Zim17/Tim15 links mitochondrial iron–sulfur cluster biosynthesis to nuclear genome stability

Genomic instability is related to a wide-range of human diseases. Here, we show that mitochondrial iron–sulfur cluster biosynthesis is important for the maintenance of nuclear genome stability in Saccharomyces cerevisiae. Cells lacking the mitochondrial chaperone Zim17 (Tim15/Hep1), a component of the iron–sulfur biosynthesis machinery, have limited respiration activity, mimic the metabolic response to iron starvation and suffer a dramatic increase in nuclear genome recombination. Increased oxidative damage or deficient DNA repair do not account for the observed genomic hyperrecombination. Impaired cell-cycle progression and genetic interactions of ZIM17 with components of the RFC-like complex involved in mitotic checkpoints indicate that replicative stress causes hyperrecombination in zim17Δ mutants. Furthermore, nuclear accumulation of pre-ribosomal particles in zim17Δ mutants reinforces the importance of iron–sulfur clusters in normal ribosome biosynthesis. We propose that compromised ribosome biosynthesis and cell-cycle progression are interconnected, together contributing to replicative stress and nuclear genome instability in zim17Δ mutants.

PGC-1α induces mitochondrial and myokine transcriptional programs and lipid droplet and glycogen accumulation in cultured human skeletal muscle cells

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a chief activator of mitochondrial and metabolic programs and protects against atrophy in skeletal muscle (skm). Here we tested whether PGC-1α overexpression could restructure the transcriptome and metabolism of primary cultured human skm cells, which display a phenotype that resembles the atrophic phenotype. An oligonucleotide microarray analysis was used to reveal the effects of PGC-1α on the whole transcriptome. Fifty-three different genes showed altered expression in response to PGC-1α: 42 upregulated and 11 downregulated. The main gene ontologies (GO) associated with the upregulated genes were mitochondrial components and processes and this was linked with an increase in COX activity, an indicator of mitochondrial content. Furthermore, PGC-1α enhanced mitochondrial oxidation of palmitate and lactate to CO2, but not glucose oxidation. The other most significantly associated GOs for the upregulated genes were chemotaxis and cytokine activity, and several cytokines, including IL-8/CXCL8, CXCL6, CCL5 and CCL8, were within the most highly induced genes. Indeed, PGC-1α highly increased IL-8 cell protein content. The most upregulated gene was PVALB, which is related to calcium signaling. Potential metabolic regulators of fatty acid and glucose storage were among mainly regulated genes. The mRNA and protein level of FITM1/FIT1, which enhances the formation of lipid droplets, was raised by PGC-1α, while in oleate-incubated cells PGC-1α increased the number of smaller lipid droplets and modestly triglyceride levels, compared to controls. CALM1, the calcium-modulated δ subunit of phosphorylase kinase, was downregulated by PGC-1α, while glycogen phosphorylase was inactivated and glycogen storage was increased by PGC-1α. In conclusion, of the metabolic transcriptome deficiencies of cultured skm cells, PGC-1α rescued the expression of genes encoding mitochondrial proteins and FITM1. Several myokine genes, including IL-8 and CCL5, which are known to be constitutively expressed in human skm cells, were induced by PGC-1α.

Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes

Yeast cells contain a family of three monothiol glutaredoxins: Grx3, 4, and 5. Absence of Grx5 leads to constitutive oxidative damage, exacerbating that caused by external oxidants. Phenotypic defects associated with the absence of Grx5 are suppressed by overexpression ofSSQ1 and ISA2, two genes involved in the synthesis and assembly of iron/sulfur clusters into proteins. Grx5 localizes at the mitochondrial matrix, like other proteins involved in the synthesis of these clusters, and the mature form lacks the first 29 amino acids of the translation product. Absence of Grx5 causes: 1) iron accumulation in the cell, which in turn could promote oxidative damage, and 2) inactivation of enzymes requiring iron/sulfur clusters for their activity. Reduction of iron levels in grx5 null mutants does not restore the activity of iron/sulfur enzymes, and cell growth defects are not suppressed in anaerobiosis or in the presence of disulfide reductants. Hence, Grx5 forms part of the mitochondrial machinery involved in the synthesis and assembly of iron/sulfur centers.

The Armc10/SVH gene: Genome context, regulation of mitochondrial dynamics and protection against Aβ-induced mitochondrial fragmentation

Mitochondrial function and dynamics are essential for neurotransmission, neural function and neuronal viability. Recently, we showed that the eutherian-specific Armcx gene cluster (Armcx1-6 genes), located in the X chromosome, encodes for a new family of proteins that localise to mitochondria, regulating mitochondrial trafficking. The Armcx gene cluster evolved by retrotransposition of the Armc10 gene mRNA, which is present in all vertebrates and is considered to be the ancestor gene. Here we investigate the genomic organisation, mitochondrial functions and putative neuroprotective role of the Armc10 ancestor gene. The genomic context of the Armc10 locus shows considerable syntenic conservation among vertebrates, and sequence comparisons and CHIP-data suggest the presence of at least three conserved enhancers. We also show that the Armc10 protein localises to mitochondria and that it is highly expressed in the brain. Furthermore, we show that Armc10 levels regulate mitochondrial trafficking in neurons, but not mitochondrial aggregation, by controlling the number of moving mitochondria. We further demonstrate that the Armc10 protein interacts with the KIF5/Miro1-2/Trak2 trafficking complex. Finally, we show that overexpression of Armc10 in neurons prevents A beta-induced mitochondrial fission and neuronal death. Our data suggest both conserved and differential roles of the Armc10/Armcx gene family in regulating mitochondrial dynamics in neurons, and underscore a protective effect of the Armc10 gene against A beta-induced toxicity. Overall, our findings support a further degree of regulation of mitochondrial dynamics in the brain of more evolved mammals.

The Armc10/SVH gene: Genome context, regulation of mitochondrial dynamics and protection against Aβ-induced mitochondrial fragmentation

Mitochondrial function and dynamics are essential for neurotransmission, neural function and neuronal viability. Recently, we showed that the eutherian-specific Armcx gene cluster (Armcx1-6 genes), located in the X chromosome, encodes for a new family of proteins that localise to mitochondria, regulating mitochondrial trafficking. The Armcx gene cluster evolved by retrotransposition of the Armc10 gene mRNA, which is present in all vertebrates and is considered to be the ancestor gene. Here we investigate the genomic organisation, mitochondrial functions and putative neuroprotective role of the Armc10 ancestor gene. The genomic context of the Armc10 locus shows considerable syntenic conservation among vertebrates, and sequence comparisons and CHIP-data suggest the presence of at least three conserved enhancers. We also show that the Armc10 protein localises to mitochondria and that it is highly expressed in the brain. Furthermore, we show that Armc10 levels regulate mitochondrial trafficking in neurons, but not mitochondrial aggregation, by controlling the number of moving mitochondria. We further demonstrate that the Armc10 protein interacts with the KIF5/Miro1-2/Trak2 trafficking complex. Finally, we show that overexpression of Armc10 in neurons prevents A beta-induced mitochondrial fission and neuronal death. Our data suggest both conserved and differential roles of the Armc10/Armcx gene family in regulating mitochondrial dynamics in neurons, and underscore a protective effect of the Armc10 gene against A beta-induced toxicity. Overall, our findings support a further degree of regulation of mitochondrial dynamics in the brain of more evolved mammals.

Effect of vaccination against gonadotrophin-releasing hormone, using Improvac®, on growth performance, body composition, behaviour and acute phase proteins

The objective of this study was to evaluate the effect of vaccination against GnRH on performance traits, pig behaviour and acute phase proteins. A total of 120 pigs (36 non-castrated males, NCM; 36 males to be vaccinated, IM; 24 castratedmales, CM; and 24 females, FE)were controlled in groups of 12 in pens with feeding stations allowing the recording of individual feed intake. The two vaccinations (Improvac®) were applied at a mean age of 77 and 146 days. All pigswere individually weighed every 3 weeks from the mean ages of 74 to 176 days and backfat thickness (BT) and loinmuscle depth (LD) were also recorded ultrasonically. Twelve group-housed pigs for each treatment were video recorded during 2 consecutive days at weeks 9, 11, 20, 21, 23 and 25 of age to score the number of inactive or active pigs in each treatment group by scan sampling. Aggressive behaviour by the feeder and away from the feeder, and mounting behaviour was also scored by focal sampling. Blood samples from 12 NCM, 12 CM and 12 IM were taken to determine the concentration of circulating acute phase protein Pig-MAP atweeks 1, 2, 4, 11, 13, 21 and 25 of age. After slaughter, the number of skin lesions on the left half carcasswas scored. IMpresented overall a higher growth rate and daily feed intake compared to NCM (Pb0.05),whereas their feed conversion ratios did not differ significantly. In comparison with CM, IM presented a better feed conversion ratio (Pb0.05), since their overall dailyweight gaindid not differ significantly, butIM ate less. Final leanmeat percentage of IM and CM was lower compared to that of NCM (Pb0.05). Activity, mounting and aggressive behaviour of NCM was higher than in IM, CM and FE after the second vaccination. Pig-MAP concentrationswere significantly elevated just after surgical castrationand after bothadministrations of the vaccine (Pb0.05), but concentrations subsequently decreased throughout time. Skin lesions of NCM were significantly higher compared to that of IM and FE (Pb0.05). The effects of vaccination were especially remarkable after the second dose, when the higher feed intake and lower activity of IM compared to NCMmight result in higher final body weight and more fat. Results from this study indicate that some welfare aspects such as a reduced aggression and mounting behaviour may be improved by vaccination against GnRH, together with productive benefits like adequate feed conversion ratio and daily weight gain.

Biophysical characterization of tubulin tyrosine ligase-like proteins on microtubule dynamics

Report for the scientific sojourn carried out at the Cell Biology and Biophysics Unit from the National Institutes of Health, from 2010 to 2012.

Distributed all-atom simulations of intrinsically unstructured proteins

The Computational Biophysics Group at the Universitat Pompeu Fabra (GRIB-UPF) hosts two unique computational resources dedicated to the execution of large scale molecular dynamics (MD) simulations: (a) the ACMD molecular-dynamics software, used on standard personal computers with graphical processing units (GPUs); and (b) the GPUGRID. net computing network, supported by users distributed worldwide that volunteer GPUs for biomedical research. We leveraged these resources and developed studies, protocols and open-source software to elucidate energetics and pathways of a number of biomolecular systems, with a special focus on flexible proteins with many degrees of freedom. First, we characterized ion permeation through the bactericidal model protein Gramicidin A conducting one of the largest studies to date with the steered MD biasing methodology. Next, we addressed an open problem in structural biology, the determination of drug-protein association kinetics; we reconstructed the binding free energy, association, and dissaciociation rates of a drug like model system through a spatial decomposition and a Makov-chain analysis. The work was published in the Proceedings of the National Academy of Sciences and become one of the few landmark papers elucidating a ligand-binding pathway. Furthermore, we investigated the unstructured Kinase Inducible Domain (KID), a 28-peptide central to signalling and transcriptional response; the kinetics of this challenging system was modelled with a Markovian approach in collaboration with Frank Noe’s group at the Freie University of Berlin. The impact of the funding includes three peer-reviewed publication on high-impact journals; three more papers under review; four MD analysis components, released as open-source software; MD protocols; didactic material, and code for the hosting group.

Impaired fast-spiking, suppressed cortical inhibition and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins

Voltage-gated K+ channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to −10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 −/− mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing.

SelenoDB 1.0 : a database of selenoprotein genes, proteins and SECIS elements

Selenoproteins are a diverse group of proteinsusually misidentified and misannotated in sequencedatabases. The presence of an in-frame UGA (stop)codon in the coding sequence of selenoproteingenes precludes their identification and correctannotation. The in-frame UGA codons are recodedto cotranslationally incorporate selenocysteine,a rare selenium-containing amino acid. The developmentof ad hoc experimental and, more recently,computational approaches have allowed the efficientidentification and characterization of theselenoproteomes of a growing number of species.Today, dozens of selenoprotein families have beendescribed and more are being discovered in recentlysequenced species, but the correct genomic annotationis not available for the majority of thesegenes. SelenoDB is a long-term project that aims toprovide, through the collaborative effort of experimentaland computational researchers, automaticand manually curated annotations of selenoproteingenes, proteins and SECIS elements. Version 1.0 ofthe database includes an initial set of eukaryoticgenomic annotations, with special emphasis on thehuman selenoproteome, for immediate inspectionby selenium researchers or incorporation into moregeneral databases. SelenoDB is freely available athttp://www.selenodb.org.

A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins

Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the cis-acting SECIS RNA hairpin in the 3′UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the identification of a K (lysine)-rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding.

Reassessing the role of mitochondrial DNA mutations in autism spectrum disorder

Background: There is increasing evidence that impairment of mitochondrial energy metabolism plays an important role in the pathophysiology of autism spectrum disorders (ASD; OMIM number: 209850). A significant proportion of ASD cases display biochemical alterations suggestive of mitochondrial dysfunction and several studies have reported that mutations in the mitochondrial DNA (mtDNA) molecule could be involved in the disease phenotype. Methods: We analysed a cohort of 148 patients with idiopathic ASD for a number of mutations proposed in the literature as pathogenic in ASD. We also carried out a case control association study for the most common European haplogroups (hgs) and their diagnostic single nucleotide polymorphisms (SNPs) by comparing cases with 753 healthy and ethnically matched controls.Results: We did not find statistical support for an association between mtDNA mutations or polymorphisms and ASD.Conclusions: Our results are compatible with the idea that mtDNA mutations are not a relevant cause of ASD and the frequent observation of concomitant mitochondrial dysfunction and ASD could be due to nuclear factors influencing mitochondrion functions or to a more complex interplay between the nucleus and the mitochondrion/mtDNA.

Assessment of microbial community structure changes by amplified ribosomal DNA restriction analysis (ARDRA)

Amplified ribosomal DNA restriction analysis (ARDRA) is a simple method based on restriction endonuclease digestion of the amplified bacterial 16S rDNA. In this study we have evaluated the suitability of this method to detect differences in activated sludge bacterial communities fed on domestic or industrial wastewater, and subject to different operational conditions. The ability of ARDRA to detect these differences has been tested in modified Ludzack-Ettinger (MLE) configurations. Samples from three activated sludge wastewater treatment plants (WWTPs) with the MLE configuration were collected for both oxic and anoxic reactors, and ARDRA patterns using double enzyme digestions AluI+MspI were obtained. A matrix of Dice similarity coefficients was calculated and used to compare these restriction patterns. Differences in the community structure due to influent characteristics and temperature could be observed, but not between the oxic and anoxic reactors of each of the three MLE configurations. Other possible applications of ARDRA for detecting and monitoring changes in activated sludge systems are also discussed

Phenotypic plasticity in the Caribbean sponge Callyspongia vaginalis (Porifera: Haplosclerida)

Sponge morphological plasticity has been a long-standing source of taxonomic difficulty. In the Caribbean, several morphotypes of the sponge Callyspongia vaginalis have been observed. To determine the taxonomic status of three of these morphotypes and their relationship with the congeneric species C. plicifera and C. fallax, we compared the spicule composition, spongin fiber skeleton and sequenced fragments of the mitochondrial genes 16S and COI and nuclear genes 28S and 18S ribosomal RNA. Phylogenetic analyses with ribosomal markers 18S and 28S rRNA confirmed the position of our sequences within the Callyspongiidae. None of the genetic markers provided evidence for consistent differentiation among the three morphotypes of C. vaginalis and C. fallax, and only C. plicifera stood as a distinct species. The 16S mtDNA gene was the most variable molecular marker for this group, presenting a nucleotide variability (π = 0.024) higher than that reported for COI. Unlike recent studies for other sponge genera, our results indicate that species in the genus Callyspongia maintain a high degree of phenotypic plasticity, and that morphological characteristics may not reflect reproductive boundaries in C. vaginalis.

Mitochondrial Fatty Acid Oxidation in Obesity

Significance: Current lifestyles with high-energy diets and little exercise are triggering an alarming growth in obesity. Excess of adiposity is leading to severe increases in associated pathologies, such as insulin resistance, type 2 diabetes, atherosclerosis, cancer, arthritis, asthma, and hypertension. This, together with the lack of efficient obesity drugs, is the driving force behind much research. Recent Advances: Traditional anti-obesity strategies focused on reducing food intake and increasing physical activity. However, recent results suggest that enhancing cellular energy expenditure may be an attractive alternative therapy. Critical Issues: This review evaluates recent discoveries regarding mitochondrial fatty acid oxidation (FAO) and its potential as a therapy for obesity. We focus on the still controversial beneficial effects of increased FAO in liver and muscle, recent studies on how to potentiate adipose tissue energy expenditure, and the different hypotheses involving FAO and the reactive oxygen species production in the hypothalamic control of food intake. Future Directions: The present review aims to provide an overview of novel anti-obesity strategies that target mitochondrial FAO and that will definitively be of high interest in the future research to fight against obesity-related disorders. Antioxid. Redox Signal. 00, 000000.