Effects of Chelating Compounds on Mobilization and Phytoextraction of Copper and Lead in Contaminated Soils

Six chelating compounds: ethylenediamine-tetraacetic aci

A geostatistical framework for incorporating transport information in estimating the distribution of a groundwater contaminant plume

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/95253/1/wrcr10904.pd

Assembly-dependent translation of subunits 6 (Atp6) and 9 (Atp9) of ATP synthase in yeast mitochondria

The yeast mitochondrial ATP synthase is an assembly of 28 subunits of 17 types of which 3 (subunits 6, 8, and 9) are encoded by mitochondrial genes, while the 14 others have a nuclear genetic origin. Within the membrane domain (FO) of this enzyme, the subunit 6 and a ring of 10 identical subunits 9 transport protons across the mitochondrial inner membrane coupled to ATP synthesis in the extra-membrane structure (F1) of ATP synthase. As a result of their dual genetic origin, the ATP synthase subunits are synthesized in the cytosol and inside the mitochondrion. How they are produced in the proper stoichiometry from two different cellular compartments is still poorly understood. The experiments herein reported show that the rate of translation of the subunits 9 and 6 is enhanced in strains with mutations leading to specific defects in the assembly of these proteins. These translation modifications involve assembly intermediates interacting with subunits 6 and 9 within the final enzyme and cis-regulatory sequences that control gene expression in the organelle. In addition to enabling a balanced output of the ATP synthase subunits, these assembly-dependent feedback loops are presumably important to limit the accumulation of harmful assembly intermediates that have the potential to dissipate the mitochondrial membrane electrical potential and the main source of chemical energy of the cell

Biogenese de l’ATP synthase mitochondriale et des dysfonctions générant des maladies

Mitochondrial F1FO-ATP synthase produces most of the cellular energy in aerobiceukaryotes under the form of ATP in the process of oxidative phosphorylation. This enzymehas in most species a double genetic origin, nuclear and mitochondrial. In the first part of thiswork, I describe the construction of yeast models of ATP synthase mutations in themitochondrial ATP6 gene, that have been found in patients presenting with neurologicaldisorders (9185T>C and 9191T>C) and in tumors (8716A>G, 8914C>A, 8932C>T,8953A>G and 9131T>C). The ATP6 gene encodes an essential subunit (called a/6) of theATP synthase proton-translocating domain (FO). The 9185T>C mutation had no effect on theassembly of ATP synthase, but reduces the rate of ATP synthesis by 30%. The 9191T>Cmutation almost completely prevented incorporation of the subunit a/6 into the ATP synthase.The five mutations found in tumors had modest, if at all, effect, indicating that thesemutations probably do not favor tumorigenesis, as was hypothesized. In the second part of mythesis, I studied the regulation of synthesis of subunits a/6 and 9 in yeast mitochondria. Thesubunit 9 is present in 10 copies forming a ring that interacts with subunit 6. Protonmovements through the FO induce the rotation of the subunit 9-ring, which results inconformational changes that promote ATP synthesis in the catalytic sector (F1) of ATPsynthase. I discovered mechanisms that enable the coupling of the synthesis of these proteinsto their assembly, as a means to ensure the production of subunits 6 and 9 in the rightstoichiometry and to avoid accumulation of potentially harmful assembly intermediates of theATP synthase.La F1FO-ATP synthase mitochondriale produit la majorité de l’énergie cellulaire chezles eucaryotes aérobes sous forme d’ATP par le processus des oxydations phosphorylantes.Chez la plupart des espèces, cette enzyme possède une origine génétique double, nucléaire etmitochondriale. Dans la première partie de ce travail, je décris la construction de modèles delevure de mutations du gène mitochondrial ATP6 de l’ATP synthase découvertes chez despatients atteints de maladies neurologiques (9185T>C and 9191T>C) ou dans des tumeurs(8716A>G, 8914C>A, 8932C>T, 8953A>G and 9131T>C). Le gène ATP6 code une sousunitéessentielle (a/6) du domaine FO de l’ATP synthase. J’ai trouvé que la mutation 9185T>Cn’affecte pas l’assemblage de l’ATP synthase, mais conduit à une diminution de la vitesse desynthèse d’ATP d’environ 30%. La mutation 9191T>C empêche presque entièrementl’incorporation de la sous-unité a/6 dans l’ATP synthase. Les cinq mutations identifiées dansles tumeurs ont un effet modeste à nul, indiquant que ces mutations ne favorisent pas latumorigenèse en affectant le processus énergétique mitochondrial, comme évoquéprécédemment. J’ai ensuite étudié la régulation de la synthèse des sous-unités a/6 et 9 dans lesmitochondries de levures. La sous-unité 9 est présente sous la forme d’un anneau de 10 copiesqui interagit avec la sous-unité 6. Durant la catalyse, la rotation de cet anneau provoque deschangements conformationnels favorisant la synthèse d’ATP dans le secteur F1 de l’ATPsynthase. Je montre que la synthèse de ces protéines est couplée à leur assemblage, demanière à ce qu’elles soient produites dans une stoechiométrie adéquate et pour éviterl’accumulation d’intermédiaires d’ATP synthase potentiellement délétère

Biogenesis of mitochondrial ATP synthase and its dysfunction leading to diseases

La F1FO-ATP synthase mitochondriale produit la majorité de l’énergie cellulaire chezles eucaryotes aérobes sous forme d’ATP par le processus des oxydations phosphorylantes.Chez la plupart des espèces, cette enzyme possède une origine génétique double, nucléaire etmitochondriale. Dans la première partie de ce travail, je décris la construction de modèles delevure de mutations du gène mitochondrial ATP6 de l’ATP synthase découvertes chez despatients atteints de maladies neurologiques (9185T>C and 9191T>C) ou dans des tumeurs(8716A>G, 8914C>A, 8932C>T, 8953A>G and 9131T>C). Le gène ATP6 code une sousunitéessentielle (a/6) du domaine FO de l’ATP synthase. J’ai trouvé que la mutation 9185T>Cn’affecte pas l’assemblage de l’ATP synthase, mais conduit à une diminution de la vitesse desynthèse d’ATP d’environ 30%. La mutation 9191T>C empêche presque entièrementl’incorporation de la sous-unité a/6 dans l’ATP synthase. Les cinq mutations identifiées dansles tumeurs ont un effet modeste à nul, indiquant que ces mutations ne favorisent pas latumorigenèse en affectant le processus énergétique mitochondrial, comme évoquéprécédemment. J’ai ensuite étudié la régulation de la synthèse des sous-unités a/6 et 9 dans lesmitochondries de levures. La sous-unité 9 est présente sous la forme d’un anneau de 10 copiesqui interagit avec la sous-unité 6. Durant la catalyse, la rotation de cet anneau provoque deschangements conformationnels favorisant la synthèse d’ATP dans le secteur F1 de l’ATPsynthase. Je montre que la synthèse de ces protéines est couplée à leur assemblage, demanière à ce qu’elles soient produites dans une stoechiométrie adéquate et pour éviterl’accumulation d’intermédiaires d’ATP synthase potentiellement délétèresMitochondrial F1FO-ATP synthase produces most of the cellular energy in aerobiceukaryotes under the form of ATP in the process of oxidative phosphorylation. This enzymehas in most species a double genetic origin, nuclear and mitochondrial. In the first part of thiswork, I describe the construction of yeast models of ATP synthase mutations in themitochondrial ATP6 gene, that have been found in patients presenting with neurologicaldisorders (9185T>C and 9191T>C) and in tumors (8716A>G, 8914C>A, 8932C>T,8953A>G and 9131T>C). The ATP6 gene encodes an essential subunit (called a/6) of theATP synthase proton-translocating domain (FO). The 9185T>C mutation had no effect on theassembly of ATP synthase, but reduces the rate of ATP synthesis by 30%. The 9191T>Cmutation almost completely prevented incorporation of the subunit a/6 into the ATP synthase.The five mutations found in tumors had modest, if at all, effect, indicating that thesemutations probably do not favor tumorigenesis, as was hypothesized. In the second part of mythesis, I studied the regulation of synthesis of subunits a/6 and 9 in yeast mitochondria. Thesubunit 9 is present in 10 copies forming a ring that interacts with subunit 6. Protonmovements through the FO induce the rotation of the subunit 9-ring, which results inconformational changes that promote ATP synthesis in the catalytic sector (F1) of ATPsynthase. I discovered mechanisms that enable the coupling of the synthesis of these proteinsto their assembly, as a means to ensure the production of subunits 6 and 9 in the rightstoichiometry and to avoid accumulation of potentially harmful assembly intermediates of theATP synthase

Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C

Mutations in the human mitochondrial ATP6 gene encoding ATP synthase subunit a/6 (referred to as Atp6p in yeast) are at the base of neurodegenerative disorders like Neurogenic Ataxia and Retinitis Pigmentosa (NARP), Leigh syndrome (LS), CharcoteMarieeTooth (CMT), and ataxia telangiectasia. In previous studies, using the yeast Saccharomyces cerevisiae as a model we were able to better define how several of these mutations impact the ATP synthase. Here we report the construction of yeast models of two other ATP6 pathogenic mutations, T9185C and T9191C. The first one was reported as conferring a mild, sometimes reversible, CMT clinical phenotype; the second one has been described in a patient presenting with severe LS. We found that an equivalent of the T9185C mutation partially impaired the functioning of yeast ATP synthase, with only a 30% deficit in mitochondrial ATP production. An equivalent of the mutation T9191C had much more severe effects, with a nearly complete block in yeast Atp6p assembly and an >95% drop in the rate of ATP synthesis. These findings provide a molecular basis for the relative severities of the diseases induced by T9185C and T9191C

Yeast models of mutations in the mitochondrial ATP6 gene found in human cancer cells

Since the discovery of somatic mtDNA mutations in tumor cells, multiple studies have focused on establishing a causal relationship between those changes and alterations in energy metabolism, a hallmark of cancer cells. Yet the consequences of these mutations on mitochondrial function remain largely unknown. In this study, Saccharomyces cerevisiae has been used as a model to investigate the functional consequences of four cancer-associated missense mutations (8914CNA, 8932CNT, 8953ANG, 9131TNC) found in the mitochondrial MT-ATP6 gene. This gene encodes the a-subunit of F1FO-ATP synthase, which catalyzes the last steps of ATP production in mitochondria. Although the four studied mutations affected well-conserved residues of the a-subunit, only one of them (8932CNT) had a significant impact on mitochondrial function, due to a less efficient incorporation of the asubunit into ATP synthase. Our findings indicate that these ATP6 genetic variants found in human tumors are neutral mitochondrial genome substitutions with a limited, if any, impact on the energetic function of mitochondria

The Nucleocapsid Protein of Human Coronavirus NL63

<div><p>Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the <i>Coronavirinae</i> subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.</p></div

References to sequence and structural data on polypeptides used to design the expression constructs of HCoV-NL63 nucleocapsid N- and C- terminal domains.

<p>References to sequence and structural data on polypeptides used to design the expression constructs of HCoV-NL63 nucleocapsid N- and C- terminal domains.</p

N protein and CTD are able to form dimers.

<p>Molecular weight values for WT nucleocapsid protein and its domains, as determined using mass spectrometry. Experimental details are provided in the text.</p><p>N protein and CTD are able to form dimers.</p