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Probing Mitochondrial DNA Structure with Mitochondria-Targeted DNA MethyltransferasesRebelo, Adriana 18 December 2009 (has links)
The mitochondria contain their own genome, which is organized in a dynamic high-order nucleoid structure consisting of several copies of mitochondrial DNA (mtDNA) molecules associated with proteins. The mitochondrial nucleoids are the units of mtDNA inheritance, and are the sites of mtDNA transcription, replication and maintenance. Therefore, the integrity of mitochondrial nucleoids is a key determinant of mitochondrial metabolism and the bioenergetic state of the cell. Deciphering the interaction of mtDNA with proteins in nucleoprotein complexes is fundamental to understand the mechanisms of mtDNA segregation leading to mitochondrial dysfunction and to develop therapies to treat diseases associated with mtDNA mutations. The work presented in this dissertation provides essential insights into the dynamics of mtDNA interaction with nucleoid proteins. In order to unveil the organization of the mitochondrial genome, we have mapped major regulatory regions of the mtDNA in vivo using mitochondrial-targeted DNA methyltransferases. In chapter 2, we have demonstrated that DNA methyltranferases are powerful tools in probing mtDNA-protein interactions in living cells. The DNA methyltransferases' accessibility to their cognate sites in the mtDNA is negatively correlated with the frequency and binding strength that protein factors occupy a specific site. Our results show that the transcription termination region (TERM) within the tRNALeu(UUR) gene is consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mTERF1 (mitochondrial transcription termination factor 1). DNA methyltransferases have also been shown to be effective in detecting changes in mitochondrial nucleoid architecture due to nucleoid remodeling. We were able to determine changes in the packaging state of mitochondrial nucleoids by monitoring changes in mtDNA accessibility. The impact of altered levels of major nucleoid proteins was assessed by monitoring changes in mtDNA methylation pattern. We observed a more condensed nucleoid state causing a decrease in mtDNA methylation when the levels of the mitochondrial transcription factor A (TFAM) were altered. Changes in mtDNA methylation pattern were also evident when cells were treated with ethidium bromide (EtBr) and hydrogen peroxide. The mtDNA nucleoids adopted a less compact state during rapid mtDNA replication after EtBr treatment. In contrast, we observed a more compact mtDNA, less accessible to DNA methyltransferase after hydrogen peroxide treatment. Our results indicate that mitochondrial nucleoids are not static, but are constantly been modulated in response to factors that affect the nucleoid environment. In chapter 3, we identified the in vivo DNA binding sites of major transcription regulatory proteins, TFAM and mTERF3 using a targeted gene methylation (TAGM) strategy. In this approach, the mtDNA binding protein is fused to a DNA methyltransferase as an attempt to selectively methylate the sites adjacent to the protein target DNA region. Knowledge on how proteins interact with the mtDNA in high-order structures, which function as a mitochondrial genetic unit, will help elucidate the segregation and accumulation of mutated mtDNA in diseased tissues.
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Recherche des partenaires d'ATAD3 et étude de l'effet de l'invalidation sur la différenciation adipocytaire / Research of ATAD3 partners and study of the effect of invalidation on the adipocyte differentiation.Li, Shuijie 06 July 2012 (has links)
Le projet principal a consisté à étudier le rôle d'ATAD3 dans un modèle de différenciation cellulaire en culture, les cellules adipocytaires 3T3-L1 induites par l'insuline. L'induction de ces cellules provoque la mise en route du programme d'adipogenèse qui précède la synthèse de gouttelettes de triglycérides (lipogenèse). Nous avons observé qu'au cours de la différenciation, une synthèse et un remodelage du réseau mitochondrial ont lieu. ATAD3, exprimée dans les pré-adipocytes, s'accumule avant la synthèse et le remodelage du réseau mitochondrial et est maintenue élevée tout au long de la différenciation. Nous avons réalisé des lignées invalidées constitutivement pour ATAD3 (siRNA) et étudié la différenciation de ces cellules. Nous avons montré que l'invalidation d'ATAD3 inhibe la lipogenèse et le remodelage du réseau mitochondrial sans affecter la signalisation par l'insuline ni la synthèse des protéines mitochondriales. Le phénotype a été récupéré par transfection d'ATAD3 et une complémentation par Drp1 a été mise en évidence. Ces résultats montrent qu'ATAD3 est nécessaire et limitant dans la différenciation adipocytaire. Trois projets annexes ont complétés ce travail de thèse : Le premier a consisté à contribuer à la purification d'ATAD3 humaine, produite chez la levure. Ces expériences n'ont pas permis de révéler l'activité ATPasique d'ATAD3 mais ont permis d'obtenir une quantité significative de protéine recombinante qui pourra servir à la production d'anticorps ainsi qu'à d'autres applications. Le second a consisté à étudier les isoformes d'ATAD3 chez l'homme et les rongeurs. Ces expériences ont permis de révéler l'existence de plusieurs isoformes tissulaires. Un troisième projet a consisté à identifier des partenaires protéiques d'ATAD3 afin de tenter de comprendre la fonction de cette protéine. A l'aide de sondes protéiques d'ATAD3 radiomarquées (transcription/traduction in vitro), nous avons réalisé des tests d'interaction de type Far Western. Ces expériences ont aboutis à quelques candidats potentiels interagissant avec les parties N- ou C-terminale d'ATAD3. / ATAD3 is a vital inner membrane mitochondrial ATPase with unknown function but widely expressed in animals and plants. The main project was to study the role of ATAD3 in a cellular model of differentiation, the one used being adipocyte-like 3T3-L1 cells induced by insuline. Induction of these cells provokes the start of the adipogenesis program which precedes lipid droplets synthesis (lipogenesis). We observed that along differentiation, a synthesis and a remodelling of mitochondria occur. ATAD3, which is expressed in pre-adipocytes, is increased before lipid droplets accumulation and before mitochondrial synthesis and remodelling and is maintained all along differntiation. We have generated cell lines with constitutive invalidation of ATAD3 (siRNA) and studied the differentiation of these cells. We have shown that ATAD3 invalidation inhibits lipogenesis and mitochondrial remodelling without any inhibition of insulin transduction signal neither inhibition of mitochondrial protein synthesis. The phenotype was rescued by ATAD3 transfection and complemented by over-expression of Drp1. These results show that ATAD3 is a necessary and limiting factor in adypocyte differentiation and lipogenesis. Three parallel projects completed this thesis work: The first was to contribute to purification of recombinant human ATAD3 produced in yeast. These experiments didn't allow us to reveal the ATPase activity of ATAD3 but allowed us to obtain a significant quantity of purified protein that will be useful to generate antibodies and also for others applications. The second was to study ATAD3 isoforms expression profiles in human and rodent samples. These experiments allowed the description of several isoforms of ATAD3 with tissue-specific expression. A third project was the identification of ATAD3 protein partners in order to try to understand its function. Using labelled ATAD3 protein probes (in vitro transcription/traduction), we performed interaction tests by Far Western. These experiments led to the identification of seve
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Investigation of SHOT1-binding ATPases in Arabidopsis thalianaZelman, Sam 18 December 2020 (has links)
Mitochondria play critical roles not only in primary metabolism as a central organelle for ATP generation, but also in responding to abiotic stresses. We identified a mutation in the MTERF18 (Mitochondrial Transcription Termination factor)/SHOT1 (Suppressor of hot1-4 1) gene in Arabidopsis thaliana that enables plants to better tolerate heat and oxidative stresses, presumably due to reduced oxidative damage, but the exact molecular mechanism of the heat tolerance is unknown. In order to reveal the stress tolerance mechanisms of mterf18/shot1 mutations, it is critical to understand the molecular defects of the mutant and to identify the molecular targets of the MTERF18/SHOT1 protein. MTERF18/SHOT1, a mitochondrial matrix protein, was found to bind to membrane-spanning mitochondrial AAA+ proteins homologous to ATAD3a of humans and other multicellular eukaryotes. A. thaliana has four ATAD3a homologues in two clades, and plants require one gene from each clade for viability. Previous studies of the topology and ATPase activity of ATAD3a suggest a role in endoplasmic reticulum (ER)-mitochondria contact sites. These sites are poorly defined in plants, and their relationship to heat stress tolerance is intriguing. To better understand ATAD3 function I expressed and purified the soluble, matrix-located, catalytic C-terminal ATPase domain of these proteins in order to assay their ATPase activity and oligomerization states. Transgenic plants with fluorescently labelled ER and mitochondria have been generated to observe effects of the MTERF18/SHOT1 mutation on ER-mitochondria dynamics. These studies of the four ATAD3 proteins will provide insights into ER-mitochondrial contact sites in plants, and into their link to MTERF18/SHOT1 and heat stress tolerance. I also provide a review of our current knowledge of ER-mitochondria contact site protein components in plants with reference to these proteins in A. thaliana.
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Recherche des partenaires d'ATAD3 et étude de l'effet de l'invalidation sur la différenciation adipocytaireLi, Shuijie 06 July 2012 (has links) (PDF)
Le projet principal a consisté à étudier le rôle d'ATAD3 dans un modèle de différenciation cellulaire en culture, les cellules adipocytaires 3T3-L1 induites par l'insuline. L'induction de ces cellules provoque la mise en route du programme d'adipogenèse qui précède la synthèse de gouttelettes de triglycérides (lipogenèse). Nous avons observé qu'au cours de la différenciation, une synthèse et un remodelage du réseau mitochondrial ont lieu. ATAD3, exprimée dans les pré-adipocytes, s'accumule avant la synthèse et le remodelage du réseau mitochondrial et est maintenue élevée tout au long de la différenciation. Nous avons réalisé des lignées invalidées constitutivement pour ATAD3 (siRNA) et étudié la différenciation de ces cellules. Nous avons montré que l'invalidation d'ATAD3 inhibe la lipogenèse et le remodelage du réseau mitochondrial sans affecter la signalisation par l'insuline ni la synthèse des protéines mitochondriales. Le phénotype a été récupéré par transfection d'ATAD3 et une complémentation par Drp1 a été mise en évidence. Ces résultats montrent qu'ATAD3 est nécessaire et limitant dans la différenciation adipocytaire. Trois projets annexes ont complétés ce travail de thèse : Le premier a consisté à contribuer à la purification d'ATAD3 humaine, produite chez la levure. Ces expériences n'ont pas permis de révéler l'activité ATPasique d'ATAD3 mais ont permis d'obtenir une quantité significative de protéine recombinante qui pourra servir à la production d'anticorps ainsi qu'à d'autres applications. Le second a consisté à étudier les isoformes d'ATAD3 chez l'homme et les rongeurs. Ces expériences ont permis de révéler l'existence de plusieurs isoformes tissulaires. Un troisième projet a consisté à identifier des partenaires protéiques d'ATAD3 afin de tenter de comprendre la fonction de cette protéine. A l'aide de sondes protéiques d'ATAD3 radiomarquées (transcription/traduction in vitro), nous avons réalisé des tests d'interaction de type Far Western. Ces expériences ont aboutis à quelques candidats potentiels interagissant avec les parties N- ou C-terminale d'ATAD3.
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Exploring Knockdown Phenotypes and Interactions between ATAD3 Proteins in Arabidopsis thalianaGordon, Eli S 20 October 2021 (has links) (PDF)
Mitochondria are required for a diverse array of cellular functions and processes. ATAD3 (ATPase family AAA domain containing protein 3) proteins are newly discovered mitochondrial membrane proteins in Arabidopsis thaliana. Homologous to ATAD3A in metazoans, Co-Immunoprecipitation/Mass spectrometry and genomic analysis identified a four ATAD3A homologues in A. thaliana. The four A. thaliana proteins are referred to as ATAD3A1 (At3g03060), ATAD3A2 (At5g16930), ATAD3B1 (At2g18330), and ATAD3B2 (At4g36580). Studies in metazoans indicate that ATAD3A localizes to Mitochondria-ER contact sites and is involved in a variety of processes required for proper mitochondrial function, but ATAD3A proteins are poorly defined in plants. ATAD3A is a mitochondrial membrane protein with unique topology. It comprises an N-terminal DUF (Domain of unknown function) domain that contains two transmembrane sequences the are inserted or interact with both the inner and outer mitochondrial membranes, two coiled-coil domains thought to help in oligomerization, and a region that is exposed to the cytosol, proposed to interact with the ER. It has a C-terminal AAA domain exposed to the mitochondrial matrix. ATAD3 proteins in A. thaliana have undergone two gene-duplication events, resulting in two clades, both of which are required for plant viability. I created artificial microRNA to knockdown expression of ATAD3A1 in the atad3b1 mutant background to assess the growth and mitochondrial phenotypes and found these plants displayed delayed and deficient growth and deformed mitochondria. I utilized Bi-Molecular Complementation Fluorescence and Laser-Scanning Confocal Microscopy to assess oligomeric patterns of A. thaliana ATAD3 proteins in vivo and discovered that ATAD3 proteins hetero-oligomerize with each other. I also created multiple constructs encoding ATAD3A1 fusion proteins to elucidate the amino acid sequence required to target ATAD3A1 to the mitochondria, and ATAD3A1 fusions with TurboID to identify protein-protein interactions using proximity-based labeling.
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