La phosphodiesterase specifique du GMP cyclique du batonnet retinien. Liaison a la membrane et activation parla transducine

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Cadmium, ATPase-P , Levure (Du transport à la toxicité )

Mes travaux ont consisté en l'étude fonctionnelle de l'A TPase-CdL+, CadA de Listeria monocytogenes e't en compréhension des mécanismes de toxicité et de réponse vis-à-vis du cadmium chez Saccharomyces cerevisiae. 'originalité de ce travail repose sur l'utilisation d'un phénotype de sensibilité au cadmium associé à l'expression de CadA chez S. cerevisiae. Ce phénotype s'est révélé être un système de criblage pertinent pour l'identification des acides aminés essentiels au fonctionnement de CadA et un système d'étude de la toxicité du cadmium chez la levure. L'ATPase-P CadA transporte les ions Cd2+ d'un côté à l'autre d'une membrane en utilisant l'hydrolyse de l'ATPomme source d'énergie. La mutagenèse dirigée des acides aminés membranaires de CadA a permis de mettre en évidence que 2 ions Cd2+ seraient transportés par A TP hydrolysé et que les sites de transport du métal impliquent les cystéines et la proline du motif CPC (TM6), un aspartate (TM8), un glutamate (TM4) et une méthionine (TM3). l'expression de CadA rend la levure S. cerevisiae hypersensible au cadmium. Une souche sauvage croît en présence de lOOIlM de cadmium alors que la souche exprimant CadA est incapable de croître en présence de IIlM de cadmium. Ce phénotype a permis de montrer que l'entrée du cadmium se fait par un transporteur de manganèse et que la sensibilité au cadmium résulte d'une accumulation de cadmium dans le réticulum endoplasmique (RE), accumulation réalisée par CadA. Dans le RE, le cadmium altère le processus de repliement, ce à quoi la levure répond en déclenchant la réponse UPR. Cette réponse, aussi observée chez une levure normale fait du RE une cible majeure de la toxicité du cadmium.Two projects has been developed during my PhD. One consisting in the functional study of CadA, the Cd2+ -A TPase from Listeria monocytogenes, the other one was focused on the toxicity of cadmium and the associated response of the yeast Saccharomyces cerevisiae. This two studies used a a phenotype of sensitivity to cadmium induced by CadA expression in yeast. This phenotype was used as a screening tool to identify essential amino acids of Cd transport byCadA and to study cadmium toxicity and the corresponding yeast cellular response. CadA actively transports Cd using A TP hydrolysis as energy source. Directed mutagenesis of the membranous amino-acids revealed that Cd transport pathway implied four transmembrane segments (Tm) and more precisely the cysteine C354, C356 and pro line P355 of the CPC motif located in Tm6, aspartate D692 in Tm8, glutamate EI64 in Tm4 and methionine Ml49 in Tm5. From our studies, 2 Cd ions would be translocated for each hydrolysis A TP. Expression of CadA in the yeast Saccharomyces cerevisiae induces an hypersensitivity to Cd. A wild type cell can grow up to IOOIlM cadmium whereas CadA expressing yeast cannot grow with IIlM cadmium in the culture medium. This cadmium sensitivity was due to the localisation of CadA in the endoplasmic reticulum membrane. Transport of cadmium in this compartment poduces an accumulation of misfolded proteins that induces the Unfolded Protein Response (UPR). As UPR also occurs in a wild type yeast exposed to low Cd concentration, one can point out endoplasmic reticulum as a extremely sensitive cellular compartment. UPR also appears as an early response to Cd as it happens far before any visible signs oftoxicity.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

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Cadmium

RSC Metallobiology Series No. 2International audienceCadmium is not an essential element for life. It is geologically marginal but anthropogenic activities have contributed significantly to its dispersion in the environment and to cadmium exposure of living species. The natural speciation of the divalent cation Cd2+ is dominated by its high propensity to bind to sulfur ligands, but Cd2+ may also occupy sites providing imidazole and carboxylate ligands. It binds to cell walls by passive adsorption (bio-sorption) and it may interact with surface receptors. Cellular uptake can occur by ion mimicry through a variety of transporters of essential divalent cations, but not always. Once inside cells, Cd2+ preferentially binds to thiol-rich molecules. It can accumulate in intracellular vesicles. It may also be transported over long distances within multicellular organisms and be trapped in locations devoid of efficient excretion systems. These locations include the renal cortex of animals and the leaves of hyper-accumulating plants. No specific regulatory mechanism monitors Cd2+ cellular concentrations. Thiol recruitment by cadmium is a major interference mechanism with many signalling pathways that rely on thiolate-disulfide equilibria and other redox-related processes. Cadmium thus compromises the antioxidant intracellular response that relies heavily on molecules with reactive thiolates. These biochemical features dominate cadmium toxicity, which is complex because of the diversity of the biological targets and the consequent pleiotropic effects. This chapter compares the cadmium-handling systems known throughout phylogeny and highlights the basic principles underlying the impact of cadmium in biology

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

Biophysical and structural characterization of the putative nickel chaperone CooT from Carboxydothermus hydrogenoformans.

International audienceCarboxydothermus hydrogenoformans is a model microorganism for the study of [NiFe]-CODH, a key enzyme of carbon cycle in anaerobic microorganisms. The enzyme possesses a unique active site (C-cluster), constituted of a distorted [NiFe3S4] cubane linked to a mononuclear Fe(II) center. Both the biogenesis of the C-cluster and the activation of CODH by nickel insertion remain unclear. Among the three accessory proteins thought to play a role in this latter step (CooC, CooJ, and CooT), CooT is identified as a nickel chaperone involved in CODH maturation in Rhodospirillum rubrum. Here, we structurally and biophysically characterized a putative CooT protein present in C. hydrogenoformans (pChCooT). Despite the low sequence homologies between CooT from R. rubrum (RrCooT) and pChCooT (19% sequence identity), the two proteins share several similarities, such as their overall structure and a solvent-exposed Ni(II)-binding site at the dimer interface. Moreover, the X-ray structure of pChCooT reveals the proximity between the histidine 55, a potential nickel-coordinating residue, and the cysteine 2, a highly conserved key residue in Ni(II)-binding

High-affinity iron and calcium transport pathways are involved in U(VI) uptake in the budding yeast Saccharomyces cerevisiae

International audienceUranium (U) is a naturally-occurring radionuclide that is toxic for all living organisms. To date, the mechanisms of U uptake are far from being understood. Here we provide a direct characterization of the transport machineries capable of transporting U, using the yeast Saccharomyces cerevisiae as a unicellular eukaryote model. First, we evidenced a metabolism-dependent U transport in yeast. Then, competition experiments with essential metals allowed us to identify calcium, iron and copper entry pathways as potential routes for U uptake. The analysis of various metal transport mutants revealed that mutant affected in calcium (mid1Δ and cch1Δ) and Fe(III) (ftr1Δ) transport, exhibited highly reduced U uptake rates and accumulation, demonstrating the implication of the calcium channel Mid1/Cch1 and the iron permease Ftr1 in U uptake. Finally, expression of the Mid1 gene into the mid1Δ mutant restored U uptake levels of the wild type strain, underscoring the central role of the Mid1/Cch1 calcium channel in U absorption process in yeast. Our results also open up the opportunity for rapid screening of U-transporter candidates by functional expression in yeast, before their validation in more complex higher eukaryote model systems

Functional characterization of the Ca2+-ATPase SMA1 from Schistosoma mansoni.

International audienceSchistosoma mansoni is a parasite that causes bilharzia, a neglected tropical disease affecting hundreds of millions of people each year worldwide. In 2012, S. mansoni had been identified as the only invertebrate possessing two SERCA-type Ca2+-ATPases, SMA1 and SMA2. However, our analysis of recent genomic data shows that the presence of two SERCA pumps is rather frequent in parasitic flatworms. To understand the reasons of this redundancy in S. mansoni , we compared SMA1 and SMA2 at different levels. In terms of sequence and organization, the genes SMA1 and SMA2 are similar, suggesting that they might be the result of a duplication event. At the protein level, SMA1 and SMA2 only slightly differ in length and in the sequence of the nucleotide binding domain. To get functional information on SMA1, we produced it in an active form in S. cerevisiae , as previously done for SMA2. Using phosphorylation assays from ATP, we demonstrated that like SMA2, SMA1 bound calcium in a cooperative mode with an apparent affinity in the micromolar range. We also showed that SMA1 and SMA2 had close sensitivities to cyclopiazonic acid but different sensitivities to thapsigargin, two specific inhibitors of SERCA pumps. On the basis of transcriptomic data available in GeneDB, we hypothesize that SMA1 is a housekeeping Ca2+-ATPase whereas SMA2 might be required in particular striated-like muscles like those present the tail of the cercariae, the infecting form of the parasite

Cd2+ and the N-terminal metal-binding domain protect the putative membranous CPC motif of the Cd2+-ATPase of Listeria monocytogenes.

CadA, the Cd(2+)-ATPase of Listeria monocytogenes, contains four cysteine residues: two in the CTNC (Cys-Thr-Asn-Cys) sequence in the cytoplasmic metal-binding domain (MBD), and two in the CPC (Cys-Pro-Cys) sequence in the membrane domain. Taking advantage of DeltaMBD, a truncated version of CadA that lacks the MBD but which still acts as a functional Cd(2+)-ATPase [Bal, Mintz, Guillain and Catty (2001) FEBS Lett. 506, 249-252], we analysed the role of the membrane cysteine residues (studied using DeltaMBD) separately from that of the cysteine residues of the MBD, which were studied using full-length CadA. The role of the cysteines was assessed by reacting DeltaMBD and CadA with N -ethylmaleimide (NEM), an SH-specific reagent, in the presence or absence of Cd(2+). We show here that (i) in both DeltaMBD and CadA, the cysteine residues in the CPC motif are essential for phosphorylation; (ii) in both proteins, Cd(2+) protects against alkylation by NEM; and (iii) in the absence of Cd(2+), the MBD of CadA also protects against alkylation by NEM. Our results suggest that the CPC motif is present in the membrane Cd(2+) transport site(s) and that the MBD protects these site(s)