Farnesol induces the transcriptional accumulation of the Aspergillus nidulans Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase

Farnesol (FOH) is a non-sterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. It has been shown previously that FOH triggers morphological features characteristic of apoptosis in the filamentous fungus Aspergillus nidulans. Here, we investigate which pathways are influenced through FOH by examining the transcriptional profile of A. nidulans exposed to this isoprenoid. We observed decreased mRNA abundance of several genes involved in RNA processing and modification, transcription, translation, ribosomal structure and biogenesis, amino acid transport and metabolism, and ergosterol biosynthesis. We also observed increased mRNA expression of genes encoding a number of mitochondrial proteins and characterized in detail one of them, the aifA, encoding the Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase. The Delta aifA mutant is more sensitive to FOH (about 8.0% and 0% survival when exposed to 10 and 100 mu M FOH respectively) than the wild type (about 97% and 3% survival when exposed to 10 and 100 mu M FOH respectively). These results suggest that AifA is possibly important for decreasing the effects of FOH and reactive oxygen species. Furthermore, we showed an involvement of autophagy and protein kinase C in A. nidulans FOH-induced apoptosis.

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.

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.

CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice have a mutation within the gene encoding cytosolic carboxypeptidase 1 (CCP1/Nna1), which has homology to metallocarboxypeptidases. To assess the function of CCP1/Nna1, quantitative proteomics and peptidomics approaches were used to compare proteins and peptides in mutant and wild-type mice. Hundreds of peptides derived from cytosolic and mitochondrial proteins are greatly elevated in pcd mouse hypothalamus, amygdala, cortex, prefrontal cortex, and striatum. However, the major proteins detected on 2-D gel electrophoresis were present in mutant and wild-type mouse cortex and hypothalamus at comparable levels, and proteasome activity is normal in these brain regions of pcd mice, suggesting that the increase in cellular peptide levels in the pcd mice is due to reduced degradation of the peptides downstream of the proteasome. Both nondegenerating and degenerating regions of pcd mouse brain, but not wild-type mouse brain, show elevated autophagy, which can be triggered by a decrease in amino acid levels. Taken together with previous studies on CCP1/Nna1, these data suggest that CCP1/Nna1 plays a role in protein turnover by cleaving proteasome-generated peptides into amino acids and that decreased peptide turnover in the pcd mice leads to cell death.-Berezniuk, I., Sironi, J., Callaway, M. B., Castro, L. M., Hirata, I. Y., Ferro, E. S., Fricker, L. D. CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice. FASEB J. 24, 1813-1823 (2010). www.fasebj.org

The mitochondrial genome of the phytopathogenic basidiomycete Moniliophthora perniciosa is 109 kb in size and contains a stable integrated plasmid

We present here the sequence of the mitochondrial genome of the basidiomycete phytopathogenic hemibiotrophic fungus Moniliophthora perniciosa, causal agent of the Witches` Broom Disease in Theobroma cacao. The DNA is a circular molecule of 109103 base pairs, with 31.9 % GC, and is the largest sequenced so far. This size is due essentially to the presence of numerous non-conserved hypothetical ORFs. It contains the 14 genes coding for proteins involved in the oxidative phosphorylation, the two rRNA genes, one ORF coding for a ribosomal protein (rps3), and a set of 26 tRNA genes that recognize codons for all amino acids. Seven homing endonucleases are located inside introns. Except atp8, all conserved known genes are in the same orientation. Phylogenetic analysis based on the cox genes agrees with the commonly accepted fungal taxonomy. An uncommon feature of this mitochondrial genome is the presence of a region that contains a set of four, relatively small, nested, inverted repeats enclosing two genes coding for polymerases with an invertron-type structure and three conserved hypothetical genes interpreted as the stable integration of a mitochondrial linear plasmid. The integration of this plasmid seems to be a recent evolutionary event that could have implications in fungal biology. This sequence is available under GenBank accession number AY376688. (c) 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

On the mechanisms of phenothiazine-induced mitochondrial permeability transition: Thiol oxidation, strict Ca(2+) dependence, and cyt c release

Phenothiazines (PTZ) are drugs widely used in the treatment of schizophrenia. Trifluoperazine, a piperazinic PTZ derivative, has been described as inhibitor of the mitochondrial permeability transition (MPT). We reported previously the antioxidant activity of thioridazine at relatively low concentrations associated to the inhibition of the MPT (Brit. J. Pharmacol., 2002;136:136-142). In this study, it was investigated the induction of MPT by PTZ derivatives at concentrations higher than 10 mu M focusing on the molecular mechanism involved. PTZ promoted a dose-response mitochondrial swelling accompanied by mitochondrial transmembrane potential dissipation and calcium release, being thioridazine the most potent derivative. PTZ-induced MPT was partially inhibited by CsA or Mg(2+) and completely abolished by the abstraction of calcium. The oxidation of reduced thiol group of mitochondrial membrane proteins by PTZ was upstream the VIP opening and it was not sufficient to promote the opening of PTP that only occurred when calcium was present in the mitochondrial matrix. EPR experiments using DMPO as spin trapping excluded the participation of reactive oxygen species on the PTZ-induced MPT. Since 117 give rise to cation radicals chemically by the action of peroxidases and cyanide inhibited the PTZ-induced swelling, we propose that VIZ bury in the inner mitochondrial membrane and the chemically generated 117 cation radicals modify specific thiol groups that in the presence of Ca(2+) result in MPT associated to cytochrome c release. These findings contribute for the understanding of mechanisms of MET induction and may have implications for the cell death induced by PTZ. (C) 2010 Elsevier Inc. 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.

Mitochondrial ATP-sensitive K(+) channels as redox signals to liver mitochondria in response to hypertriglyceridemia

We have recently demonstrated that hypertriglyceridemic (HTG) mice present both elevated body metabolic rates and mild mitochondrial uncoupling in the liver owing to stimulated activity of the ATP-sensitive potassium channel (mitoK(ATP)). Because lipid excess normally leads to cell redox imbalance, we examined the hepatic oxidative status in this model. Cell redox imbalance was evidenced by increased total levels of carbonylated proteins, malondialdehydes, and GSSG/GSH ratios in HTG livers compared to wild type. In addition, the activities of the extramitochondrial enzymes NADPH oxidase and xanthine oxidase were elevated in HTG livers. In contrast, Mn-superoxide dismutase activity and content, a mitochondrial matrix marker, were significantly decreased in HTG livers. isolated HTG liver mitochondria presented lower rates of H(2)O(2) production, which were reversed by mitoK(ATP) antagonists. In vivo antioxidant treatment with N-acetylcysteine decreased both mitoKATP activity and metabolic rates in HTG mice. These data indicate that high levels of triglycerides increase reactive oxygen generation by extramitochondrial enzymes that promote MitoK(ATP) activation. The mild uncoupling mediated by mitoK(ATP) increases metabolic rates and protects mitochondria against oxidative damage. Therefore, a biological role for mitoK(ATP) is a redox sensor is shown here for the first time in an in vivo model of systemic and cellular lipid excess, (C) 2009 Elsevier Inc. All rights reserved.

Stoichiometry and thermodynamics of the interaction between the C-terminus of human 90 kDa heat shock protein Hsp90 and the mitochondrial translocase of outer membrane Tom70

A large majority of the 1000-1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a K(D) of 360 30 nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated. (C) 2011 Elsevier Inc. All rights reserved.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples

Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male). For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.

The mitochondrial genome of the stingless bee Melipona bicolor (Hymenoptera, Apidae, Meliponini): sequence, gene organization and a unique tRNA translocation event conserved across the tribe Meliponini

At present a complete mtDNA sequence has been reported for only two hymenopterans, the Old World honey bee, Apis mellifera and the sawfly Perga condei. Among the bee group, the tribe Meliponini (stingless bees) has some distinction due to its Pantropical distribution, great number of species and large importance as main pollinators in several ecosystems, including the Brazilian rain forest. However few molecular studies have been conducted on this group of bees and few sequence data from mitochondrial genomes have been described. In this project, we PCR amplified and sequenced 78% of the mitochondrial genome of the stingless bee Melipona bicolor (Apidae, Meliponini). The sequenced region contains all of the 13 mitochondrial protein-coding genes, 18 of 22 tRNA genes, and both rRNA genes (one of them was partially sequenced). We also report the genome organization (gene content and order), gene translation, genetic code, and other molecular features, such as base frequencies, codon usage, gene initiation and termination. We compare these characteristics of M. bicolor to those of the mitochondrial genome of A. mellifera and other insects. A highly biased A+T content is a typical characteristic of the A. mellifera mitochondrial genome and it was even more extreme in that of M. bicolor. Length and compositional differences between M. bicolor and A. mellifera genes were detected and the gene order was compared. Eleven tRNA gene translocations were observed between these two species. This latter finding was surprising, considering the taxonomic proximity of these two bee tribes. The tRNA Lys gene translocation was investigated within Meliponini and showed high conservation across the Pantropical range of the tribe.