Bases moléculaires de la réactivité croisée des cupines 11S

International audienc

Ligands specify estrogen receptor alpha nuclear localization and degradation

Abstract Background The estrogen receptor alpha (ERα) is found predominately in the nucleus, both in hormone stimulated and untreated cells. Intracellular distribution of the ERα changes in the presence of agonists but the impact of different antiestrogens on the fate of ERα is a matter of debate. Results A MCF-7 cell line stably expressing GFP-tagged human ERα (SK19 cell line) was created to examine the localization of ligand-bound GFP-ERα. We combined digitonin-based cell fractionation analyses with fluorescence and immuno-electron microscopy to determine the intracellular distribution of ligand-bound ERα and/or GFP-ERα. Using fluorescence- and electron microscopy we demonstrate that both endogenous ERα and GFP-ERα form numerous nuclear focal accumulations upon addition of agonist, 17β-estradiol (E2), and pure antagonists (selective estrogen regulator disruptor; SERD), ICI 182,780 or RU58,668, while in the presence of partial antagonists (selective estrogen regulator modulator; SERM), 4-hydroxytamoxifen (OHT) or RU39,411, diffuse nuclear staining persisted. Digitonin based cell fractionation analyses confirmed that endogenous ERα and GFP-ERα predominantly reside in the nuclear fraction. Overall ERα protein levels were reduced after estradiol treatment. In the presence of SERMs ERα was stabilized in the nuclear soluble fraction, while in the presence of SERDs protein levels decreased drastically and the remaining ERα was largely found in a nuclear insoluble fraction. mRNA levels of ESR1 were reduced compared to untreated cells in the presence of all ligands tested, including E2. E2 and SERDs induced ERα degradation occurred in distinct nuclear foci composed of ERα and the proteasome providing a simple explanation for ERα sequestration in the nucleus. Conclusions Our results indicate that chemical structure of ligands directly affect the nuclear fate and protein turnover of the estrogen receptor alpha independently of their impact on transcription. These findings provide a molecular basis for the selection of antiestrogen compounds issue from pharmacological studies aimed at improving treatment of breast cancer.</p

Prédiction in silico de l'allergénicité des protéines

International audienc

Entomophagie et risque allergique

International audienceThough traditionally confined to some Asian, African and South American countries, consumption of edible insects, known as entomophagy, is gradually spreading to the USA and European countries. Although it remains rather limited, essentially for psychological reasons, in some European countries entomophagy is developing with the emergence of companies dedicated to the mass production of edible insects, together with the opening of restaurants specialized in menus featuring such insects. In spite of the nutritional interest and apparent safety of eating edible insects, it is advisable that we be aware of the allergic risk, which this may represent for people allergic to shellfish, mollusks or house dust mites. Various panallergens such as tropomyosin and arginine kinase, which are common to insects, crustaceans, mollusks, dust mites and nematodes, can be responsible for the cross-reactivity between these organisms of different origins. In addition to these panallergens, other allergens more specifically associated with insects could likewise trigger allergic reactions. However, these allergens are still not well known and remain to be identified and characterized. In the meantime and because of the existence of cross-reactive allergens in insects, it seems wise to advise individuals known to be allergic to shellfish or mollusks to avoid eating edible insects.Traditionnellement confinée à différents pays d’Asie, d’Afrique et d’Amérique du Sud, la consommation d’insectes ou entomophagie commence à s’étendre à l’Europe et aux États-Unis. Bien que très limitée, surtout pour des raisons psychologiques, l’entomophagie tend à se développer avec l’émergence, dans différents pays d’Europe, d’une production industrielle d’insectes comestibles, associée à l’ouverture de restaurants spécialisés dans des menus à base d’insectes. Malgré l’intérêt nutritionnel et l’apparente innocuité des insectes comestibles, il convient d’apprécier le risque allergénique qu’ils peuvent représenter pour des sujets allergiques aux crustacés, aux acariens ou aux mollusques. Divers pan-allergènes tels que la tropomyosine ou l’arginine-kinase, communs aux insectes, aux crustacés, aux acariens, aux mollusques et aux nématodes, pourraient être responsables de réactions croisées entre ces organismes d’origine différente. D’autres allergènes, plus spécifiques des insectes, pourraient également déclencher des réactions allergiques. Ces allergènes restent encore très mal connus et demandent à être identifiés et caractérisés. Dans cette attente et en raison de l’existence d’allergènes croisants chez les insectes, il paraît prudent de conseiller aux personnes allergiques aux crustacés ou aux mollusques, d’éviter de consommer ce genre de nourriture

Allergénicité des protéines édulcorantes: What about the allergenicity of sweet-tasting proteins?

International audiencePlant proteins with sweet-tasting properties are increasingly used as substitutes for low-calorie sweeteners in the food industry. Thaumatin, the sweet protein isolated from the aril of the katemfe fruit (Thaumatococcus daniellii), is widely used as a natural sweetener. Two other low-calorie sweeteners with enhanced sweet-tasting properties, brazzein and monellin, which are isolated from the fruits of Pentadiplandra brazzeana and Dioscoreophyllum cumminsii, respectively, are still waiting to be produced on a large scale as recombinant sweet proteins for food industry. Although these sweet-tasting proteins are constitutively expressed in fruits, most of them consist of PR-proteins whose synthesis is strongly enhanced as a result of infection of the plant by phytopathogenic micro-organisms, fungi or molds. Both the sequence and structural similarities which the sweet proteins share with allergens, e.g., Art v 1 for brazzein, Mus a 4 and Ole a 13 for thaumatin, Ana o 3 and Pis v 1 for mabinlin, and Gly m Kunitz trypsin-inhibitor for monellin, suggest some possible allergenic propensity for these plant proteins. However, their allergenic potential following ingestion still remains to be demonstrated unambiguously.Des protéines végétales à propriétés édulcorantes sont utilisées dans différents secteurs de l’alimentation humaine où elles remplacent les édulcorants de synthèse. C’est le cas de la thaumatine, protéine édulcorante extraite de l’arille des fruits du katemfe (Thaumatococcus daniellii). Deux autres protéines édulcorantes à pouvoir sucrant élevé, la brazzéine des fruits de Pentadiplandra brazzeana et la monelline des fruits de Dioscoreophyllum cumminsii, sont également disponibles, mais leur production industrielle n’est pas encore programmée. Bien qu’elles soient exprimées de façon constitutive dans les fruits, la plupart de ces protéines correspondent à des protéines de défense de la plante ou protéines PR, dont la synthèse est exacerbée lorsque la plante est en conditions de stress, lors d’une attaque fongique, par exemple. Les homologies de séquence, et surtout de structure, que ces protéines édulcorantes partagent avec des allergènes avérés, avec Art v 1 pour la brazzéine, avec Mus a 4 ou Ole e 13 pour la thaumatine, avec Ana o 3 ou Pis v 1 pour la mabinline, avec l’inhibiteur de Kunitz du soja pour la monelline, suggèrent une possible allergénicité de ces protéines végétales. Néanmoins, leur potentiel allergénique reste à démontrer lors de leur consommation alimentaire

Les allergènes croisants des insectes comestibles

International audienc

L’association Alt a 1 (Alternaria)–Act d 2 (kiwi) : origine et pertinence clinique possible

International audienceThe Alt a 1 allergen of Alternaria was reported recently to interact with the Act d 2 allergen which is found inside the pulp of kiwi fruit. This association is due to an as yet unknown interaction between these two allergens. Preceding to this interaction, Alt a 1 has to spread from the spores of Alternaria which were germinating on the surface of the fruit and then migrate into the pulp of the fruit where it associates with Act d 2. Such migration requires that Alt a 1 has the capacity to pass through the kiwi's exterior wall and then to pass through the plasma membrane of the pulp cells. To react with the kiwi's pulp cells, Alt a 1 enters the fruit via the lenticels that are scattered on its surface and then enters the pulp cells using plasma membrane protein transporters. Once inside these cells, Alt a 1 can associate with Act d 2 by means of electrostatic, hydrophilic and/or hydrophobic interactions, as suggested by in silico molecular docking experiments. In addition to this protein–protein interaction, the glycan moiety of N-glycosylated Alt a 1 could be recognized by the electro-negatively charged groove of Act d 2, resulting in an enhanced Alt a 1–Act d 2 association. As with kiwi fruit, Alt a 1 might associate with PR5 thaumatin-like proteins occurring in other fruits, such as cherry (Pru av 2), apple (Mal d 2), and banana (Mus a 4). The potential clinical relevance of this association is illustrated by the high frequency of cosensitizations observed between Act d 2 and Alt a 1 in ISAC microarray assays; up to 85% of patients sensitized to Act d 2 are also sensitized to Alt a 1. Conversely, only 39% of patients sensitized to Alt a 1 are also cosensitized to Act d 2.Récemment, l’association de l’allergène Act d 2 du kiwi avec l’allergène Alt a 1 d’Alternaria a été mise en évidence dans la pulpe du fruit. Cette association repose sur une interaction entre les deux allergènes dont le mécanisme reste encore mal connu. L’allergène Alt a 1 diffuse à partir de la paroi des spores d’Alternaria germant à la surface du fruit, pour migrer dans la pulpe du fruit où il s’associe avec Act d 2. Cette pénétration de l’allergène dans le fruit implique nécessairement un franchissement des parois et des membranes cellulaires du fruit par l’allergène. Alt a 1 emprunterait les lenticelles (stomates morts) pour franchir les parois cellulaires et des transporteurs membranaires spécifiques pour pénétrer dans les cellules de la pulpe. L’association des deux allergènes au sein de la pulpe s’effectuerait grâce à des interactions électrostatiques, hydrophiles et hydrophobes comme le suggèrent des expériences d’ancrage moléculaire in silico. Une reconnaissance des chaînes glycaniques d’Alt a 1 par la crevasse catalytique d’Act d 2, pourrait également intervenir dans cette association. D’autres fruits comme la banane ou la pomme, riches en protéines PR5 thaumatin-like, pourraient présenter ce type d’association. La pertinence clinique possible de cette association repose sur l’observation d’une fréquence élevée de cosensibilisations entre Act d 2 et Alt a 1 : 85 % des patients sensibilisés à Act d 2 le sont également à Alt a 1. Par contre, seulement 39 % des patients sensibilisés à Alt a 1 le sont également à Act d 2

Molecular and Biochemical Analysis of the Estrogenic and Proliferative Properties of Vitamin E Compounds

International audienceTocols are vitamin E compounds that include tocopherols (TPs) and tocotrienols (TTs). These lipophilic compounds are phenolic antioxidants and are reportedly able to modulate estrogen receptor β (ERβ). We investigated the molecular determinants that control their estrogenicity and effects on the proliferation of breast cancer cells. Docking experiments highlighted the importance of the tocol phenolic groups for their interaction with the ERs. Binding experiments confirmed that they directly interact with both ERα and ERβ with their isoforms showing potencies in the following order: δ-tocols > γ-tocols > α-tocols. We also found that tocols activated the transcription of an estrogen-responsive reporter gene that had been stably transfected into cells expressing either ERα or ERβ. The role of the phenolic group in tocol-ER interaction was further established using δ-tocopherylquinone, the oxidized form of δ-TP, which had no ER affinity and did not induce ER-dependent transcriptional modulation. Tocol activity also required the AF1 transactivation domain of ER. We found that both δ-TP and δ-TT stimulated the expression of endogenous ER-dependent genes. However, whereas δ-TP induced the proliferation of ER-positive breast cancer cells but not ER-negative breast cancer cells, δ-TT inhibited the proliferation of both ER-positive and ER-negative breast cancer cells. These effects of δ-TT were found to act through the down regulation of HMG-CoA reductase (HMGR) activity, establishing that ERs are not involved in this effect. Altogether, these data show that the reduced form of δ-TP has estrogenic properties which are lost when it is oxidized, highlighting the importance of the redox status in its estrogenicity. Moreover, we have shown that δ-TT has antiproliferative effects on breast cancer cells independently of their ER status through the inhibition of HMGR. These data clearly show that TPs can be discriminated from TTs according to their structure

Les allergènes croisants des insectes comestibles

International audienceThe increasing production and consumption of insect proteins as substitutes for meat proteins in European countries raises the question of their safety in terms of risk of allergy, of bacteriological and parasitological infection, and of toxicological contamination. The close relationship between insects with other arthropods (schrimps, dust mites) suggests the occurrence of an allergenic risk associated to entomophagy. In this respect, a few cases of anaphylactic reactions following the consumption of edible insects have been reported in the literature. However, the responsible allergens still remain poorly investigated, like most of the other insect proteins. Potential allergens have been recently identified in various species of edible insects like the yellow mealworm (Tenebrio molitor) and the silkworm (Bombyx mori). These allergenic proteins have been identified owing to their IgE-binding cross-reactivity toward patient sera allergic to shrimps and dust mites. The IgE-binding cross-reacting allergens of edible insects essentially correspond to enzymes (α-amylase, arginine kinase, chitinase, glutathione-S-transferase, triose phosphate isomerase, trypsin), hæmolyphatic proteins (hæmocyanin, hexamerin), and muscle proteins (actin, sarcoplasmic Ca-binding protein, myosin, tropomyosin, troponin). All of these potential allergens consist of ubiquitous proteins sharing three-dimensional structures and functions, which have been readily conserved during the evolution of arthropods and molluscs. Accordingly, most of these conserved structures and functions also occur in the allergenic proteins of both the German (Blattella germanica) and American (Periplaneta americana) cockroaches. However, the occurrence of specific allergens in edible insects has been not yet reported.Le recours aux protéines d’insectes comme substituts des protéines d’origine animale dans l’alimentation commence à s’intensifier dans plusieurs pays de l’Union européenne. La production (entomoculture) et la consommation (entomophagie) des insectes comestibles posent la question de leur innocuité en termes de risque allergique, microbiologique, parasitologique et toxicologique. La parenté étoite des insectes avec d’autres arthropodes (crustacés, acariens) suggère l’existence d’un risque allergique associé à l’entomophagie, d’autant que des cas d’anaphylaxie consécutifs à la consommation d’insectes ont été rapportés dans la littérature. Les allergènes responsables sont encore mal connus, comme d’ailleurs les protéines des insectes dont la diversité reste à analyser. Cependant, plusieurs allergènes potentiels ont été identifiés dans différentes espèces d’insectes comestibles. Ceux du ver de farine (Tenebrio molitor) et du ver à soie (Bombyx mori) ont été particulièrement analysés. Ces allergènes ont été identifiés à l’aide de sérums de patients allergiques aux crustacés ou aux acariens. Ces allergènes croisants correspondent essentiellement à des enzymes (α-amylase, arginine kinase, chitinase, glutathion-S-transférase, triose phosphate isomérase, trypsine), des protéines circulantes (hémocyanine, hexamérine) et des protéines musculaires (actine, sarcoplasmic Ca-binding protein, myosine, tropomyosine, troponine). Tous ces allergènes sont des protéines ubiquitaires dont les structures et les fonctions ont été parfaitement conservées au cours de l’évolution des arthropodes et des mollusques. On les retrouve en particulier dans les allergènes de contact des blattes Européenne (Blattella germanica) et Américaine (Periplaneta americana). Par contre, l’existence d’allergènes spécifiques des insectes comestibles n’a pas encore été rapportée