Purification of mitochondrial RNase P in A. nidulans

Résumé La ribonucléase P (RNase P) est une ribonucléoprotéine omniprésente dans tous les règnes du vivant, elle est responsable de la maturation en 5’ des précurseurs des ARNs de transfert (ARNts) et quelques autres petits ARNs. L’enzyme est composée d'une sous unité catalytique d'ARN (ARN-P) et d'une ou de plusieurs protéines selon les espèces. Chez les eucaryotes, l’activité de la RNase P cytoplasmique est distincte de celles des organelles (mitochondrie et chloroplaste). Chez la plupart des espèces, les ARN-P sont constituées de plusieurs éléments structuraux secondaires critiques conservés au cours de l’évolution. En revanche, au niveau de la structure, une réduction forte été observé dans la plupart des mtARN-Ps. Le nombre de protéines composant la RNase P est extrêmement variable : une chez les bactéries, environ quatre chez les archéobactéries, et dix chez la forme cytoplasmique des eucaryotes. Cet aspect est peu connu pour les formes mitochondriales. Dans la plupart des cas, l’identification de la mtRNase P est le résultat de longues procédures de purification comprenant plusieurs étapes dans le but de réduire au minimum le nombre de protéines requises pour l’activité (exemple de la levure et A. nidulans). Cela mène régulièrement à la perte de l’activité et de l’intégrité des complexes ribonucléo-protéiques natifs. Dans ce travail, par l’utilisation de la technique de BN-PAGE, nous avons développé une procédure d’enrichissement de l’activité RNase P mitochondriale native, donnant un rendement raisonnable. Les fractions enrichies capables de cette activité enzymatique ont été analysées par LC/MS/MS et les résultats montrent que l’holoenzyme de la RNase P de chacune des fractions contient un nombre de protéines beaucoup plus grand que ce qui était connue. Nous suggérons une liste de protéines (principalement hypothétiques) qui accompagnent l’activité de la RNase P. IV De plus, la question de la localisation de la mtRNase P de A. nidulans a été étudiée, selon nos résultats, la majorité de la mtRNase P est attachée á la membrane interne de la mitochondrie. Sa solubilisation se fait par l’utilisation de différents types de détergent. Ces derniers permettent l’obtention d’un spectre de complexes de la RNase P de différentes tailles.

Evaluation of mitochondrial function in a model of developmental programming of hypertension associated with transient neonatal oxygen exposure

UNE EXPOSITION NÉONATALE À L’OXYGÈNE MÈNE À DES MODIFICATIONS DE LA FONCTION MITOCHONDRIALE CHEZ LE RAT ADULTE Introduction: L’exposition à l’oxygène (O2) des ratons nouveau-nés a des conséquences à l’âge adulte dont une hypertension artérielle (HTA), une dysfonction vasculaire, une néphropénie et des indices de stress oxydant. En considérant que les reins sont encore en développement actif lors des premiers jours après la naissance chez les rats, jouent un rôle clé dans le développement de l’hypertension et qu’une dysfonction mitochondriale est associé à une augmentation du stress oxydant, nous postulons que les conditions délétères néonatales peuvent avoir un impact significatif au niveau rénal sur la modulation de l’expression de protéines clés du fonctionnement mitochondrial et une production mitochondriale excessive d’espèces réactives de l’ O2. Méthodes: Des ratons Sprague-Dawley sont exposés à 80% d’O2 (H) ou 21% O2 (Ctrl) du 3e au 10e jr de vie. En considérant que plusieurs organes des rats sont encore en développement actif à la naissance, ces rongeurs sont un modèle reconnu pour étudier les complications d’une hyperoxie néonatale, comme celles liées à une naissance prématurée chez l’homme. À 4 et à 16 semaines, les reins sont prélevés et les mitochondries sont extraites suivant une méthode d’extraction standard, avec un tampon contenant du sucrose 0.32 M et différentes centrifugations. L’expression des protéines mitochondriales a été mesurée par Western blot, tandis que la production d’ H202 et les activités des enzymes clés du cycle de Krebs ont été évaluées par spectrophotométrie. Les résultats sont exprimés par la moyenne ± SD. Résultats: Les rats mâles H de 16 semaines (n=6) présentent une activité de citrate synthase (considéré standard interne de l’expression protéique et de l’abondance mitochondriales) augmentée (12.4 ± 8.4 vs 4.1 ± 0.5 μmole/mL/min), une diminution de l’activité d’aconitase (enzyme sensible au redox mitochondrial) (0.11 ± 0.05 vs 0.20 ± 0.04 μmoles/min/mg mitochondrie), ainsi qu’une augmentation dans la production de H202 (7.0 ± 1.3 vs 5.4 ± 0.8 ρmoles/mg protéines mitochondriales) comparativement au groupe Ctrl (n=6 mâles et 4 femelles). Le groupe H (vs Ctrl) présente également une diminution dans l’expression de peroxiredoxin-3 (Prx3) (H 0.61±0.06 vs. Ctrl 0.78±0.02 unité relative, -23%; p<0.05), une protéine impliquée dans l’élimination d’ H202, de l’expression du cytochrome C oxidase (Complexe IV) (H 1.02±0.04 vs. Ctrl 1.20±0.02 unité relative, -15%; p<0.05), une protéine de la chaine de respiration mitochondriale, tandis que l’expression de la protéine de découplage (uncoupling protein)-2 (UCP2), impliquée dans la dispersion du gradient proton, est significativement augmentée (H 1.05±0.02 vs. Ctrl 0.90±0.03 unité relative, +17%; p<0.05). Les femelles H (n=6) (vs Ctrl, n=6) de 16 semaines démontrent une augmentation significative de l’activité de l’aconitase (0.33±0.03 vs 0.17±0.02 μmoles/min/mg mitochondrie), de l’expression de l’ATP synthase sous unité β (H 0.73±0.02 vs. Ctrl 0.59±0.02 unité relative, +25%; p<0.05) et de l’expression de MnSOD (H 0.89±0.02 vs. Ctrl 0.74±0.03 unité relative, +20%; p<0.05) (superoxide dismutase mitochondriale, important antioxidant), tandis que l’expression de Prx3 est significativement réduite (H 1.1±0.07 vs. Ctrl 0.85±0.01 unité relative, -24%; p<0.05). À 4 semaines, les mâles H (vs Ctrl) présentent une augmentation significative de l’expression de Prx3 (H 0.72±0.03 vs. Ctrl 0.56±0.04 unité relative, +31%; p<0.05) et les femelles présentent une augmentation significative de l’expression d’UCP2 (H 1.22±0.05 vs. Ctrl 1.03±0.04 unité relative, +18%; p<0.05) et de l’expression de MnSOD (H 1.36±0.01 vs. 1.19±0.06 unité relative, +14%; p<0.05). Conclusions: Une exposition néonatale à l’O2 chez le rat adulte mène à des indices de dysfonction mitochondriale dans les reins adultes, associée à une augmentation dans la production d’espèces réactives de l’oxygène, suggérant que ces modifications mitochondriales pourraient jouer un rôle dans l’hypertension artérielle et d’un stress oxydant, et par conséquent, être un facteur possible dans la progression vers des maladies cardiovasculaires. Mots-clés: Mitochondries, Reins, Hypertension, Oxygène, Stress Oxydant, Programmation

The pro-inflammatory oxidant hypochlorous acid induces Bax-dependent mitochondrial permeabilisation and cell death through AIF-/EndoG-dependent pathways

At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H2O2) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown. We determined the molecular mechanisms by which HOCl induced cell death in human mesenchymal progenitor cells (MPCs) differentiated into a chondrocytic phenotype as a model of human cartilage cells and show that HOCl induced rapid Bax conformational change, mitochondrial permeability and release of intra-mitochondrial pro-apoptotic proteins which resulted in nuclear translocation of AIF and EndoG. siRNA-mediated knockdown of Bax substantially prevented mitochondrial permeability, release of intra-mitochondrial pro-apoptotic proteins. Cell death was inhibited by siRNA-mediated knockdown of Bax, AIF or EndoG. Although we observed several biochemical markers of apoptosis, caspase activation was not detected either by western blotting, fluorescence activity assays or by using caspase inhibitors to inhibit cell death. This was further supported by findings that (1) in vitro exposure of recombinant human caspases to HOCl caused significant inhibition of caspase activity and (2) the addition of HOCl to staurosporine-treated MPCs inhibited the activity of cellular caspases. Our results show for the first time that HOCl induced Bax-dependent mitochondrial permeability which led to cell death without caspase activity by processes involving AIF/EndoG-dependent pathways. Our study provides a novel insight into the potential mechanisms of cell death in the inflamed human joint. (c) 2006 Elsevier Inc. All rights reserved.

c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in mitochondrion-dependent apoptosis

The molecular mechanisms underlying the initiation and control of the release of cytochrome c during mitochondrion-dependent apoptosis are thought to involve the phosphorylation of mitochondrial Bcl-2 and Bcl-x(L). Although the c-Jun N-terminal kinase (JNK) has been proposed to mediate the phosphorylation of Bcl-2/Bcl-x(L) the mechanisms linking the modification of these proteins and the release of cytochrome c remain to be elucidated. This study was aimed at establishing interdependency between JNK signalling and mitochondrial apoptosis. Using an experimental model consisting of isolated, bioenergetically competent rat brain mitochondria, these studies show that (i) JNK catalysed the phosphorylation of Bcl-2 and Bcl-x(L) as well as other mitochondrial proteins, as shown by two-dimensional isoelectric focusing/SDS/PAGE; (ii) JNK-induced cytochrome c release, in a process independent of the permeability transition of the inner mitochondrial membrane (imPT) and insensitive to cyclosporin A; (iii) JNK mediated a partial collapse of the mitochondrial inner-membrane potential (Deltapsim) in an imPT- and cyclosporin A-independent manner; and (iv) JNK was unable to induce imPT/swelling and did not act as a co-inducer, but as an inhibitor of Ca-induced imPT. The results are discussed with regard to the functional link between the Deltapsim and factors influencing the permeability transition of the inner and outer mitochondrial membranes. Taken together, JNK-dependent phosphorylation of mitochondrial proteins including, but not limited to, Bcl-2/Bcl-x(L) may represent a potential of the modulation of mitochondrial function during apoptosis.

Phylogenetic relationships among species of Anopheles (Nyssorhynchus) (Diptera, Culicidae) based on nuclear and mitochondrial gene sequences

Phylogenetic relationships among 21 species of mosquitoes in subgenus Nyssorhynchus were inferred from the nuclear white and mitochondrial NADH dehydrogenase subunit 6 (ND6) genes. Bayestan phylogenetic methods found that none of the three Sections within Nyssorhynchus (Albimanus, Argyritarsis, Myzorhynchella) were supported in all analyses, although Myzorhynchella was found to be monophyletic at the combined genes Within the Albimanus Section the monophyly of the Stroder Subgroup was strongly supported and within the Myzorhynchella Section Anopheles anrunesi and An lutzu formed a strongly supported monophyletic group The epidemiologically significant Albitarsis Complex showed evidence of paraphyly (relative to An lanet-Myzorhynchella) and discordance across gene trees, and the previously synonomized species of An. dunhami and An goeldii were recovered as sister species Finally, there was evidence of complexes in several species, including An antunesi, An deaneorum, and An. strodei (c) 2010 Elsevier B.V. All rights reserved

Phylogeny, species limits, and biogeography of the Brazilian lizards of the genus Eurolophosaurus (Squamata : Tropiduridae) as inferred from mitochondrial DNA sequences

Phylogenetic relationships and divergence times for 10 populations of the three recognized ""species"" of Brazilian lizards of genus Eurolophosaurus were estimated from 1229 bp of cyt b, COI, 12S, and 16S rRNA mitochondrial gene segments. Eurolophosaurus is monophyletic and the basal split within the genus separates E divaricatus from a clade comprising E amathites and E nanuzae. Three populations of E divaricatus, which occurs along the western bank of Rio S (a) over tildeo Francisco, were consistently grouped together. Oil the east bank of the river, E amathites and E nanuzae from state of Bahia were recovered as the sister group of E nanuzae populations from state of Minas Gerais. The paraphyly of E nanuzae and the high divergence levels among populations of E divaricatus strongly suggest that species limits in Eurolophosaurus should be revised. Even considering an extreme evolutionary rate of 2.8% sequence divergence per million years for the four gene segments analyzed together, E. divaricatus would have separated from the two other species by at least 5.5 my ago, and E. amathites from E nanuzae populations from Bahia and Minas Gerais, respectively, by 1.5 and 3.5 my. The paleolacustrine hypothesis and changes in the course of the river potentially explain faunal divergence in the area, but divergences are much older than previously admitted. (C) 2007 Elsevier Inc. All rights reserved.

Role of the Mitochondrial Mutations, m. 827A > G and the Novel m. 7462C > T, in the Origin of Hearing Loss

Samples from 30 deaf probands exhibiting features suggestive of syndromic mitochondrial deafness or from families with maternal transmission of deafness were selected for investigation of mutations in the mitochondrial genes MT-RNR1 and MT-TS1. Patients with mutation m. 1555A>G had been previously excluded from this sample. In the MT-RNR1 gene, five probands presented the m. 827A>G sequence variant, of uncertain pathogenicity. This change was also detected in 66 subjects of an unaffected control sample of 306 Brazilian individuals from various ethnic backgrounds. Given its high frequency, we consider it unlikely to have a pathogenic role on hereditary deafness. As to the MT-TS1 gene, one proband presented the previously known pathogenic m. 7472insC mutation and three probands presented a novel variant, m. 7462C>T, which was absent from the same control sample of 306 individuals. Because of its absence in control samples and association with a family history of hearing impairment, we suggest it might be a novel pathogenic mutation.

Genetic structure, phylogeny, and biogeography of Brazilian eyelid-less lizards of genera Calyptommatus and Nothobachia (Squamata, Gymnophthalmidae) as inferred from mitochondrial DNA sequences

Calyptommatus and Nothobachia genera of gymnophthalmid lizards are restricted to sandy open habitats on Sao Francisco River margins, northeastern Brazil. Phylogenetic relationships and geographic distribution of the four recognized species of Calyptommatus were analyzed from partial mitochondrial cyt b, 12S, and 16S rRNA genes sequencing, taking allopatric populations of the monotypic Nothobachia ablephara as the outgroup. In Calyptommatus a basal split separated C. sinebrachiatus, a species restricted to the eastern bank of the river, from the three other species. In this clade, C. confusionibus, found on western margin, was recovered as the sister group of the two other species, C. leiolepis and C. nicterus, from opposite margins. According to approximate date estimations, C. sinebrachiatus would have separated from the other congeneric species by 4.4-6.5 my, and C. nicterus, also from eastern bank, would be diverging by 1.8-2.6 my from C. leiolepis, the sister species on the opposite margin. C. confusionibus and C. leiolepis, both from western sandy areas, would be differentiating by 2.8-5.0 my. Divergence times of about 3.0-4.0 my were estimated for allopatric populations of Nothobachia restricted to western margin. Significant differences in 16S rRNA secondary structure relatively to other vertebrates are reported. Distinct evolutionary patterns are proposed for different taxa in those sandy areas, probably related to historical changes in the course of Sao Francisco River. (C) 2010 Elsevier Inc. All rights reserved.

The Trypanosoma cruzi proteins TcCox10 and TcCox15 catalyze the formation of heme A in the yeast Saccharomyces cerevisiae

Trypanosoma cruzi, the etiologic agent for Chagas` disease, has requirements for several cofactors, one of which is heme. Because this organism is unable to synthesize heme, which serves as a prosthetic group for several heme proteins (including the respiratory chain complexes), it therefore must be acquired from the environment. Considering this deficiency, it is an open question as to how heme A, the essential cofactor for eukaryotic CcO enzymes, is acquired by this parasite. In the present work, we provide evidence for the presence and functionality of genes coding for heme O and heme A synthases, which catalyze the synthesis of heme O and its conversion into heme A, respectively. The functions of these T. cruzi proteins were evaluated using yeast complementation assays, and the mRNA levels of their respective genes were analyzed at the different T. cruzi life stages. It was observed that the amount of mRNA coding for these proteins changes during the parasite life cycle, suggesting that this variation could reflect different respiratory requirements in the different parasite life stages.

Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats

The genetic diversity and phylogeographical patterns of Trypanosoma species that infect Brazilian bats were evaluated by examining 1043 bats from 63 species of seven families captured in Amazonia, the Pantanal, Cerrado and the Atlantic Forest biomes of Brazil. The prevalence of trypanosonne-infected bats, as estimated by haemoculture, was 12.9%, resulting in 77 Cultures of isolates, most morphologically identified as Trypanosoma cf. cruzi, classified by barcoding using partial sequences from ssrRNA gene into the subgenus Schizotrypanum and identified as T. cruzi (15), T cruzi marinkellei (37) or T. cf. dionisii (25). Phylogenetic analyses using nuclear ssrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and mitochondrial cytochrome b (Cyt b) gene sequences generated three clades, which clustered together forming the subgenus Schizotrypanum. In addition to vector association, bat trypanosomes were related by the evolutionary history, ecology and phylogeography of the bats. Tryponosoma cf. dionisii trypanosomes (32.4%) infected 12 species from four bat families captured in all biomes, from North to South Brazil, and clustered with T. dionisii from Europe despite being separated by some genetic distance. Trypanosoma cruzi marinkellei (49.3%) was restricted to phyllostomid bats from Amazonia to the Pantanal (North to Central). Trypanosoma cruzi (18.2%) was found mainly in vespertilionid and phyllostomid bats from the Pantanal/Cerrado and the Atlantic Forest (Central to Southeast), with a few isolates from Amazonia. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

The mitochondrial view of Blastocladiella emersonii

The mitochondrial genome of the chytrid Blastocladiella emersonii was sequenced and annotated, revealing the complete set of oxidative phosphorylation genes and tRNAs/rRNAs necessary for the translation process. Phylogenetic reconstructions reinforce the proposal of the new phylum Blastocladiomycota. Evidences of gene duplication due to inserted elements suggest shared susceptibility to gene invasion/exchange between chytrids and zygomycetes. The gene content of B. emersonii is very similar to Allomyces macrogynus but the content of intronic and changeable elements is much lower suggesting a stronger resistance to this kind of exchange. in addition, a total of 401 potential nuclear transcripts encoding mitochondrial proteins were obtained after B. emersonii EST database scanning using Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana data as probes and TargetP tool to find N-terminal mitochondrial signal in translated sequences. (c) 2008 Elsevier B.V. All rights reserved.

trans, trans-2,4-decadienal induces mitochondrial dysfunction and oxidative stress

Lipid peroxidation produces a large number of reactive aldehydes as secondary products. We have previously shown that the reaction of cytochrome c with trans,trans-2, 4-decadienal (DDE), an aldehyde generated as a product of lipid peroxidation in cell membranes, results in the formation of adducts. Mass spectrometry analysis indicated that His-33, Lys-39, Lys-72 and Lys-100 in cytochrome c were modified by DDE. In the present work, we investigated the effect of DDE on isolated rat liver mitochondria. DDE (162 mu M) treatment increases the rate of mitochondrial oxygen consumption. Extensive mitochondrial swelling upon treatment with DDE (900 nM-162 mu M) was observed by light scattering and transmission electron microscopy experiments. DDE-induced loss of inner mitochondrial membrane potentials, monitored by safranin O fluorescence, was also observed. Furthermore, DDE-treated mitochondria showed an increase in lipid peroxidation, as monitored by MDA formation. These results suggest that reactive aldehydes promote mitochondrial dysfunction.

The role of mitochondrial DNA damage in the citotoxicity of reactive oxygen species

Mitochondria contain their own genome, a small circular molecule of around 16.5 kbases. The mitochondrial DNA (mtDNA) encodes for only 13 polypeptides, but its integrity is essential for mitochondrial function, as all 13 proteins are regulatory subunits of the oxidative phosphorylation complexes. Nonetheless, the mtDNA is physically associated with the inner mitochondrial membrane, where the majority of the cellular reactive oxygen species are generated. In fact, the mitochondrial DNA accumulates high levels of oxidized lesions, which have been associated with several pathological and degenerative processes. The cellular responses to nuclear DNA damage have been extensively studied, but so far little is known about the functional outcome and cellular responses to mtDNA damage. In this review we will discuss the mechanisms that lead to damage accumulation and the in vitro models we are establishing to dissect the cellular responses to oxidative damage in the mtDNA and to sort out the differential cellular consequences of accumulation of damage in each cellular genome, the nuclear and the mitochondrial genome.

The mitochondrial transcription factor A functions in mitochondrial base excision repair

Mitochondrial transcription factor A (TFAM) is an essential component of mitochondrial nucleoids TFAM plays an important role in mitochondrial transcription and replication TFAM has been previously reported to inhibit nucleotide excision repair (NER) in vitro but NER has not yet been detected in mitochondria, whereas base excision repair (BER) has been comprehensively characterized in these organelles The BER proteins are associated with the inner membrane in mitochondria and thus with the mitochondrial nucleoid, where TFAM is also situated However, a function for TFAM in BER has not yet been investigated This study examines the role of TFAM in BER In vitro studies with purified recombinant TFAM indicate that it preferentially binds to DNA containing 8-oxoguanines, but not to abasic sites, uracils, or a gap in the sequence TFAM inhibited the in vitro incision activity of 8-oxoguanine DNA glycosylase (OGG1), uracil-DNA glycosylase (UDG), apurinic endonuclease 1 (APE1), and nucleotide incorporation by DNA polymerase gamma (pol gamma) On the other hand, a DNA binding-defective TFAM mutant, L58A, showed less inhibition of BER in vitro Characterization of TFAM knockdown (KD) cells revealed that these lysates had higher 8oxoG incision activity without changes in alpha OGG1 protein levels TFAM KD cells had mild resistance to menadione and increased damage accumulation in the mtDNA when compared to the control cells In addition, we found that the tumor suppressor p53, which has been shown to interact with and alter the DNA binding activity of TFAM, alleviates TFAM-Induced inhibition of BER proteins Together, the results suggest that TFAM modulates BER in mitochondria by virtue of its DNA binding activity and protein interactions Published by Elsevier B V

Mitochondrial ion transport pathways: Role in metabolic diseases

Mitochondria are the central coordinators of energy metabolism and alterations in their function and number have long been associated with metabolic disorders such as obesity, diabetes and hyperlipidemias. Since oxidative phosphorylation requires an electrochemical gradient across the inner mitochondrial membrane, ion channels in this membrane certainly must play an important role in the regulation of energy metabolism. However, in many experimental settings, the relationship between the activity of mitochondrial ion transport and metabolic disorders is still poorly understood. This review briefly summarizes some aspects of mitochondrial H(+) transport (promoted by uncoupling proteins, UCPs). Ca(2+) and K(+) uniporters which may be determinant in metabolic disorders. (C) 2009 Elsevier B.V. All rights reserved.