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Lipoylation and assembly of a 2-oxoacid dehydrogenase multienzyme complex from thermoplasma acidophilumPosner, Mareike January 2009 (has links)
Energy generating processes like the citric acid cycle are a pivotal part of metabolism. Members of the 2-oxoacid dehydrogenase multienzyme complex (OADHC) superfamily feed into and act within the citric acid cycle. OADHCs are composed of three enzymes: 2-oxoacid decarboxylase (E1), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). Covalent attachment of lipoic acid (LA) to E2 is essential for overall OADHC activity. Although thought to be absent in Archaea, it has recently been found that Thermoplasma acidophilum has all the components for an active recombinant OADHC (Heath et al., 2007). Recent studies have further suggested that Tp. acidophilum may have an enzyme to covalently attach LA to E2 (Sun et al., 2007; McManus et al., 2006). This work describes the cloning and recombinant expression of the Thermoplasma lipoate protein ligase (Tp. LplA), its C-terminal domain and a fusion protein composed of the above two proteins. Both proteins are required for lipoylation of E2 in vitro. For the first time, in vivo lipoylation of E2 in Tp. acidophilum cell cultures is also being reported. The effect of lipoylation and temperature on the Thermoplasma OADHC assembly has also been studied. This study revealed the temperature dependence of the E2 core and the whole complex assembly. These findings are in line with the optimum growth temperature of Tp. acidophilum. Dynamic light scattering and analytical ultracentrifugation were used to determine the molecular mass of whole OADHC. The molecular mass was determined to be 5 MDa with an octahedral geometry of the E2 core. The results of this work strengthen the assumption that these enzyme systems may have had or potentially have a role in the Archaea. This may hold further clues to the evolutionary relationship between the three kingdoms of life and the role of OADHCs/lipoylation in the Archaea. The temperature dependent assembly of the complex and thermostability of these proteins may also provide a model to study thermostability and protein-protein interactions at high temperatures.
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Homeostasis of endocytic and autophagic systems insights from the host-pathogen interaction /Cianciola, Nicholas L. January 2009 (has links)
Thesis (Ph. D.)--Case Western Reserve University, 2009. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references.
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Exploration d'anomalies mitochondriales dans les fibroblastes de patients atteints de déficit dans les voies de biogenèse des centres fer-soufre ou de synthèse de l'acide lipoïque / Investigation of Mitochondrial Dysfunctions in Fibroblasts of Patients with Deficiency in Iron-Sulfur Cluster Biogenesis or Lipoic Acid Synthesis PathwaysLebigot, Elise 31 January 2019 (has links)
Le but de cette thèse est d’étudier les modifications biochimiques mitochondriales liées à un défaut de lipoylation des protéines dans les fibroblastes de 14 patients. Ces patients sont porteurs d’une mutation dans un gène codant une des protéines impliquées soit dans la synthèse de l’acide lipoïque (LIPT1, LIPT2) soit dans la voie de biogenèse des centres Fe-S mitochondriale (FDX1L, ISCA1, ISCA2, IBA57, NFU1, BOLA3). La voie de biogenèse des centres Fe-S est nécessaire à la maturation des protéines Fe-S mitochondriales, dont la lipoic acid synthase (LIAS).Ces travaux ont permis d’étudier notamment un deuxième cas de déficit en FDX1L ainsi qu’un patient porteur d’une nouvelle mutation dans ISCA1. Les déficits dans la voie de la biogenèse des centres Fe-S observés chez les patients étudiés affectent principalement la maturation des protéines mitochondriales à centre [4Fe-4S] dont l’aconitase mitochondriale, les complexes I et II de la chaîne respiratoire et la LIAS, induisant ainsi un défaut de lipoylation d’enzymes clés du métabolisme énergétique (PDHc, KGDHc). Aucune atteinte du réseau mitochondrial ni de variations du stress oxydatif n’ont pu être mises en évidence. Finalement, l’ajout d’acide lipoïque exogène n’améliore pas les déficits observés.Les profils d’expression des protéines dans les fibroblastes des patients suggèrent que les protéines NFU1, BOLA3 et IBA57 ainsi que ISCA1, ISCA2 et IBA57 coopèrent entre elles de manière complexe. / The aim of this work is to study mitochondrial dysfunctions related to a defect of protein lipoylation in fibroblasts of 14 patients. These patients carry a point mutation in a gene encoding for a protein involved either in lipoic acid biosynthesis (LIPT1 or LIPT2) or in the mitochondrial pathway devoted to iron-sulfur cluster biogenesis (FDX1L, ISCA1, ISCA2, IBA57, NFU1, BOLA3) essential for maturation of mitochondrial Fe-S proteins such as lipoic acid synthase (LIAS). This work describes the second case of FDX1L deficiency and a patient with a new mutation in ISCA1 gene.We found that mitochondrial [4Fe-4S] proteins (mitochondrial aconitase, complexes I and II of the respiratory chain and LIAS) are mainly affected in fibroblasts of patients with defect in the mitochondrial Fe-S maturation pathway. Secondary, LIAS dysfunction leads to decreased lipoylation of PDHc and KGDHc, complexes involved in energy metabolism. Neither mitochondrial network nor oxidative stress biomarkers was modified in our study. Addition of exogenous lipoic acid did not rescue the mitochondrial deficiency.Protein expression profiles obtained in fibroblasts of patients suggest that NFU1, BOLA3 and IBA57 and also ISCA1, ISCA2 and IBA57 could function and interact together to form protein complexes.
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Proteomic Analysis Reveals a Novel Function of the Kinase Sat4p in Saccharomyces cerevisiae MitochondriaGey, Uta, Czupalla, Cornelia, Hoflack, Bernard, Krause, Udo, Rödel, Gerhard 07 May 2015 (has links) (PDF)
The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt) localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE) approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p) and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p). The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p), Lys114 (Kgd2p) and Lys102 (Gcv3p), respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.
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Proteomic Analysis Reveals a Novel Function of the Kinase Sat4p in Saccharomyces cerevisiae MitochondriaGey, Uta, Czupalla, Cornelia, Hoflack, Bernard, Krause, Udo, Rödel, Gerhard 07 May 2015 (has links)
The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt) localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE) approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p) and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p). The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p), Lys114 (Kgd2p) and Lys102 (Gcv3p), respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.
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