Cd2+- or Hg2+-binding proteins can replace the Cu+-chaperone Atx1 in delivering Cu+ to the secretory pathway in yeast

AbstractCopper delivery to Ccc2 – the Golgi Cu+-ATPase – was investigated in vivo, replacing the Cu+-chaperone Atx1 by various structural homologues in an atx1-Δ yeast strain. Various proteins, displaying the same ferredoxin-like fold and (M/L)(T/S)CXXC metal-binding motif as Atx1 and known as Cu+-, Cd2+- or Hg2+-binding proteins were able to replace Atx1. Therefore, regardless of their original function, these proteins could all bind copper and transfer it to Ccc2, suggesting that Ccc2 is opportunistic and can interact with many different proteins to gain Cu+. The possible role of electrostatic potential surfaces in the docking of Ccc2 with these Atx1-homologues is discussed

Sushi in the United States, 1945-1970

Sushi first achieved widespread popularity in the United States in the mid-1960s. Many accounts of sushi’s US establishment foreground the role of a small number of key actors, yet underplay the role of a complex web of large-scale factors that provided the context in which sushi was able to flourish. This article critically reviews existing literature, arguing that sushi’s US popularity arose from contingent, long-term, and gradual processes. It examines US newspaper accounts of sushi during 1945–1970, which suggest the discursive context for US acceptance of sushi was considerably more propitious than generally acknowledged. Using California as a case study, the analysis also explains conducive social and material factors, and directs attention to the interplay of supply- and demand-side forces in the favorable positioning of this “new” food. The article argues that the US establishment of sushi can be understood as part of broader public acceptance of Japanese cuisine

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Etude de l ATPase cuivre eucaryote Ccc2 de Saccharomyces cerevisiae (de la localisation à la fonction )

Dans cette thèse, nous avons abordé l'étude du fonctionnement de Ccc2, l'ATPase à cuivre de S. Cerevisiae sous deux aspects. Le premier, cellulaire, concerne une étude de sa localisation et de sa fonction; le second, biochimique, est une étude du mécanisme du transport du cuivre par l'ATPase. L'étude de la distribution intra-cellulaire de Ccc2 montre que l'ATPase n'est pas confinée dans l'appareil de Golgi, mais qu'elle est distribuée dans tous les compartiments de la voie sécrétoire, ainsi qu'à la membrane de la vacuole. Cette dernière localisation, qui n'avait jamais été décrite, nous a permis de montrer la participation de Ccc2 à l'accumulation de cuivre dans la vacuole. Il s'agit du premier transporteur actif découvert pour cette fonction. D'autre part, l'étude biochimique nous a permis de mettre en évidence la fixation de deux cuivres dans le domaine nucléotidique isolé. Deux acides aminés importants pour cette liaison du cuivre ont été identifiés, les cystéines 708 et 718. Reste à découvrir le rôle de ces sites dans le transport du cuivre par Ccc2.This thesis investigated two aspects of Ccc2, S. Cerevisiae copper ATPase. One is cellular and concerns the localization and the cellular function of Ccc2; the second one is biochemical and deals with the mechanism of copper transport by Ccc2. We found out that Ccc2 localizes at all the secretory pathway compartments and at the membrane of the vacuole. The latter result is the first evidence for a new function of Ccc2, that is to store copper into the vacuole lumen. Ccc2 is the first copper active transporter found in a vacuole. Our biochemical study shows that 2 coppers atoms bind to the isolated nucleotide domain of Ccc2. Two cysteines, C708 and C718, were identify as being part of the copper binding sites. Further studies will determine the function of these copper binding sites in the mechanism of copper transport by Ccc2.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Chelation therapy in Wilson's disease: from D-penicillamine to the design of selective bioinspired intracellular Cu(I) chelators

International audienceWilson's disease is an orphan disease due to copper homeostasis dysfunction. Mutations of the ATP7B gene induces an impaired functioning of a Cu-ATPase, impaired Cu detoxification in the liver and copper overload in the body. Indeed, even though copper is an essential element, which is used as cofactor by many enzymes playing vital roles, it becomes toxic when in excess as it promotes cytotoxic reactions leading to oxidative stress. In this perspective, human copper homeostasis is first described in order to explain the mechanisms promoting copper overload in Wilson's disease. We will see that the liver is the main organ for copper distribution and detoxification in the body. Nowadays this disease is treated life-long by systemic chelation therapy, which is not satisfactory in many cases. Therefore the design of more selective and efficient drugs is of great interest. A strategy to design more specific chelators to treat localized copper accumulation in the liver will then be presented. In particular we will show how bioinorganic chemistry may help in the design of such novel chelators by taking inspiration from the biological copper cell transporters

Convoyage et transfert du cuivre(I) à l'appareil de Golgi dans saccharomyces cerevisiae (rôle de la métallo-chaperonne Atx1 et de l'ATPase Ccc2)

L'objectif de cette thèse réside dans l'étude du convoyage du cu(I) à l'intérieur de l'appareil de golgi dans la levure saccharomyces cerevisiae. La métallo-chaperonne ATX1 séquestre le cu(I) cytosolique puis le transfère à l'extremité N-terminale de l'atpase CCC2, localisée dans les membranes du golgi. Ce domaine N- terminal est constitué de deux domaines de liaison aux métaux lourds, dénommés M1 et M2, qui sont homologues à ATX1. Des tests de complémentation dans une souche ATX1 ont montré que des protéines analogues à ATX1, de par un repliement babbab et la présence d'un motif de liaison aux métaux lourds CXXC (M1 et M2 produit en protéines solubles par exemple) pouvaient se substituer à ATX1. Afin d'améliorer la sensibilité du test phénotypique, un nouveau vecteur d'expression a été créé qui permet l'expression de CCC2 en infime quantité. En utilisant ce nouveau système d'expression, des tests de complémentation ont été réalisés dans une soucheATX1 CCC2. La co-expression d'ATX1 (ou d'un homologue) avec des protéines CCC2 modifiées sur leur extrémité n- terminale a permis de caractériser in vivo le mécanisme de transfert du cu(I) entre les deux protéines partenaires. Ainsi, ATX1 et certains homologues délivrent le cu(I) indifféremment à M1 ou à M2 de CCC2. Néanmoins, les homologues sont moins efficaces qu' ATX1. Cette différence de fonctionnalité semble dépendre de la flexibilite de la boucle de liaison au métal des homologues, ainsi que de leurs surfaces de potentiel électrostatique. Le cuivre lié sur l'extrémité N-terminale de l'atpase est ensuite transféré à un site de liaison qui reste à définir, localisé potentiellement sur la grande boucle cytoplasmique.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Identification et caractérisation des sites de transport de CadA, l'ATPase-cadmium de listeria monocytogenes

L'ATPase-Cd2+ CadA de L. monocytogenes appartient à la famille des ATPases P1 qui assurent le transport d'ions lourds à travers unel membrane. Au cours de cette thèse, nous avons montré que l'expression de CadA dans S. cerevisiae induit un phénotype de sensibilité au Cd2+ et que le Cd-ATP pouvait remplacer le Mg-ATP dans le cycle enzymatique de la protéine. Nous avons aussi montré que quatre hélices transmembranaires (3, 4, 6 et 8) constitueraient la voie de passage du Cd2+ dans CadA. Au sein de ces hélices, les acides aminés M149, C354 et T684 pourraient faire partie du site de liaison tandis que E164 et C356 pourraient être importants dans le processus de dissociation du métal. P355 et D692 seraient nécessaires à la phosphorylation. La caractérisation fonctionnelle d'ATPases chimériques a mis en évidence la possibilité d'échanger le domaine de phosphorylation entre différentes ATPases de type P.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Zinc and copper toxicity in host defense against pathogens: Mycobacterium tuberculosis as a model example of an emerging paradigm

International audienc

Dissecting the role of the N-terminal metal-binding domains in activating the yeast copper ATPase in vivo

International audienceIn yeast, copper delivery to the trans-Golgi network involves interactions between the metallo-chaperone Atx1 and the N-terminus of Ccc2, the P-type ATPase responsible for copper transport across trans-Golgi network membranes. Disruption of the Atx1–Ccc2 route leads to cell growth arrest in a copper-and-iron-limited medium, a phenotype allowing complementation studies. Coexpression of Atx1 and Ccc2 mutants in an atx1Δccc2Δ strain allowed us to study in vivo Atx1–Ccc2 and intra-Ccc2 domain–domain interactions, leading to active copper transfer into the trans-Golgi network. The Ccc2 N-terminus encloses two copper-binding domains, M1 and M2. We show that in vivo Atx1–M1 or Atx1–M2 interactions activate Ccc2. M1 or M2, expressed in place of the metallo-chaperone Atx1, were not as efficient as Atx1 in delivering copper to the Ccc2 N-terminus. However, when the Ccc2 N-terminus was truncated, these independent metal-binding domains behaved like functional metallo-chaperones in delivering copper to another copper-binding site in Ccc2 whose identity is still unknown. Therefore, we provide evidence of a dual role for the Ccc2 N-terminus, namely to receive copper from Atx1 and to convey copper to another domain of Ccc2, thereby activating the ATPase. At variance with their prokaryotic homologues, Atx1 did not activate the Ccc2-derived ATPase lacking its N-terminus

A possible regulatory role for the metal-binding domain of CadA, the Listeria monocytogenes Cd2+-ATPase

AbstractUsing the baculovirus/Sf9 expression system, we produced CadA and ΔMBD, a metal-binding domain, truncated CadA. Both proteins had the expected properties of P-type ATPases: ATP-induced Cd2+ accumulation, Cd2+-sensitive ATP and Pi phosphorylation and ATPase activity. ΔMBD displayed lower initial transport velocity as well as lower maximal ATPase activity than CadA. MBD truncation flattened the Cd2+ dependence of the ATPase activity and increased apparent Cd2+ affinity, suggesting a positive cooperativity between MBD and membranous transport sites. We propose that occupancy of MBD by Cd2+ modulates CadA activity