaf temperature is significantly lower under LEDs. This raises the question of whether LED lighting has a positive impact on the reduction in water consumption during lettuce production. In this paper, we investigated this question and found that the water consumption of lettuce produced under LEDs was significantly lower (−15%) than under HPS without loss of yield. We also found that supplementary lighting increases the concentrations of caffeoylquinic acid, dicaffeoyltartaric acid, dicaffeoylquinic acid and that of the total phenolic compounds in lettuce leaves by 61%, 39%, 163% and 38%, respectively. Only the LED fixture was also efficient enough to increase the concentration of caffeoyltartaric acid (+24%). Most of the phenolic compounds showed a very strong positive correlation with the chlorophyll concentration in lettuce, which predominated in the leaves exposed to the LED lighting. Based on these facts, we conclude that by optimizing the light composition, more sustainable plant production, higher concentrations of chlorophyll and some phenolic compounds are possible. Bundesministerium für Bildung und Forschung

Summary Light quality affects plant morphology. Hence, light quality is an important factor in the cultivation of ornamental plants. The recent advances in LED technology allow producers to adjust light quality to manipulate plant growth. For growers of ornamental plants the primary aim regarding plant morphology is to keep plants short and compact with attractive flowers. However, data on the effect of light quality on plant morphology are still sparse. This study provides data on the effect of different LED light regimes on height and fresh weight of shoot and flower, leaf area and compactness of the model plant Helianthus annuus. Regression analysis of these data shows that red light promotes compact growth.

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti(4+)-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO(2) enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti(4+)-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 μg of a HeLa cell lysate digest. In comparison, ∼ 2000 unique phosphopeptides could be identified by Ti(4+)-IMAC with HFP and close to 3000 by TiO(2). We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti(4+)-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 μg of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti(4+)-IMAC is particularly advantageous for phosphopeptides with multiple basic residues.

SummaryZP3, a major component of the zona pellucida (ZP) matrix coating mammalian eggs, is essential for fertilization by acting as sperm receptor. By retaining a propeptide that contains a polymerization-blocking external hydrophobic patch (EHP), we determined the crystal structure of an avian homolog of ZP3 at 2.0 Å resolution. The structure unveils the fold of a complete ZP domain module in a homodimeric arrangement required for secretion and reveals how EHP prevents premature incorporation of ZP3 into the ZP. This suggests mechanisms underlying polymerization and how local structural differences, reflected by alternative disulfide patterns, control the specificity of ZP subunit interaction. Close relative positioning of a conserved O-glycan important for sperm binding and the hypervariable, positively selected C-terminal region of ZP3 suggests a concerted role in the regulation of species-restricted gamete recognition. Alternative conformations of the area around the O-glycan indicate how sperm binding could trigger downstream events via intramolecular signaling.

The estrogen receptors (ERs) are ligand-dependent transcription factors that activate transcription by binding to estrogen response elements. Estrogen-mediated effects are tissue- and cell type-specific, determined by the cofactor recruitment to the ERs among other factors. To understand these differences in estrogen action, it is important to identify the various compositions of the ER complexes (ER receptosomes). In this report, we describe a fast and efficient method for the isolation of the ERalpha receptosome for proteomics analysis. Using immobilized estrogen response element on a Sepharose column in combination with two-dimensional electrophoresis and MALDI-TOF MS, significant amounts of proteins could be isolated and identified. Differences in ERalpha complex composition with the ER ligands 17beta-estradiol, 4-hydroxytamoxifen, and ICI-182,780 could also be observed. Thus, this approach provides an easy and relevant way of identifying ERalpha cofactor and transcription factor recruitment under different conditions.

This study reports on the spatiotemporal dynamics of the expression of estrogen receptors (ERs) in the mouse central nervous system (CNS) during the early postnatal and the peripubertal period. At postnatal day 7 (P7), neurons with strong nuclear immunostaining for both ERalpha and ERbeta1 were widely distributed throughout the brain. Sucrose density gradient sedimentation followed by western blotting supported the histochemical evidence for high levels of both ERs at P7. Over the following 2 days, there was a rapid downregulation of ERs. At P9, ERalpha expression was visible only in the hypothalamic area. Decline in ERbeta1 expression was slower than that of ERalpha, and ERalpha-negative, ERbeta1-positive cells were observed in the dentate gyrus and walls of third ventricle. Between P14 and P35, ERs were undetectable except for the hypothalamic area. As before P7, the ovary does not produce estrogen but does produce 5alpha-androstane-3beta, 17beta-diol (3betaAdiol), an estrogenic metabolite of dihydrotestosterone, we examined the effects of high levels of 3betaAdiol in the postnatal period. We used CYP7B1 knockout mice which cannot hydroxylate and inactivate 3betaAdiol. The brains of these mice are abnormally large with reduced apoptosis. In the early postnatal period, there was 1-week delay in the timing of the reduction in ER expression in the brain. These data reveal that the time when ERs might be activated in the brain is limited to the first 8 postnatal days. In addition, the importance of aromatase has to be reconsidered as the alternative estrogen, 3betaAdiol, is important in neuronal function in the postnatal brain.

Two estrogen receptors (ERα and ERβ) are found throughout the mammary gland. Evidence indicates that, while ERα transduces proliferation signals, ERβ opposes this effect and is necessary for epithelial differentiation. Using mouse mammary epithelial cells, we have previously shown that activation of ERβ opposes ERα-induced proliferation and increases apoptosis. Furthermore, stable knockdown of ERβ resulted in loss of growth contact inhibition. In this work, we report that loss of ERβ is associated with a decrease of E-cadherin protein levels through different posttranscriptional regulatory mechanisms. Ligand activation of ERα induced E-cadherin extracellular shedding and internalization only in the absence of ERβ, followed by lysosomal degradation. Loss of ERβ also led to an increase of E-cadherin uptake in a ligand-independent manner through mechanisms that required caveolae formation. Proteasome activity was necessary for both mechanisms to operate. Increased E-cadherin internalization correlated with the up-regulation of β-catenin transcriptional activity and impaired morphogenesis on Engelbreth-Holm-Swarm matrix. Taken together, these results emphasize the role of epithelial ERβ in maintaining cell adhesion and a differentiated phenotype and highlight the potential importance of ERβ for the design of specific agonists for use in breast cancer therapy. [Cancer Res 2008;68(21):8695–704]

Uromodulin (or Tamm-Horsfall protein) is the most abundant protein in human urine under physiological conditions. Little is known about the molecular mechanism of uromodulin secretion. By extensive Mass Spectrometry analyses we mapped the C-termini of human and murine urinary proteins demonstrating that urinary uromodulin is generated by a conserved C-terminal proteolytic cleavage and retains its entire ZP domain. (c) 2008 Elsevier Inc. All rights reserved.

Interstitial cystitis/painful bladder syndrome is a disease seen mostly in women, and symptoms tend to be worse premenopausally or during ovulation. The four cardinal symptoms of interstitial cystitis/painful bladder syndrome are bladder pain, urgency, frequency, and nocturia. Estrogen has been implicated in the etiology of this disease, but the role of the two estrogen receptors (ER), ERα and ERβ, has not been investigated. We found that, in the bladders of WT mice, ERβ is expressed in the basal cell layer of the urothelium. Bladders of male ERβ −/− mice were intact and morphologically indistinguishable from those of their WT littermates. However, in female ERβ −/− mice, there was ulceration and atrophy of bladder urothelium concomitant with infiltration of γδ T cells concentrated in the areas of atrophy and shedding of urothelium. The data support the idea that activated γδ T cells are causing the damage to the urothelium. The hyperactivity of T cells may be because of an imbalance between ERα and ERβ signaling in female ERβ −/− mice. Our data suggest that reduced ERβ signaling might have a role in the pathogenesis of interstitial cystitis, and ERβ could be a candidate for a target of medical therapy.

The human mitochondrial transcription termination factor (mTERF) is a 39-kDa protein that terminates transcription at the 3'-end of the 16 S rRNA gene and thereby controls expression of the ribosomal transcription unit of mitochondrial DNA. The transcription termination activity of human mTERF has been notoriously difficult to study in vitro, and it has been suggested that the activity of the protein is regulated by posttranslational modifications or by protein polymerization. We here characterize the activity of recombinant human mTERF expressed in insect cells. We observed that mTERF efficiently promotes sequence-specific termination in a completely recombinant and highly purified in vitro system for mitochondrial transcription. The termination activity has a distinct polarity, and we observed complete transcription termination when the mTERF-binding site is oriented in a forward position relative the heavy strand promoter but only partial transcription termination when the binding site is in the reverse position. We analyzed the biochemical characteristics of the active mTERF protein and found that it is a stable monomer at physiological salt concentration. Structural analysis, including phosphostaining, two-dimensional electrophoresis, and electrospray mass spectrometry, detected no evidence of phosphorylation. We conclude that the monomeric human mTERF is fully active in its non-phosphorylated form and that the protein does not require additional cellular factors to terminate mitochondrial transcription in vitro.

The report of elevated acrylamide levels in some foods raised an international health alarm, because acrylamide probably has carcinogenic, neurotoxic, and genotoxic properties. However, data on the bioavailability of acrylamide from food matrices in humans are limited. In particular, only little is known about the interactions of acrylamide with food ingredients. Using a human intestine model (Caco-2 cells), this study shows that acrylamide monomers are highly bioavailable and pass the cell monolayer via passive diffusion. Furthermore, acrylamide binds to dietary proteins such as chicken egg albumin under intestinal and cooking conditions. This binding reduces the concentration of acrylamide monomers and leads to a reduced uptake by Caco-2 cells. Hence, it is concluded that a protein-rich diet may reduce acrylamide uptake.

Lupinus albus cell cultures secrete a large set of hydrolases into their medium with a small number of highly abundant proteins. We have investigated the protein composition of the medium with two different methods, two-dimensional gel electrophoresis-electrospray ionisation tandem mass spectrometry (ESI-MS-MS) and enzymatic analysis. The proteomic approach revealed the presence of several abundant proteins that had been overlooked using standard enzyme assays, e.g. subtilisin-like protease, glucan 1,3-beta-glucosidase, alpha-amylase, chitinase, thaumatin-like protein, and a secretory pathogenesis-related protein. Several low-abundant proteins were readily detectable by enzymatic assays (peroxidases, phosphatase), but could not be found by ESI-MS-MS. Both data sets support the assumed lytic function of the medium, which appears to be similar to that of the plant vacuole.

Crystal and small-angle X-ray scattering structures of full-length human SUFU alone and in complex with the conserved SYGHL motif from GLI transcription factors show major conformational changes associated with binding and reveal an intrinsically disordered region crucial for pathway activation., Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU–GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU–GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

Acrylamide is a reactive neurotoxin with a high intestinal bioavailability. Recently we have shown that under the pH regime of the gut acrylamide can react with proteins and that this reaction reduces the uptake of acrylamide in a gut model. On the other hand, using radioactive labeled acrylamide, Bjellaas et al. [Toxicol. Sci. 100, 374-380 (2007)] showed that in vivo the vast majority of orally administered acrylamide is absorbed and excreted as N-acetyl-S- (3-amino-3-oxopropyl)-cysteine with the urine. Therefore, we tested whether intestinal proteases can degrade a protein with acrylamide bound to cysteine residues. Furthermore we tested whether the product of this reaction, S-(3-amino-3-oxopropyl)-cysteine, can pass the intestinal barrier. Here we showed that S-(3-amino-3-oxopropyl)-cysteine is indeed a product of proteolytic degradation of acrylamide-treated proteins. Using Caco-2 cells as a gut model, we further showed that the non-protein amino acid S-(3-amino-3-oxopropyl)-cysteine is a substrate for the neutral and cationic amino acid transporter system. Hence we concluded that protein-bound acrylamide can be released in the intestine and that the resulting product S-(3-amino-3-oxopropyl)-cysteine is transported through the intestinal barrier and later excreted via the urine.

The first step in mammalian fertilization, where both species specificity and the one-sperm-per-egg norm are enforced, is recognition between coat proteins in the membrane around the egg (the zona pellucida) and sperm. The mouse zona pellucida protein ZP3 is the primary receptor for sperm and its most conserved domain (ZP-N) is also found in many different extracellular proteins with a wide range of biological functions. The crystal structure of the ZP-N domain of ZP3 has now been determined. ZP-N adopts an immunoglobulin-like fold but shares little similarity with other immunoglobulin-like domains, marking it out as a new subtype of this superfamily. As well as providing a glimpse of mammalian fertilization at atomic resolution, the structure of ZP3 ZP-N is of relevance for reproductive medicine and also for diseases such as non-syndromic deafness or renal and vascular disorders, where ZP-N containing proteins are involved. Species-specific recognition between the egg extracellular matrix (zona pellucida) and sperm is the first step of mammalian fertilization. This paper reports the 2.3 A resolution structure of the 'zona pellucida filament' of the egg, which act as sperm receptors. The structure supports the presence of ZP–N repeats within the amino-terminal region of ZP2 and other vertebrate zona pellucida/vitelline proteins, and has implications for egg coat architecture and the post-fertilization block to polyspermy and speciation. Species-specific recognition between the egg extracellular matrix (zona pellucida) and sperm is the first, crucial step of mammalian fertilization1. Zona pellucida filament components ZP3 and ZP2 act as sperm receptors, and mice lacking either of the corresponding genes produce oocytes without a zona pellucida and are completely infertile2. Like their counterparts in the vitelline envelope of non-mammalian eggs and many other secreted eukaryotic proteins, zona pellucida subunits polymerize using a ‘zona pellucida (ZP) domain’ module3,4,5, whose conserved amino-terminal part (ZP-N) was suggested to constitute a domain of its own6. No atomic structure has been reported for ZP domain proteins, and there is no structural information on any conserved vertebrate protein that is essential for fertilization and directly involved in egg–sperm binding. Here we describe the 2.3 angstrom (A) resolution structure of the ZP-N fragment of mouse primary sperm receptor ZP3. The ZP-N fold defines a new immunoglobulin superfamily subtype with a β-sheet extension characterized by an E′ strand and an invariant tyrosine residue implicated in polymerization. The structure strongly supports the presence of ZP-N repeats within the N-terminal region of ZP2 and other vertebrate zona pellucida/vitelline envelope proteins, with implications for overall egg coat architecture, the post-fertilization block to polyspermy and speciation. Moreover, it provides an important framework for understanding human diseases caused by mutations in ZP domain proteins and developing new methods of non-hormonal contraception.

Monosaccharide transporter (MST) genes of Lupinus polyphyllus and L. albus were cloned, expressed and characterised. The isolation and functional characterisation of a cDNA clone and its corresponding genomic clone of a sugar transporter from L. polyphyllus (LpSTP1) is reported. Phylogenetic comparison of the nucleic and amino acid sequences showed the highest similarity to the AtSTP1 gene from Arabidopsis thaliana, which encodes a high affinity sugar transporter. The similar topology as well as the substrate specificity and expression pattern of LpSTP1 encoded protein additionally support the high similarity to the AtSTP1 gene product. The 1,590 bp LpSTP1 cDNA clone was heterologously expressed in yeast resulting in a fully functional specific sugar transporter. This transformation restored the viability of a yeast deletion mutant, which is devoid of all intrinsic MSTs and thus unable to take up and grow on hexose-containing media. The LpSTP1 protein is postulated to be a high-affinity MST since it supported growth best on media containing 0.2% hexose. Tissue-specific expression of LaSTP1 in L. albus was assayed by real-time PCR, which revealed that the lupin STP1 is mainly expressed in flower buds, flowers and young leaves. The results suggest that the main role of LaSTP1 is to catalyse monosaccharide import in sink tissues to meet increased carbohydrate demand during plant development.

Recently, Yang et al. reported that estrogen receptor beta (ERbeta) is a mitochondrial protein rather than a nuclear receptor. Because this claim would lead to a significant change in our understanding of estrogen signaling, we have attempted to reproduce the MALDI-TOF data of Yang et al. We separated proteins extracted from mouse liver mitochondria by SDS-PAGE and analysed a gel band covering the molecular weight range of 50-65 kDa by MALDI-TOF/TOF. Analysis of the data with the MASCOT database algorithm provided no evidence for the presence of ERbeta in the mitochondria. If we search (as the authors did) with only the peptide masses which match to tryptic fragments of ERbeta, ERbeta is identified with a significant score of 69. However, fragmentation of these peptides shows that they are not from ERbeta. Our conclusion is that ERbeta cannot be identified by MALDI-TOF from a mixture of mitochondrial proteins resolved on SDS-PAGE.

Several papers report that the colon is one of the tissues regulated by estrogen receptor (ER)β. To better understand the physiological role of ERβ in colonic tissue, we have compared morphology, proliferation, and differentiation of colonic epithelium in ERβ −/− mice and WT littermates. BrdUrd labeling revealed that the number of proliferating cells was higher in ERβ −/− mice and that the migration of labeled cells toward the luminal surface was faster in ERβ −/− mice than in WT littermates. Additionally, in the absence of ERβ, there was a decrease in apoptosis, which was measured by immunohistochemical staining of cleaved caspase-3. The state of differentiation of the colonic epithelial cells was studied by using epithelial markers. In ERβ −/− mice, there was a significant decrease in the expression of the differentiation marker cytokeratin (CK)20 and in the cellular adhesion molecules α-catenin (an adherens junction protein) and plectin (a hemidesmosomal protein). These changes were also evident by electron microscopy as abnormalities in tight junctions and in the number and shape of desmosomes in ERβ −/− mice. These findings suggest a role for ERβ in the organization and architectural maintenance of the colon. Furthermore, our results indicate that the rapidly proliferating cells of the colonic epithelium in ERβ −/− mice are lost by increased shedding and not by increased apoptosis. In this way, hyperproliferative cells that lack ERβ do not form hyperplastic lesions and do not accumulate in the superficial epithelium.

Abnormally large, heavy brain with a decreased number of apoptotic cells in CYP7B1 knockout mice

Acrylamide is an α,β-unsaturated vinyl monomer that causes cytotoxicity due to its alkylating properties. In recent years several proteins have been identified that are alkylated by acrylamide in vivo. This finding might explain the neurotoxic effects of acrylamide in humans. However, the list of potential acrylamide target proteins is far from being complete. In particular, the proteins that mediate the cytotoxicity of acrylamide in cell cultures remained unknown. Here we identify two novel acrylamide target proteins in human cell cultures (Jurkat, HepG2 and Caco-2), adenosine deaminase and thioredoxin