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Structural and Functional Studies on Human Mitochondrial Iron-Sulfur Cluster BiosynthesisTsai, Chi-Lin 2011 May 1900 (has links)
Iron-sulfur (Fe-S) clusters are critical protein cofactors found in all life forms. In
eukaryotes, a well-conserved biosynthetic pathway located in the mitochondria is used to
assemble Fe-S clusters. Although proteins required for Fe-S cluster biosynthesis have
been identified, their precise function and mechanism remain elusive. In this study,
biochemical and biophysical methods are applied to understand molecular details for the
core components of the human Fe-S cluster biosynthesis: Nfs1, Isd11, Isu2, and frataxin
(Fxn). Nfs1 is a cysteine desulfurase that converts cysteine into alanine and transfers the
sulfur to a scaffold protein Isu2 for Fe-S clusters. Fxn depletion is associated with the
neurodegenerative disease Friedreich’s ataxia (FRDA), and results in a complicated
phenotype that includes loss of Fe-S clusters.
The results presented here provide the first in vitro evidence for a stable protein
complex that exists in at least two forms: an inactive complex with Nfs1, Isd11, and Isu2
(SDU) components and an active form that also includes Fxn (SDUF). Fxn binding
dramatically changes the catalytic efficiency (kcat/KM) of Nfs1 from 25 to 10,100 M-1s-1 and enhances the rate of Fe-S cluster biosynthesis 25 fold. Oxidizing conditions diminish
the levels of both complex formation and Fxn-based activation, whereas Fe2 further
stimulates Nfs1 activity. Mutagenesis coupled to enzyme kinetics indicate that one of the
three conserved cysteines (C104) on Isu2 accepts the sulfane sulfur from Nfs1 and that
this transfer event likely requires prior binding of Fxn. In vitro interrogation of FRDA
I154F and W155R and related Fxn variants revealed the binding affinity to SDU
followed the trend Fxn ~ I154F > W155F > W155A ~ W155R. The Fxn variants also
have diminished ability to facilitate both sulfur transfer and Fe-S cluster assembly. Fxn
crystallographic structures reveal specific rearrangements associated with the loss of
function. Importantly, the weaker binding and lower activity of the W155R variant
compared to I154F explains the earlier onset and more severe disease progression.
Finally, these experimental results coupled with computational docking studies suggest a
model for how human Fxn functions as an allosteric activator and triggers sulfur transfer
and Fe-S cluster assembly.
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Etude du rôle de la frataxine bactérienne CyaY chez Escherichia coli / Study of bacterial frataxin CyaY in Escherichia coliRoche, Béatrice 01 December 2015 (has links)
Les protéines à centre Fe-S sont impliquées dans de nombreux processus cellulaires. In vivo, la formation des centres Fe-S est réalisée par des machineries multi-protéiques dont ISC et SUF, conservées chez les eucaryotes et les procaryotes. D’autres composants participent à la formation des centres Fe-S chez les eucaryotes, comme la frataxine (FXN). La FXN est une protéine présente chez l’homme, les plantes, la levure ou encore les bactéries à Gram négatif. Chez les eucaryotes, l’absence de FXN conduit à des phénotypes drastiques comme une accumulation de fer dans la mitochondrie, une diminution drastique de l’activité d’enzymes à centre Fe-S ou encore des dommages oxydatifs. Chez l’homme, un déficit en FXN est responsable d’une maladie neurodégénérative, l’ataxie de Friedreich. A la différence des eucaryotes, chez les procaryotes comme Escherichia coli, l’absence de CyaY, homologue bactérien de la FXN, ne conduit à aucun des phénotypes évoqués ci-dessus.Durant ma thèse, je me suis intéressée au rôle de CyaY chez E. coli. J’ai montré que, in vivo, CyaY favorise la formation des centres Fe-S via la machinerie ISC. Un lien génétique entre CyaY et IscX a également pu être établi, montrant que ces deux protéines participent à la formation des centres Fe-S in vivo. Je me suis ensuite intéressée aux bases moléculaires pouvant expliquer la différence entre les phénotypes liés à l’absence de FXN chez les eucaryotes et les procaryotes. J’ai montré que le résidu 108 de IscU joue un rôle clé pour la dépendance de CyaY. Enfin, pour mieux comprendre le rôle de CyaY chez E. coli, j’ai réalisé une approche globale en caractérisant le transcriptome du mutant ∆cyaY. / Fe-S cluster containing proteins are involved in many cellular processes such as respiration, DNA repair or gene regulation. In vivo, Fe-S cluster biogenesis is catalysed by specific protein machineries, ISC and SUF, conserved in both eukaryotes and prokaryotes. Frataxin (FXN) is a small protein found in humans, plants, yeast and Gram negative bacteria. In eukaryotes, a defect in FXN leads to drastic phenotypes such as mitochondrial iron accumulation, drastic decrease of Fe-S cluster protein activity, sensitivity to oxidants. In humans, FXN deficiency is responsible for the neurodegenerative disease, Friedreich’s ataxia. In prokaryotes like E. coli, a defect in CyaY, the bacterial FXN homolog, does not lead to significant phenotypes compared to the wild-type strain. During my thesis, I investigated the role of the bacterial FXN CyaY in E. coli. I showed that, in vivo, CyaY assisted the ISC-catalyzed Fe-S cluster biogenesis. A genetic link was also observed between cyaY and iscX, demonstrating that these proteins participate in Fe-S cluster biogenesis. In a second part, I investigated the differences between the impact of the eukaryotic versus prokaryotic FXN. I showed that the IscU 108th residue is crucial for the CyaY-dependency. Finally, I used a transcriptomic approach to test whether CyaY has a global role in E. coli.
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