Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Clinical response to chemotherapy in oesophageal adenocarcinoma patients is linked to defects in mitochondria

Chemotherapeutic drugs kill cancer cells, but it is unclear why this happens in responding patients but not in non-responders. Proteomic profiles of patients with oesophageal adenocarcinoma may be helpful in predicting response and selecting more effective treatment strategies. In this study, pretherapeutic oesophageal adenocarcinoma biopsies were analysed for proteomic changes associated with response to chemotherapy by MALDI imaging mass spectrometry. Resulting candidate proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and investigated for functional relevance in vitro. Clinical impact was validated in pretherapeutic biopsies from an independent patient cohort. Studies on the incidence of these defects in other solid tumours were included. We discovered that clinical response to cisplatin correlated with pre-existing defects in the mitochondrial respiratory chain complexes of cancer cells, caused by loss of specific cytochrome c oxidase (COX) subunits. Knockdown of a COX protein altered chemosensitivity in vitro, increasing the propensity of cancer cells to undergo cell death following cisplatin treatment. In an independent validation, patients with reduced COX protein expression prior to treatment exhibited favourable clinical outcomes to chemotherapy, whereas tumours with unchanged COX expression were chemoresistant. In conclusion, previously undiscovered pre-existing defects in mitochondrial respiratory complexes cause cancer cells to become chemosensitive: mitochondrial defects lower the cells' threshold for undergoing cell death in response to cisplatin. By contrast, cancer cells with intact mitochondrial respiratory complexes are chemoresistant and have a high threshold for cisplatin-induced cell death. This connection between mitochondrial respiration and chemosensitivity is relevant to anticancer therapeutics that target the mitochondrial electron transport chain.

Changes in mitochondrial enzymatic activities of monocytes during prolonged hypobaric hypoxia and influence of antioxidants: A randomized controlled study

OBJECTIVES Exposure to high altitudes is associated with oxidative cellular damage due to the increased level of reactive oxygen and nitrogen species and altered activity of antioxidant systems. Subjects were submitted to prolonged hypoxia, to evaluate changes in mitochondrial enzyme activities of monocytes and their attenuation by supplementation with antioxidants. METHODS Twelve subjects were randomly assigned to receive antioxidant supplements or placebo prior to and during an expedition to Pik Lenin (7145 m). Monocytes were isolated from blood samples to determine the activity of mitochondrial enzymes cytochrome c oxidase and citrate synthase at 490 m (baseline) and at the altitudes of 3550 m, 4590 m, and 5530 m. RESULTS An increase in citrate synthase activity at all altitudes levels was observed. Hypoxia induced an increase in the activity of cytochrome c oxidase only at 4590 m. Neither citrate synthase activity nor cytochrome c oxidase activity differed between the subjects receiving antioxidant supplements and those receiving placebo. CONCLUSIONS Hypoxia leads to an increase in citrate synthase activity of monocyte mitochondria as a marker of mitochondrial mass, which is not modified by antioxidant supplementation. The increase in mitochondrial mass may represent a compensatory mechanism to preserve oxidative phosphorylation of monocytes at high altitudes.

Function of cardiolipin in mitochondria of Saccharomyces cerevisiae

Cardiolipin and its precursor phosphatidylglycerol, phospholipids found uniquely in membranes engaged in oxidative phosphorylation, play important roles in multimeric complexes of the energy transducing system (ETS) associated with the inner mitochondrial membrane. A combined molecular genetic and biochemical approach was used to more precisely define the role of cardiolipin in cell processes. ^ Strains of yeast Saccharomyces cerevisiae unable to synthesize cardiolipin because of the crd1Δ allele (encodes cardiolipin synthase) with different phenotypes were analyzed to determine which phenotypes are due to lack of cardiolipin. We concluded that many of the severe phenotypes ascribed to cells lacking cardiolipin, particularly when grown at 37°C, are because of the synergistic interaction of the crd1Δ mutation with the reduced expression of the PET56 gene which encodes a component essential for the formation of functional mitochondrial ribosomes. We also demonstrate that much of the reduced mitochondrial function in crd1Δ is because of reduced expression of ETS components at elevated temperature. ^ A crd1Δ mutant of S. cerevisiae has less severe physiological changes than strains lacking both phosphatidylglycerol and cardiolipin due to an increased level of phosphatidylglycerol, which might partially substitute for the cardiolipin-requiring functions. By varying the level of cardiolipin, we were able to correlate phenotypes in a dose-dependent manner with the level of cardiolipin to support more strongly an involvement of cardiolipin in a particular cellular process. There is almost complete lack of a supercomplex composed of cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) in extracts of cardiolipin-lacking mitochondria when compared to wild type cells and the level of supercomplex varies in proportion to the cardiolipin levels. Reduced cardiolipin levels also compromise the growth properties of yeast in a dose-dependent manner suggesting that the loss in growth efficiency is related to a role of cardiolipin that cannot be replaced by phosphatidylglycerol. An independent kinetic approach was performed to compare organization of the respiratory chain in wild-type and cardiolipin-lacking mitochondria. Cardiolipin-lacking mitochondria display kinetic properties for electron transfer between complexes III and IV via cytochrome c consistent with cytochrome c being a freely diffusible carrier, confirming complexes III and IV exist as individual complexes and not associated into a supercomplex in cardiolipin-lacking mitochondria. ^

Apparent mtDNA heteroplasmy in Alzheimer’s disease patients and in normals due to PCR amplification of nucleus-embedded mtDNA pseudogenes

In an unprecedented finding, Davis et al. [Davis, R. E., Miller, S., Herrnstadt, C., Ghosh, S. S., Fahy, E., Shinobu, L. A., Galasko, D., Thal, L. J., Beal, M. F., Howell, N. & Parker, W. D., Jr. (1997) Proc. Natl. Acad. Sci. USA 94, 4526–4531] used an unusual DNA isolation method to show that healthy adults harbor a specific population of mutated mitochondrial cytochrome c oxidase (COX) genes that coexist with normal mtDNAs. They reported that this heteroplasmic population was present at a level of 10–15% in the blood of normal individuals and at a significantly higher level (20–30%) in patients with sporadic Alzheimer’s disease. We provide compelling evidence that the DNA isolation method employed resulted in the coamplification of authentic mtDNA-encoded COX genes together with highly similar COX-like sequences embedded in nuclear DNA (“mtDNA pseudogenes”). We conclude that the observed heteroplasmy is an artifact.

The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation

The expression of DCC (deleted in colorectal cancer) is often markedly reduced in colorectal and other cancers. However, the rarity of point mutations identified in DCC coding sequences and the lack of a tumor predisposition phenotype in DCC hemizygous mice have raised questions about its role as a tumor suppressor. DCC also mediates axon guidance and functions as a dependence receptor; such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand. We now show that DCC drives cell death independently of both the mitochondria-dependent pathway and the death receptor/caspase-8 pathway. Moreover, we demonstrate that DCC interacts with both caspase-3 and caspase-9 and drives the activation of caspase-3 through caspase-9 without a requirement for cytochrome c or Apaf-1. Hence, DCC defines an additional pathway for the apoptosome-independent caspase activation.

Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.

As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain.

Genetic diversity and susceptibility to Vip3Aa20 protein in Brazilian populations of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae)

Helicoverpa armigera (Hübner) was officially reported in Brazil in 2013. This species is closely related to Helicoverpa zea (Boddie) and has caused significant crop damage in Brazil. The use of genetically modified crops expressing insecticidal protein from Bacillus thuringiensis (Berliner) has been one of the control tactics for managing these pests. Genetically modified maize expressing Vip3Aa20 was approved to commercial use in Brazil in 2009. Understanding the genetic diversity and the susceptibility to B. thuringiensis proteins in H. armigera and H. zea populations in Brazil are crucial for establishing Insect Resistance Management (IRM) programs in Brazil. Therefore, the objectives of this study were: (a) to infer demographic parameters and genetic structure of H. armigera and H. zea Brazil; (b) to assess the intra and interspecific gene flow and genetic diversity of H. armigera and H. zea; and (c) to evaluate the susceptibility to Vip3Aa20 protein in H. armigera and H. zea populations of Brazil. A phylogeographic analysis of field H. armigera and H. zea populations was performed using a partial sequence data from the cytochrome c oxidase I (COI) gene. H. armigera individuals were most prevalent on dicotyledonous hosts and H. zea individuals were most prevalent on maize crops. Both species showed signs of demographic expansion and no genetic structure. High genetic diversity and wide distribution were observed for H. armigera. A joint analysis indicated the presence of Chinese, Indian, and European lineages within the Brazilian populations of H. armigera. In the cross-species amplification study, seven microsatellite loci were amplified; and showed a potential hybrid offspring in natural conditions. Interespecific analyses using the same microsatellite loci with Brazilian H. armigera and H. zea in compare to the USA H. zea were also conducted. When analyses were performed within each species, 10 microsatellites were used for H. armigera, and eight for H. zea. We detected high intraspecific gene flow in populations of H. armigera and H. zea from Brazil and H. zea from the USA. Genetic diversity was similar for both species. However, H. armigera was more similar to H. zea from Brazil than H. zea from the USA and some putative hybrid individuals were found in Brazilian populations.Tthere was low gene flow between Brazilian and USA H. zea. The baseline susceptibility to Vip3Aa20 resulted in low interpopulation variation for H. zea (3-fold) and for H. armigera (5-fold), based on LC50. H. armigera was more tolerant to Vip3Aa20 than H. zea (≈ 40 to 75-fold, based on CL50). The diagnostic concentration for susceptibility monitoring, based on CL99, was fairly high (6,400 ng Vip3Aa20/cm2) for H. zea and not validated for H. armigera due to the high amount of protein needed for bioassays. Implementing IRM strategies to Vip3Aa20 in H. armigera and H. zea will be of a great challenge in Brazil, mainly due to the low susceptibility to Vip3Aa20 and high genetic diversity and gene flow in both species, besides a potential of hybrid individuals between H. armigera and H. zea under field conditions.

Defenses against oxidative stress in Neisseria gonorrhoeae: A system tailored for a challenging environment

Neisseria gonorrhoeae is a host-adapted pathogen that colonizes primarily the human genitourinary tract. This bacterium encounters reactive oxygen and reactive nitrogen species as a consequence of localized inflammatory responses in the urethra of males and endocervix of females and also of the activity of commensal lactobacilli in the vaginal flora. This review describes recent advances in the understanding of defense systems against oxidative stress in N. gonorrhoeae and shows that while some of its defenses have similarities to the paradigm established with Escherichia coli, there are also some key differences. These differences include the presence of a defense system against superoxide based on manganese ions and a glutathione-dependent system for defense against nitric oxide which is under the control of a novel MerR-like transcriptional regulator. An understanding of the defenses against oxidative stress in N. gonorrhoeae and their regulation may provide new insights into the ways in which this bacterium survives challenges from polymorphonuclear leukocytes and urogenital epithelial cells.

Dysfonctions mitochondriales associées à l’acidose lactique du Saguenay-Lac-Saint-Jean révélées par l’étude d’un nouveau modèle murin de la maladie

Le syndrome de Leigh version canadienne-française (LSFC) est une maladie autosomale récessive causée par une mutation du gène LRPPRC, encodant une protéine du même nom. LRPPRC est impliquée dans la traduction des gènes mitochondriaux qui encodent certains complexes de la chaine respiratoire. Les répercussions biochimiques incluent un déficit tissu spécifique de la cytochrome c oxydase (COX), principalement dans le foie et le cerveau, et la survenue de crises d’acidose fatales chez 80 % des enfants atteints avant l’âge de 3-4 ans. L’identification d’options thérapeutiques demeure encore un défi de taille et ceci est en partie relié au manque de connaissances des fonctions biologiques de LRPPRC et des mécanismes impliqués dans la pathogenèse du LSFC, au niveau des dysfonctions mitochondriales résultantes. Afin d’étudier ces mécanismes, le consortium de l’acidose lactique, dont fait partie notre laboratoire, a récemment développé un modèle murin portant une ablation de LRPPRC spécifique au foie (souris H-Lrpprc-/-). L’objectif principal est de déterminer si ce modèle reproduit le phénotype pathologique observé dans les cultures de fibroblastes humains issus de biopsies de peau de patients LSFC. Dans le cadre des travaux de ce mémoire, nous avons amorcé la caractérisation de ce nouveau modèle, en examinant le phénotype général, l’histopathologie hépatique et les fonctions mitochondriales, et en nous focalisant principalement sur les fonctions respiratoires et la capacité à oxyder divers types de substrats. Nous avons observé un retard de croissance, une hépatomégalie ainsi que plusieurs anomalies histologiques du foie chez la souris HLrpprc-/-. De plus, l’ablation de LRPPRC induit un déficit du complexe IV, mais aussi de l’ATP synthase, et affecte l’oxydation des acides gras à longues chaines. À la lumière de ces résultats, nous croyons que le modèle murin H-Lrpprc-/- contribuera à l’avancement des connaissances générales sur LRPPRC, nous permettant de mieux comprendre l’influence de la protéine sur les fonctions mitochondriales.

Dysfonctions mitochondriales associées à l’acidose lactique du Saguenay-Lac-Saint-Jean révélées par l’étude d’un nouveau modèle murin de la maladie

Le syndrome de Leigh version canadienne-française (LSFC) est une maladie autosomale récessive causée par une mutation du gène LRPPRC, encodant une protéine du même nom. LRPPRC est impliquée dans la traduction des gènes mitochondriaux qui encodent certains complexes de la chaine respiratoire. Les répercussions biochimiques incluent un déficit tissu spécifique de la cytochrome c oxydase (COX), principalement dans le foie et le cerveau, et la survenue de crises d’acidose fatales chez 80 % des enfants atteints avant l’âge de 3-4 ans. L’identification d’options thérapeutiques demeure encore un défi de taille et ceci est en partie relié au manque de connaissances des fonctions biologiques de LRPPRC et des mécanismes impliqués dans la pathogenèse du LSFC, au niveau des dysfonctions mitochondriales résultantes. Afin d’étudier ces mécanismes, le consortium de l’acidose lactique, dont fait partie notre laboratoire, a récemment développé un modèle murin portant une ablation de LRPPRC spécifique au foie (souris H-Lrpprc-/-). L’objectif principal est de déterminer si ce modèle reproduit le phénotype pathologique observé dans les cultures de fibroblastes humains issus de biopsies de peau de patients LSFC. Dans le cadre des travaux de ce mémoire, nous avons amorcé la caractérisation de ce nouveau modèle, en examinant le phénotype général, l’histopathologie hépatique et les fonctions mitochondriales, et en nous focalisant principalement sur les fonctions respiratoires et la capacité à oxyder divers types de substrats. Nous avons observé un retard de croissance, une hépatomégalie ainsi que plusieurs anomalies histologiques du foie chez la souris HLrpprc-/-. De plus, l’ablation de LRPPRC induit un déficit du complexe IV, mais aussi de l’ATP synthase, et affecte l’oxydation des acides gras à longues chaines. À la lumière de ces résultats, nous croyons que le modèle murin H-Lrpprc-/- contribuera à l’avancement des connaissances générales sur LRPPRC, nous permettant de mieux comprendre l’influence de la protéine sur les fonctions mitochondriales.

DNA barcoding: an effective tool to overcome morphological identification constraints in the assessment of the ecological quality

DNA barcoding has the potential to overcome taxonomic challenges in biological community assessments. However, fulfilling that potential requires successful amplification of a large and unbiased portion of the community. In this study, we attempted to identify mitochondrial gene cytochrome c oxidase I (COI) barcodes from 1024 benthic invertebrate specimens belonging to 54 taxa from low salinity environments of the Mira estuary and Torgal riverside (SW Portugal). Up to 17 primer pairs and several reaction conditions were attempted among specimens from all taxa, with amplification success defined as a single band of approximately 658 bp visualized on a pre-cast agarose gel, starting near the 5' end of the COI gene and suitable for sequencing. Amplification success was achieved for 99.6% of the 54 taxa, though no single primer was successful for more than 88.9% of the taxa. However, only 68.5% of the specimens within these taxa successfully amplified. Inhibition factors resulting from a non-purified DNA extracted and inexistence of species-specific primers for COI were pointed as the main reasons for an unsuccessful amplification. These results suggest that DNA barcoding can be an effective tool for application in low salinity environments where taxa such as chironomids and oligochaetes are challenging for morphological identification. Nevertheless, its implementation is not simple, as methods are still being standardized and multiple species

Molecular basis of intramolecular electron transfer in sulfite-oxidizing enzymes is revealed by high resolution structure of a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit

Sulfite-oxidizing molybdoenzymes convert the highly reactive and therefore toxic sulfite to sulfate and have been identified in insects, animals, plants, and bacteria. Although the well studied enzymes from higher animals serve to detoxify sulfite that arises from the catabolism of sulfur-containing amino acids, the bacterial enzymes have a central role in converting sulfite formed during dissimilatory oxidation of reduced sulfur compounds. Here we describe the structure of the Starkeya novella sulfite dehydrogenase, a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit, that reveals the molecular mechanism of intramolecular electron transfer in sulfite-oxidizing enzymes. The close approach of the two redox centers in the protein complex (Mo-Fe distance 16.6 angstrom) allows for rapid electron transfer via tunnelling or aided by the protein environment. The high resolution structure of the complex has allowed the identification of potential through-bond pathways for electron transfer including a direct link via Arg-55A and/or an aromatic-mediated pathway. A potential site of electron transfer to an external acceptor cytochrome c was also identified on the SorB subunit on the opposite side to the interaction with the catalytic SorA subunit.

Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning deficit and inhibiting cytochrome oxidase activity.

Previously, we developed a rat model of persistent mitochondrial dysfunction based upon the chronic partial inhibition of the mitochondrial enzyme cytochrome oxidase (EC 1.9.3.1). Continuous systemic infusion of sodium azide at approximately 1 mg/kg per hr inhibited cytochrome oxidase activity and produced a spatial learning deficit. In other laboratories, glucocorticoids have been reported to exacerbate neuronal damage from various acute metabolic insults. Therefore, we tested the hypothesis that corticosterone, the primary glucocorticoid in the rat, would potentiate the sodium azide-induced learning deficit. To this end, we first identified nonimpairing doses of sodium azide (approximately 0.75 mg/kg per hr) and corticosterone (100-mg pellet, 3-week sustained-release). We now report that chronic co-administration of these individually nonimpairing treatments produced a severe learning deficit. Moreover, the low dose of corticosterone, which did not elevate serum corticosterone, acted synergistically with sodium azide to inhibit cytochrome oxidase activity. The latter result represents a previously unidentified effect of glucocorticoids that provides a candidate mechanism for glucocorticoid potentiation of neurotoxicity induced by metabolic insult. These results may have the clinical implication of expanding the definition of hypercortisolism in patient populations with compromised oxidative metabolism. Furthermore, they suggest that glucocorticoid treatment may contribute to pathology in disease or trauma conditions that involve metabolic insult.

Cellular prion protein (PrP(C)) and superoxide dismutase (SOD) in vascular cells under oxidative stress

The PrP(C) is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP(C) with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP(C) expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrP(C) expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9 +/- 1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP(C) expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP(C) only in cells stimulated for 8 hours with the different stressors. The PrP(C) mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP(C) protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP(C). (C) 2009 Elsevier GmbH. All rights reserved.