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Find video protocols related to scientific articles indexed in Pubmed.
A mutation in the human CBP4 ortholog UQCC3 impairs complex III assembly, activity and cytochrome b stability.
Hum. Mol. Genet.
PUBLISHED: 07-09-2014
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Complex III (cytochrome bc1) is a protein complex of the mitochondrial inner membrane that transfers electrons from ubiquinol to cytochrome c. Its assembly requires the coordinated expression of mitochondrial-encoded cytochrome b and nuclear-encoded subunits and assembly factors. Complex III deficiency is a severe multisystem disorder caused by mutations in subunit genes or assembly factors. Sequence-profile-based orthology predicts C11orf83, hereafter named UQCC3, to be the ortholog of the fungal complex III assembly factor CBP4. We describe a homozygous c.59T>A missense mutation in UQCC3 from a consanguineous patient diagnosed with isolated complex III deficiency, displaying lactic acidosis, hypoglycemia, hypotonia and delayed development without dysmorphic features. Patient fibroblasts have reduced complex III activity and lower levels of the holocomplex and its subunits than controls. They have no detectable UQCC3 protein and have lower levels of cytochrome b protein. Furthermore, in patient cells, cytochrome b is absent from a high-molecular-weight complex III. UQCC3 is reduced in cells depleted for the complex III assembly factors UQCC1 and UQCC2. Conversely, absence of UQCC3 in patient cells does not affect UQCC1 and UQCC2. This suggests that UQCC3 functions in the complex III assembly pathway downstream of UQCC1 and UQCC2 and is consistent with what is known about the function of Cbp4 and of the fungal orthologs of UQCC1 and UQCC2, Cbp3 and Cbp6. We conclude that UQCC3 functions in complex III assembly and that the c.59T>A mutation has a causal role in complex III deficiency.
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MPV17L2 is required for ribosome assembly in mitochondria.
Nucleic Acids Res.
PUBLISHED: 06-19-2014
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MPV17 is a mitochondrial protein of unknown function, and mutations in MPV17 are associated with mitochondrial deoxyribonucleic acid (DNA) maintenance disorders. Here we investigated its most similar relative, MPV17L2, which is also annotated as a mitochondrial protein. Mitochondrial fractionation analyses demonstrate MPV17L2 is an integral inner membrane protein, like MPV17. However, unlike MPV17, MPV17L2 is dependent on mitochondrial DNA, as it is absent from ?(0) cells, and co-sediments on sucrose gradients with the large subunit of the mitochondrial ribosome and the monosome. Gene silencing of MPV17L2 results in marked decreases in the monosome and both subunits of the mitochondrial ribosome, leading to impaired protein synthesis in the mitochondria. Depletion of MPV17L2 also induces mitochondrial DNA aggregation. The DNA and ribosome phenotypes are linked, as in the absence of MPV17L2 proteins of the small subunit of the mitochondrial ribosome are trapped in the enlarged nucleoids, in contrast to a component of the large subunit. These findings suggest MPV17L2 contributes to the biogenesis of the mitochondrial ribosome, uniting the two subunits to create the translationally competent monosome, and provide evidence that assembly of the small subunit of the mitochondrial ribosome occurs at the nucleoid.
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Intrinsically disordered segments affect protein half-life in the cell and during evolution.
Cell Rep
PUBLISHED: 06-12-2014
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Precise control of protein turnover is essential for cellular homeostasis. The ubiquitin-proteasome system is well established as a major regulator of protein degradation, but an understanding of how inherent structural features influence the lifetimes of proteins is lacking. We report that yeast, mouse, and human proteins with terminal or internal intrinsically disordered segments have significantly shorter half-lives than proteins without these features. The lengths of the disordered segments that affect protein half-life are compatible with the structure of the proteasome. Divergence in terminal and internal disordered segments in yeast proteins originating from gene duplication leads to significantly altered half-life. Many paralogs that are affected by such changes participate in signaling, where altered protein half-life will directly impact cellular processes and function. Thus, natural variation in the length and position of disordered segments may affect protein half-life and could serve as an underappreciated source of genetic variation with important phenotypic consequences.
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The symbiotic intestinal ciliates and the evolution of their hosts.
Eur. J. Protistol.
PUBLISHED: 01-22-2014
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The evolution of sophisticated differentiations of the gastro-intestinal tract enabled herbivorous mammals to digest dietary cellulose and hemicellulose with the aid of a complex anaerobic microbiota. Distinctive symbiotic ciliates, which are unique to this habitat, are the largest representatives of this microbial community. Analyses of a total of 484 different 18S rRNA genes show that extremely complex, but related ciliate communities can occur in the rumen of cattle, sheep, goats and red deer (301 sequences). The communities in the hindgut of equids (Equus caballus, Equus quagga), and elephants (Elephas maximus, Loxodonta africanus; 162 sequences), which are clearly distinct from the ruminant ciliate biota, exhibit a much higher diversity than anticipated on the basis of their morphology. All these ciliates from the gastro-intestinal tract constitute a monophyletic group, which consists of two major taxa, i.e. Vestibuliferida and Entodiniomorphida. The ciliates from the evolutionarily older hindgut fermenters exhibit a clustering that is specific for higher taxa of their hosts, as extant species of horse and zebra on the one hand, and Africa and Indian elephant on the other hand, share related ciliates. The evolutionary younger ruminants altogether share the various entodiniomorphs and the vestibuliferids from ruminants.
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optGpSampler: an improved tool for uniformly sampling the solution-space of genome-scale metabolic networks.
PLoS ONE
PUBLISHED: 01-01-2014
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Constraint-based models of metabolic networks are typically underdetermined, because they contain more reactions than metabolites. Therefore the solutions to this system do not consist of unique flux rates for each reaction, but rather a space of possible flux rates. By uniformly sampling this space, an estimated probability distribution for each reaction's flux in the network can be obtained. However, sampling a high dimensional network is time-consuming. Furthermore, the constraints imposed on the network give rise to an irregularly shaped solution space. Therefore more tailored, efficient sampling methods are needed. We propose an efficient sampling algorithm (called optGpSampler), which implements the Artificial Centering Hit-and-Run algorithm in a different manner than the sampling algorithm implemented in the COBRA Toolbox for metabolic network analysis, here called gpSampler. Results of extensive experiments on different genome-scale metabolic networks show that optGpSampler is up to 40 times faster than gpSampler. Application of existing convergence diagnostics on small network reconstructions indicate that optGpSampler converges roughly ten times faster than gpSampler towards similar sampling distributions. For networks of higher dimension (i.e. containing more than 500 reactions), we observed significantly better convergence of optGpSampler and a large deviation between the samples generated by the two algorithms.
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Mutations in the UQCC1-Interacting Protein, UQCC2, Cause Human Complex III Deficiency Associated with Perturbed Cytochrome b Protein Expression.
PLoS Genet.
PUBLISHED: 12-01-2013
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Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.
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Human intellectual disability genes form conserved functional modules in Drosophila.
PLoS Genet.
PUBLISHED: 10-01-2013
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Intellectual Disability (ID) disorders, defined by an IQ below 70, are genetically and phenotypically highly heterogeneous. Identification of common molecular pathways underlying these disorders is crucial for understanding the molecular basis of cognition and for the development of therapeutic intervention strategies. To systematically establish their functional connectivity, we used transgenic RNAi to target 270 ID gene orthologs in the Drosophila eye. Assessment of neuronal function in behavioral and electrophysiological assays and multiparametric morphological analysis identified phenotypes associated with knockdown of 180 ID gene orthologs. Most of these genotype-phenotype associations were novel. For example, we uncovered 16 genes that are required for basal neurotransmission and have not previously been implicated in this process in any system or organism. ID gene orthologs with morphological eye phenotypes, in contrast to genes without phenotypes, are relatively highly expressed in the human nervous system and are enriched for neuronal functions, suggesting that eye phenotyping can distinguish different classes of ID genes. Indeed, grouping genes by Drosophila phenotype uncovered 26 connected functional modules. Novel links between ID genes successfully predicted that MYCN, PIGV and UPF3B regulate synapse development. Drosophila phenotype groups show, in addition to ID, significant phenotypic similarity also in humans, indicating that functional modules are conserved. The combined data indicate that ID disorders, despite their extreme genetic diversity, are caused by disruption of a limited number of highly connected functional modules.
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Genome evolution predicts genetic interactions in protein complexes and reveals cancer drug targets.
Nat Commun
PUBLISHED: 06-07-2013
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Genetic interactions reveal insights into cellular function and can be used to identify drug targets. Here we construct a new model to predict negative genetic interactions in protein complexes by exploiting the evolutionary history of genes in parallel converging pathways in metabolism. We evaluate our model with protein complexes of Saccharomyces cerevisiae and show that the predicted protein pairs more frequently have a negative genetic interaction than random proteins from the same complex. Furthermore, we apply our model to human protein complexes to predict novel cancer drug targets, and identify 20 candidate targets with empirical support and 10 novel targets amenable to further experimental validation. Our study illustrates that negative genetic interactions can be predicted by systematically exploring genome evolution, and that this is useful to identify novel anti-cancer drug targets.
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A complex V ATP5A1 defect causes fatal neonatal mitochondrial encephalopathy.
Brain
PUBLISHED: 04-18-2013
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Whole exome sequencing is a powerful tool to detect novel pathogenic mutations in patients with suspected mitochondrial disease. However, the interpretation of novel genetic variants is not always straightforward. Here, we present two siblings with a severe neonatal encephalopathy caused by complex V deficiency. The aim of this study was to uncover the underlying genetic defect using the combination of enzymatic testing and whole exome sequence analysis, and to provide evidence for causality by functional follow-up. Measurement of the oxygen consumption rate and enzyme analysis in fibroblasts were performed. Immunoblotting techniques were applied to study complex V assembly. The coding regions of the genome were analysed. Three-dimensional modelling was applied. Exome sequencing of the two siblings with complex V deficiency revealed a heterozygous mutation in the ATP5A1 gene, coding for complex V subunit ?. The father carried the variant heterozygously. At the messenger RNA level, only the mutated allele was expressed in the patients, whereas the father expressed both the wild-type and the mutant allele. Gene expression data indicate that the maternal allele is not expressed, which is supported by the observation that the ATP5A1 expression levels in the patients and their mother are reduced to ?50%. Complementation with wild-type ATP5A1 restored complex V in the patient fibroblasts, confirming pathogenicity of the defect. At the protein level, the mutation results in a disturbed interaction of the ?-subunit with the ?-subunit of complex V, which interferes with the stability of the complex. This study demonstrates the important value of functional studies in the diagnostic work-up of mitochondrial patients, in order to guide genetic variant prioritization, and to validate gene defects.
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CEP89 is required for mitochondrial metabolism and neuronal function in man and fly.
Hum. Mol. Genet.
PUBLISHED: 04-10-2013
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It is estimated that the human mitochondrial proteome consists of 1000-1500 distinct proteins. The majority of these support the various biochemical pathways that are active in these organelles. Individuals with an oxidative phosphorylation disorder of unknown cause provide a unique opportunity to identify novel genes implicated in mitochondrial biology. We identified a homozygous deletion of CEP89 in a patient with isolated complex IV deficiency, intellectual disability and multisystemic problems. CEP89 is a ubiquitously expressed and highly conserved gene of unknown function. Immunocytochemistry and cellular fractionation experiments showed that CEP89 is present both in the cytosol and in the mitochondrial intermembrane space. Furthermore, we ascertained in vitro that downregulation of CEP89 resulted in a severe decrease in complex IV in-gel activity and altered mobility, suggesting that the complex is aberrantly formed. Two-dimensional BN-SDS gel analysis revealed that CEP89 associates with a high-molecular weight complex. Together, these data confirm a role for CEP89 in mitochondrial metabolism. In addition, we modeled CEP89 loss of function in Drosophila. Ubiquitous knockdown of fly Cep89 decreased complex IV activity and resulted in complete lethality. Furthermore, Cep89 is required for mitochondrial integrity, membrane depolarization and synaptic transmission of photoreceptor neurons, and for (sub)synaptic organization of the larval neuromuscular junction. Finally, we tested neuronal Cep89 knockdown flies in the light-off jump reflex habituation assay, which revealed its role in learning. We conclude that CEP89 proteins play an important role in mitochondrial metabolism, especially complex IV activity, and are required for neuronal and cognitive function across evolution.
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Evolution of modular intraflagellar transport from a coatomer-like progenitor.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-08-2013
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The intraflagellar transport (IFT) complex is an integral component of the cilium, a quintessential organelle of the eukaryotic cell. The IFT system consists of three subcomplexes [i.e., intraflagellar transport (IFT)-A, IFT-B, and the BBSome], which together transport proteins and other molecules along the cilium. IFT dysfunction results in diseases collectively called ciliopathies. It has been proposed that the IFT complexes originated from vesicle coats similar to coat protein complex (COP) I, COPII, and clathrin. Here we provide phylogenetic evidence for common ancestry of IFT subunits and ?, ?, and ? subunits of COPI, and trace the origins of the IFT-A, IFT-B, and the BBSome subcomplexes. We find that IFT-A and the BBSome likely arose from an IFT-B-like complex by intracomplex subunit duplication. The distribution of IFT proteins across eukaryotes identifies the BBSome as a frequently lost, modular component of the IFT. Significantly, loss of the BBSome from a taxon is a frequent precursor to complete cilium loss in related taxa. Given the inferred late origin of the BBSome in cilium evolution and its frequent loss, the IFT complex behaves as a "last-in, first-out" system. The protocoatomer origin of the IFT complex corroborates involvement of IFT components in vesicle transport. Expansion of IFT subunits by duplication and their subsequent independent loss supports the idea of modularity and structural independence of the IFT subcomplexes.
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The SYSCILIA gold standard (SCGSv1) of known ciliary components and its applications within a systems biology consortium.
Cilia
PUBLISHED: 04-02-2013
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The multinational SYSCILIA consortium aims to gain a mechanistic understanding of the cilium. We utilize multiple parallel high-throughput (HTP) initiatives to develop predictive models of relationships between complex genotypes and variable phenotypes of ciliopathies. The models generated are only as good as the wet laboratory data fed into them. It is therefore essential to orchestrate a well-annotated and high-confidence dataset to be able to assess the quality of any HTP dataset. Here, we present the inaugural SYSCILIA gold standard of known ciliary components as a public resource.
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The PinkThing for analysing ChIP profiling data in their genomic context.
BMC Res Notes
PUBLISHED: 03-12-2013
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Current epigenetic research makes frequent use of whole-genome ChIP profiling for determining the in vivo binding of proteins, e.g. transcription factors and histones, to DNA. Two important and recurrent questions for these large scale analyses are: 1) What is the genomic distribution of a set of binding sites? and 2) Does this genomic distribution differ significantly from another set of sites?
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Inferring metabolic states in uncharacterized environments using gene-expression measurements.
PLoS Comput. Biol.
PUBLISHED: 01-31-2013
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The large size of metabolic networks entails an overwhelming multiplicity in the possible steady-state flux distributions that are compatible with stoichiometric constraints. This space of possibilities is largest in the frequent situation where the nutrients available to the cells are unknown. These two factors: network size and lack of knowledge of nutrient availability, challenge the identification of the actual metabolic state of living cells among the myriad possibilities. Here we address this challenge by developing a method that integrates gene-expression measurements with genome-scale models of metabolism as a means of inferring metabolic states. Our method explores the space of alternative flux distributions that maximize the agreement between gene expression and metabolic fluxes, and thereby identifies reactions that are likely to be active in the culture from which the gene-expression measurements were taken. These active reactions are used to build environment-specific metabolic models and to predict actual metabolic states. We applied our method to model the metabolic states of Saccharomyces cerevisiae growing in rich media supplemented with either glucose or ethanol as the main energy source. The resulting models comprise about 50% of the reactions in the original model, and predict environment-specific essential genes with high sensitivity. By minimizing the sum of fluxes while forcing our predicted active reactions to carry flux, we predicted the metabolic states of these yeast cultures that are in large agreement with what is known about yeast physiology. Most notably, our method predicts the Crabtree effect in yeast cells growing in excess glucose, a long-known phenomenon that could not have been predicted by traditional constraint-based modeling approaches. Our method is of immediate practical relevance for medical and industrial applications, such as the identification of novel drug targets, and the development of biotechnological processes that use complex, largely uncharacterized media, such as biofuel production.
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ODoSE: a webserver for genome-wide calculation of adaptive divergence in prokaryotes.
PLoS ONE
PUBLISHED: 01-01-2013
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Quantifying patterns of adaptive divergence between taxa is a major goal in the comparative and evolutionary study of prokaryote genomes. When applied appropriately, the McDonald-Kreitman (MK) test is a powerful test of selection based on the relative frequency of non-synonymous and synonymous substitutions between species compared to non-synonymous and synonymous polymorphisms within species. The webserver ODoSE (Ortholog Direction of Selection Engine) allows the calculation of a novel extension of the MK test, the Direction of Selection (DoS) statistic, as well as the calculation of a weighted-average Neutrality Index (NI) statistic for the entire core genome, allowing for systematic analysis of the evolutionary forces shaping core genome divergence in prokaryotes. ODoSE is hosted in a Galaxy environment, which makes it easy to use and amenable to customization and is freely available at www.odose.nl.
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Analysis of genes regulated by the transcription factor LUMAN identifies ApoA4 as a target gene in dendritic cells.
Mol. Immunol.
PUBLISHED: 08-12-2011
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Dendritic cells (DCs) are professional antigen presenting cells of the immune system that play a crucial role in initiating immune responses and maintaining self tolerance. Better understanding of the molecular basis of DC immunobiology is required to improve DC-based immunotherapies. We previously described the interaction of transcription factor LUMAN (also known as CREB3 or LZIP) with the DC-specific transmembrane protein DC-STAMP in DCs. Target genes of LUMAN and its role in DCs are currently unknown. In this study we set out to identify genes regulated by LUMAN in DCs using microarray analysis. Expression of a constitutively active form of LUMAN in mouse DC cell line D2SC/1 identified Apolipoprotein A4 (ApoA4) as its target gene. Subsequent validation experiments, bioinformatics-based promoter analysis, and silencing studies confirmed that ApoA4 is a true target gene of LUMAN in bone marrow-derived DCs (BMDCs).
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MicroRNA genes preferentially expressed in dendritic cells contain sites for conserved transcription factor binding motifs in their promoters.
BMC Genomics
PUBLISHED: 06-27-2011
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MicroRNAs (miRNAs) play a fundamental role in the regulation of gene expression by translational repression or target mRNA degradation. Regulatory elements in miRNA promoters are less well studied, but may reveal a link between their expression and a specific cell type.
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FACIL: Fast and Accurate Genetic Code Inference and Logo.
Bioinformatics
PUBLISHED: 06-08-2011
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The intensification of DNA sequencing will increasingly unveil uncharacterized species with potential alternative genetic codes. A total of 0.65% of the DNA sequences currently in Genbank encode their proteins with a variant genetic code, and these exceptions occur in many unrelated taxa.
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Restoration of complex V deficiency caused by a novel deletion in the human TMEM70 gene normalizes mitochondrial morphology.
Mitochondrion
PUBLISHED: 05-18-2011
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We report a fragmented mitochondrial network and swollen and irregularly shaped mitochondria with partial to complete loss of the cristae in fibroblasts of a patient with a novel TMEM70 gene deletion, which could be completely restored by complementation of the TMEM70 genetic defect. Comparative genomics analysis predicted the topology of TMEM70 in the inner mitochondrial membrane, which could be confirmed by immunogold labeling experiments, and showed that the TMEM70 gene is not restricted to higher multi-cellular eukaryotes. This study demonstrates that the role of complex V in mitochondrial cristae morphology applies to human mitochondrial disease pathology.
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Evidence for short-time divergence and long-time conservation of tissue-specific expression after gene duplication.
Brief. Bioinformatics
PUBLISHED: 04-22-2011
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Gene duplication is one of the main mechanisms by which genomes can acquire novel functions. It has been proposed that the retention of gene duplicates can be associated to processes of tissue expression divergence. These models predict that acquisition of divergent expression patterns should be acquired shortly after the duplication, and that larger divergence in tissue expression would be expected for paralogs, as compared to orthologs of a similar age. Many studies have shown that gene duplicates tend to have divergent expression patterns and that gene family expansions are associated with high levels of tissue specificity. However, the timeframe in which these processes occur have rarely been investigated in detail, particularly in vertebrates, and most analyses do not include direct comparisons of orthologs as a baseline for the expected levels of tissue specificity in absence of duplications. To assess the specific contribution of duplications to expression divergence, we combine here phylogenetic analyses and expression data from human and mouse. In particular, we study differences in spatial expression among human-mouse paralogs, specifically duplicated after the radiation of mammals, and compare them to pairs of orthologs in the same species. Our results show that gene duplication leads to increased levels of tissue specificity and that this tends to occur promptly after the duplication event.
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The organellar genome and metabolic potential of the hydrogen-producing mitochondrion of Nyctotherus ovalis.
Mol. Biol. Evol.
PUBLISHED: 03-04-2011
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It is generally accepted that hydrogenosomes (hydrogen-producing organelles) evolved from a mitochondrial ancestor. However, until recently, only indirect evidence for this hypothesis was available. Here, we present the almost complete genome of the hydrogen-producing mitochondrion of the anaerobic ciliate Nyctotherus ovalis and show that, except for the notable absence of genes encoding electron transport chain components of Complexes III, IV, and V, it has a gene content similar to the mitochondrial genomes of aerobic ciliates. Analysis of the genome of the hydrogen-producing mitochondrion, in combination with that of more than 9,000 genomic DNA and cDNA sequences, allows a preliminary reconstruction of the organellar metabolism. The sequence data indicate that N. ovalis possesses hydrogen-producing mitochondria that have a truncated, two step (Complex I and II) electron transport chain that uses fumarate as electron acceptor. In addition, components of an extensive protein network for the metabolism of amino acids, defense against oxidative stress, mitochondrial protein synthesis, mitochondrial protein import and processing, and transport of metabolites across the mitochondrial membrane were identified. Genes for MPV17 and ACN9, two hypothetical proteins linked to mitochondrial disease in humans, were also found. The inferred metabolism is remarkably similar to the organellar metabolism of the phylogenetically distant anaerobic Stramenopile Blastocystis. Notably, the Blastocystis organelle and that of the related flagellate Proteromonas lacertae also lack genes encoding components of Complexes III, IV, and V. Thus, our data show that the hydrogenosomes of N. ovalis are highly specialized hydrogen-producing mitochondria.
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A mutation in C2orf64 causes impaired cytochrome c oxidase assembly and mitochondrial cardiomyopathy.
Am. J. Hum. Genet.
PUBLISHED: 02-21-2011
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The assembly of mitochondrial respiratory chain complex IV (cytochrome c oxidase) involves the coordinated action of several assembly chaperones. In Saccharomyces cerevisiae, at least 30 different assembly chaperones have been identified. To date, pathogenic mutations leading to a mitochondrial disorder have been identified in only seven of the corresponding human genes. One of the genes for which the relevance to human pathology is unknown is C2orf64, an ortholog of the S. cerevisiae gene PET191. This gene has previously been shown to be a complex IV assembly factor in yeast, although its exact role is still unknown. Previous research in a large cohort of complex IV deficient patients did not support an etiological role of C2orf64 in complex IV deficiency. In this report, a homozygous mutation in C2orf64 is described in two siblings affected by fatal neonatal cardiomyopathy. Pathogenicity of the mutation is supported by the results of a complementation experiment, showing that complex IV activity can be fully restored by retroviral transduction of wild-type C2orf64 in patient-derived fibroblasts. Detailed analysis of complex IV assembly intermediates in patient fibroblasts by 2D-BN PAGE revealed the accumulation of a small assembly intermediate containing subunit COX1 but not the COX2, COX4, or COX5b subunits, indicating that C2orf64 is involved in an early step of the complex IV assembly process. The results of this study demonstrate that C2orf64 is essential for human complex IV assembly and that C2orf64 mutational analysis should be considered for complex IV deficient patients, in particular those with hypertrophic cardiomyopathy.
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TEFM (c17orf42) is necessary for transcription of human mtDNA.
Nucleic Acids Res.
PUBLISHED: 01-28-2011
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Here we show that c17orf42, hereafter TEFM (transcription elongation factor of mitochondria), makes a critical contribution to mitochondrial transcription. Inactivation of TEFM in cells by RNA interference results in respiratory incompetence owing to decreased levels of H- and L-strand promoter-distal mitochondrial transcripts. Affinity purification of TEFM from human mitochondria yielded a complex comprising mitochondrial transcripts, mitochondrial RNA polymerase (POLRMT), pentatricopeptide repeat domain 3 protein (PTCD3), and a putative DEAD-box RNA helicase, DHX30. After RNase treatment only POLRMT remained associated with TEFM, and in human cultured cells TEFM formed foci coincident with newly synthesized mitochondrial RNA. Based on deletion mutants, TEFM interacts with the catalytic region of POLRMT, and in vitro TEFM enhanced POLRMT processivity on ss- and dsDNA templates. TEFM contains two HhH motifs and a Ribonuclease H fold, similar to the nuclear transcription elongation regulator Spt6. These findings lead us to propose that TEFM is a mitochondrial transcription elongation factor.
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NDUFB7 and NDUFA8 are located at the intermembrane surface of complex I.
FEBS Lett.
PUBLISHED: 01-21-2011
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Complex I (NADH:ubiquinone oxidoreductase) is the first and largest protein complex of the oxidative phosphorylation. Crystal structures have elucidated the positions of most subunits of bacterial evolutionary origin in the complex, but the positions of the eukaryotic subunits are unknown. Based on the analysis of sequence conservation we propose intra-molecular disulfide bridges and the inter-membrane space localization of three Cx(9)C-containing subunits in human: NDUFS5, NDUFB7 and NDUFA8. We experimentally confirm the localization of the latter two, while our data are consistent with disulfide bridges in NDUFA8. We propose these subunits stabilize the membrane domain of complex I.
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Mosaic origin of the mitochondrial proteome.
Proteomics
PUBLISHED: 11-17-2010
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Although the origin of mitochondria from the endosymbiosis of an ?-proteobacterium is well established, the nature of the host cell, the metabolic complexity of the endosymbiont and the subsequent evolution of the proto-mitochondrion into all its current appearances are still the subject of discovery and sometimes debate. Here we review what has been inferred about the original composition and subsequent evolution of the mitochondrial proteome and essential mitochondrial systems. The evolutionary mosaic that currently constitutes mitochondrial proteomes contains (i) endosymbiotic proteins (15-45%), (ii) proteins without detectable orthologs outside the eukaryotic lineage (40%), and (iii) proteins that are derived from non-proteobacterial Bacteria, Bacteriophages and Archaea (15%, specifically multiple tRNA-modification proteins). Protein complexes are of endosymbiotic origin, but have greatly expanded with novel eukaryotic proteins; in contrast to mitochondrial enzymes that are both of proteobacterial and non-proteobacterial origin. This disparity is consistent with the complexity hypothesis, which argues that proteins that are a part of large, multi-subunit complexes are unlikely to undergo horizontal gene transfer. We observe that they neither change their subcellular compartments in the course of evolution, even when their genes do.
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Measuring the physical cohesiveness of proteins using physical interaction enrichment.
Bioinformatics
PUBLISHED: 08-26-2010
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Protein-protein interaction (PPI) networks are a valuable resource for the interpretation of genomics data. However, such networks have interaction enrichment biases for proteins that are often studied. These biases skew quantitative results from comparing PPI networks with genomics data. Here, we introduce an approach named physical interaction enrichment (PIE) to eliminate these biases.
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Quantitative proteome profiling of respiratory virus-infected lung epithelial cells.
J Proteomics
PUBLISHED: 04-22-2010
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Respiratory virus infections are among the primary causes of morbidity and mortality in humans. Influenza virus, respiratory syncytial virus (RSV), parainfluenza (PIV) and human metapneumovirus (hMPV) are major causes of respiratory illness in humans. Especially young children and the elderly are susceptible to infections with these viruses. In this study we aim to gain detailed insight into the molecular pathogenesis of respiratory virus infections by studying the protein expression profiles of infected lung epithelial cells. A549 cells were exposed to a set of respiratory viruses [RSV, hMPV, PIV and Measles virus (MV)] using both live and UV-inactivated virus preparations. Cells were harvested at different time points after infection and processed for proteomics analysis by 2-dimensional difference gel electrophoresis. Samples derived from infected cells were compared to mock-infected cells to identify proteins that are differentially expressed due to infection. We show that RSV, hMPV, PIV3, and MV induced similar core host responses and that mainly proteins involved in defense against ER stress and apoptosis were affected which points towards an induction of apoptosis upon infection. By 2-D DIGE analyses we have gathered information on the induction of apoptosis by respiratory viruses in A549 cells.
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Acyl-CoA dehydrogenase 9 is required for the biogenesis of oxidative phosphorylation complex I.
Cell Metab.
PUBLISHED: 03-04-2010
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Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified member of the acyl-CoA dehydrogenase family. It closely resembles very long-chain acyl-CoA dehydrogenase (VLCAD), involved in mitochondrial beta oxidation of long-chain fatty acids. Contrary to its previously proposed involvement in fatty acid oxidation, we describe a role for ACAD9 in oxidative phosphorylation. ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required for the assembly of complex I. Furthermore, ACAD9 mutations result in complex I deficiency and not in disturbed long-chain fatty acid oxidation. This strongly contrasts with its evolutionary ancestor VLCAD, which we show is not required for complex I assembly and clearly plays a role in fatty acid oxidation. Our results demonstrate that two closely related metabolic enzymes have diverged at the root of the vertebrate lineage to function in two separate mitochondrial metabolic pathways and have clinical implications for the diagnosis of complex I deficiency.
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Dominant processes during human dendritic cell maturation revealed by integration of proteome and transcriptome at the pathway level.
J. Proteome Res.
PUBLISHED: 02-06-2010
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Gene expression is commonly used to study the activation of dendritic cells (DCs) to identify proteins that determine whether these cells induce an immunostimulatory or tolerogenic immune response. RNA expression, however, does not necessarily predict protein abundance and often requires large numbers of experiments for statistical significance. Proteomics provides a direct view on protein expression but is costly and time consuming. Here, we combined a comprehensive quantitative proteome and transcriptome analysis on a single batch of immature and cytokine cocktail matured human DCs and integrated resulting data sets at the pathway level. Although overall correlation between differential mRNA and protein expression was low, correlation between components of DC relevant pathways was significantly higher. Differentially expressed proteins and genes partly mapped to identical but also to different pathway components demonstrating that RNA and protein data not only supported but also complemented each other. We identified 5 dominant pathways, which confirmed the importance of cytokines, cell adhesion, and migration in DC maturation and also indicated a fundamental role for lipid metabolism. From these pathways we extracted novel maturation markers that might improve DC vaccine design. For several of the candidate markers we confirmed widespread significance examining DCs from multiple individuals, underscoring the validity of our approach. We conclude that integration of different but related data sets at the pathway level can significantly increase the predictive power of multi "omics" analyses.
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Genome-wide profiling of p63 DNA-binding sites identifies an element that regulates gene expression during limb development in the 7q21 SHFM1 locus.
PLoS Genet.
PUBLISHED: 02-04-2010
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Heterozygous mutations in p63 are associated with split hand/foot malformations (SHFM), orofacial clefting, and ectodermal abnormalities. Elucidation of the p63 gene network that includes target genes and regulatory elements may reveal new genes for other malformation disorders. We performed genome-wide DNA-binding profiling by chromatin immunoprecipitation (ChIP), followed by deep sequencing (ChIP-seq) in primary human keratinocytes, and identified potential target genes and regulatory elements controlled by p63. We show that p63 binds to an enhancer element in the SHFM1 locus on chromosome 7q and that this element controls expression of DLX6 and possibly DLX5, both of which are important for limb development. A unique micro-deletion including this enhancer element, but not the DLX5/DLX6 genes, was identified in a patient with SHFM. Our study strongly indicates disruption of a non-coding cis-regulatory element located more than 250 kb from the DLX5/DLX6 genes as a novel disease mechanism in SHFM1. These data provide a proof-of-concept that the catalogue of p63 binding sites identified in this study may be of relevance to the studies of SHFM and other congenital malformations that resemble the p63-associated phenotypes.
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A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome.
EMBO J.
PUBLISHED: 01-25-2010
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Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes.
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The alpha-kinase family: an exceptional branch on the protein kinase tree.
Cell. Mol. Life Sci.
PUBLISHED: 09-24-2009
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The alpha-kinase family represents a class of atypical protein kinases that display little sequence similarity to conventional protein kinases. Early studies on myosin heavy chain kinases in Dictyostelium discoideum revealed their unusual propensity to phosphorylate serine and threonine residues in the context of an alpha-helix. Although recent studies show that some members of this family can also phosphorylate residues in non-helical regions, the name alpha-kinase has remained. During evolution, the alpha-kinase domains combined with many different functional subdomains such as von Willebrand factor-like motifs (vWKa) and even cation channels (TRPM6 and TRPM7). As a result, these kinases are implicated in a large variety of cellular processes such as protein translation, Mg(2+) homeostasis, intracellular transport, cell migration, adhesion, and proliferation. Here, we review the current state of knowledge on different members of this kinase family and discuss the potential use of alpha-kinases as drug targets in diseases such as cancer.
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The biological coherence of human phenome databases.
Am. J. Hum. Genet.
PUBLISHED: 07-27-2009
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Disease networks are increasingly explored as a complement to networks centered around interactions between genes and proteins. The quality of disease networks is heavily dependent on the amount and quality of phenotype information in phenotype databases of human genetic diseases. We explored which aspects of phenotype database architecture and content best reflect the underlying biology of disease. We used the OMIM-based HPO, Orphanet, and POSSUM phenotype databases for this purpose and devised a biological coherence score based on the sharing of gene ontology annotation to investigate the degree to which phenotype similarity in these databases reflects related pathobiology. Our analyses support the notion that a fine-grained phenotype ontology enhances the accuracy of phenome representation. In addition, we find that the OMIM database that is most used by the human genetics community is heavily underannotated. We show that this problem can easily be overcome by simply adding data available in the POSSUM database to improve OMIM phenotype representations in the HPO. Also, we find that the use of feature frequency estimates--currently implemented only in the Orphanet database--significantly improves the quality of the phenome representation. Our data suggest that there is much to be gained by improving human phenome databases and that some of the measures needed to achieve this are relatively easy to implement. More generally, we propose that curation and more systematic annotation of human phenome databases can greatly improve the power of the phenotype for genetic disease analysis.
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The hydrogenosomes of Psalteriomonas lanterna.
BMC Evol. Biol.
PUBLISHED: 07-20-2009
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Hydrogenosomes are organelles that produce molecular hydrogen and ATP. The broad phylogenetic distribution of their hosts suggests that the hydrogenosomes of these organisms evolved several times independently from the mitochondria of aerobic progenitors. Morphology and 18S rRNA phylogeny suggest that the microaerophilic amoeboflagellate Psalteriomonas lanterna, which possesses hydrogenosomes and elusive "modified mitochondria", belongs to the Heterolobosea, a taxon that consists predominantly of aerobic, mitochondriate organisms. This taxon is rather unrelated to taxa with hitherto studied hydrogenosomes.
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Increasing the coverage of a metapopulation consensus genome by iterative read mapping and assembly.
Bioinformatics
PUBLISHED: 06-19-2009
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Most microbial species can not be cultured in the laboratory. Metagenomic sequencing may still yield a complete genome if the sequenced community is enriched and the sequencing coverage is high. However, the complexity in a natural population may cause the enrichment culture to contain multiple related strains. This diversity can confound existing strict assembly programs and lead to a fragmented assembly, which is unnecessary if we have a related reference genome available that can function as a scaffold.
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Expansion of the human mitochondrial proteome by intra- and inter-compartmental protein duplication.
Genome Biol.
PUBLISHED: 06-09-2009
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Mitochondria are highly complex, membrane-enclosed organelles that are essential to the eukaryotic cell. The experimental elucidation of organellar proteomes combined with the sequencing of complete genomes allows us to trace the evolution of the mitochondrial proteome.
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Clustering of codons with rare cognate tRNAs in human genes suggests an extra level of expression regulation.
PLoS Genet.
PUBLISHED: 06-03-2009
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In species with large effective population sizes, highly expressed genes tend to be encoded by codons with highly abundant cognate tRNAs to maximize translation rate. However, there has been little evidence for a similar bias of synonymous codons in highly expressed human genes. Here, we ask instead whether there is evidence for the selection for codons associated with low abundance tRNAs. Rather than averaging the codon usage of complete genes, we scan the genes for windows with deviating codon usage. We show that there is a significant over representation of human genes that contain clusters of codons with low abundance cognate tRNAs. We name these regions, which on average have a 50% reduction in the amount of cognate tRNA available compared to the remainder of the gene, RTS (rare tRNA score) clusters. We observed a significant reduction in the substitution rate between the human RTS clusters and their orthologous chimp sequence, when compared to non-RTS cluster sequences. Overall, the genes with an RTS cluster have higher tissue specificity than the non-RTS cluster genes. Furthermore, these genes are functionally enriched for transcription regulation. As genes that regulate transcription in lower eukaryotes are known to be involved in translation on demand, this suggests that the mechanism of translation level expression regulation also exists within the human genome.
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The mitochondrial genomes of the ciliates Euplotes minuta and Euplotes crassus.
BMC Genomics
PUBLISHED: 05-14-2009
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There are thousands of very diverse ciliate species from which only a handful mitochondrial genomes have been studied so far. These genomes are rather similar because the ciliates analysed (Tetrahymena spp. and Paramecium aurelia) are closely related. Here we study the mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus. These ciliates are only distantly related to Tetrahymena spp. and Paramecium aurelia, but more closely related to Nyctotherus ovalis, which possesses a hydrogenosomal (mitochondrial) genome.
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Mitochondrial proteome evolution and genetic disease.
Biochim. Biophys. Acta
PUBLISHED: 03-04-2009
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Mitochondria are an essential organelle, not only to the human cell, but to all eukaryotic life. This essentiality is reflected in the large number of mutations in genes encoding mitochondrial proteins that lead to disease. Aside from their relevance to disease, mitochondria are, given their endosymbiotic origin, very interesting from an evolutionary point of view. Here, in the year that marks the bicentenary of Darwins birth and the 150th anniversary of the publication of "On the origin of species" we review approaches that implicitly or explicitly use evolutionary analyses to find new genes involved in mitochondrial disease and to predict their function and involvement in pathways. We show how the phenotypic spectrum of mitochondrial disease is linked to the evolutionary origin of mitochondrial proteins, how combinations of evolutionary data and genomics data have been used to predict the mitochondrial proteome and functional links between the mitochondrial proteins and how the evolution of the mitochondrial proteome has been used to predict new mitochondrial disease genes. For the latter we review and reanalyze the eukaryotic evolution of the NADH:ubiquinone oxidoreductase (complex I) and the proteins involved in its assembly.
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Mutations in NDUFAF3 (C3ORF60), encoding an NDUFAF4 (C6ORF66)-interacting complex I assembly protein, cause fatal neonatal mitochondrial disease.
Am. J. Hum. Genet.
PUBLISHED: 02-24-2009
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Mitochondrial complex I deficiency is the most prevalent and least understood disorder of the oxidative phosphorylation system. The genetic cause of many cases of isolated complex I deficiency is unknown because of insufficient understanding of the complex I assembly process and the factors involved. We performed homozygosity mapping and gene sequencing to identify the genetic defect in five complex I-deficient patients from three different families. All patients harbored mutations in the NDUFAF3 (C3ORF60) gene, of which the pathogenic nature was assessed by NDUFAF3-GFP baculovirus complementation in fibroblasts. We found that NDUFAF3 is a genuine mitochondrial complex I assembly protein that interacts with complex I subunits. Furthermore, we show that NDUFAF3 tightly interacts with NDUFAF4 (C6ORF66), a protein previously implicated in complex I deficiency. Additional gene conservation analysis links NDUFAF3 to bacterial-membrane-insertion gene cluster SecF/SecD/YajC and to C8ORF38, also implicated in complex I deficiency. These data not only show that NDUFAF3 mutations cause complex I deficiency but also relate different complex I disease genes by the close cooperation of their encoded proteins during the assembly process.
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Asymmetric relationships between proteins shape genome evolution.
Genome Biol.
PUBLISHED: 01-28-2009
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The relationships between proteins are often asymmetric: one protein (A) depends for its function on another protein (B), but the second protein does not depend on the first. In metabolic networks there are multiple pathways that converge into one central pathway. The enzymes in the converging pathways depend on the enzymes in the central pathway, but the enzymes in the latter do not depend on any specific enzyme in the converging pathways. Asymmetric relations are analogous to the "if->then" logical relation where A implies B, but B does not imply A (A->B).
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Discovery of a HapE mutation that causes azole resistance in Aspergillus fumigatus through whole genome sequencing and sexual crossing.
PLoS ONE
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Azole compounds are the primary therapy for patients with diseases caused by Aspergillus fumigatus. However, prolonged treatment may cause resistance to develop, which is associated with treatment failure. The azole target cyp51A is a hotspot for mutations that confer phenotypic resistance, but in an increasing number of resistant isolates the underlying mechanism remains unknown. Here, we report the discovery of a novel resistance mechanism, caused by a mutation in the CCAAT-binding transcription factor complex subunit HapE. From one patient, four A. fumigatus isolates were serially collected. The last two isolates developed an azole resistant phenotype during prolonged azole therapy. Because the resistant isolates contained a wild type cyp51A gene and the isolates were isogenic, the complete genomes of the last susceptible isolate and the first resistant isolate (taken 17 weeks apart) were sequenced using Illumina technology to identify the resistance conferring mutation. By comparing the genome sequences to each other as well as to two A. fumigatus reference genomes, several potential non-synonymous mutations in protein-coding regions were identified, six of which could be confirmed by PCR and Sanger sequencing. Subsequent sexual crossing experiments showed that resistant progeny always contained a P88L substitution in HapE, while the presence of the other five mutations did not correlate with resistance in the progeny. Cloning the mutated hapE gene into the azole susceptible akuB(KU80) strain showed that the HapE P88L mutation by itself could confer the resistant phenotype. This is the first time that whole genome sequencing and sexual crossing strategies have been used to find the genetic basis of a trait of interest in A. fumigatus. The discovery may help understand alternate pathways for azole resistance in A. fumigatus with implications for the molecular diagnosis of resistance and drug discovery.
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A mutation in the FAM36A gene, the human ortholog of COX20, impairs cytochrome c oxidase assembly and is associated with ataxia and muscle hypotonia.
Hum. Mol. Genet.
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The mitochondrial respiratory chain complex IV (cytochrome c oxidase) is a multi-subunit enzyme that transfers electrons from cytochrome c to molecular oxygen, yielding water. Its biogenesis requires concerted expression of mitochondria- and nuclear-encoded subunits and assembly factors. In this report, we describe a homozygous missense mutation in FAM36A from a patient who displays ataxia and muscle hypotonia. The FAM36A gene is a remote, putative ortholog of the fungal complex IV assembly factor COX20. Messenger RNA (mRNA) and protein co-expression analyses support the involvement of FAM36A in complex IV function in mammals. The c.154A>C mutation in the FAM36A gene, a mutation that is absent in sequenced exomes, leads to a reduced activity and lower levels of complex IV and its protein subunits. The FAM36A protein is nearly absent in patients fibroblasts. Cells affected by the mutation accumulate subassemblies of complex IV that contain COX1 but are almost devoid of COX2 protein. We observe co-purification of FAM36A and COX2 proteins, supporting that the FAM36A defect hampers the early step of complex IV assembly at the incorporation of the COX2 subunit. Lentiviral complementation of patients fibroblasts with wild-type FAM36A increases the complex IV activity as well as the amount of holocomplex IV and of individual subunits. These results establish the function of the human gene FAM36A/COX20 in complex IV assembly and support a causal role of the gene in complex IV deficiency.
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Chromatin accessibility, p300, and histone acetylation define PML-RAR? and AML1-ETO binding sites in acute myeloid leukemia.
Blood
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Chromatin accessibility plays a key role in regulating cell type specific gene expression during hematopoiesis but has also been suggested to be aberrantly regulated during leukemogenesis. To understand the leukemogenic chromatin signature, we analyzed acute promyelocytic leukemia, a subtype of leukemia characterized by the expression of RAR?-fusion proteins, such as PML-RAR?. We used nuclease accessibility sequencing in cell lines as well as patient blasts to identify accessible DNA elements and identified > 100 000 accessible regions in each case. Using ChIP-seq, we identified H2A.Z as a histone modification generally associated with these accessible regions, whereas unsupervised clustering analysis of other chromatin features, including DNA methylation, H2A.Zac, H3ac, H3K9me3, H3K27me3, and the regulatory factor p300, distinguished 6 distinct clusters of accessible sites, each with a characteristic functional makeup. Of these, PML-RAR? binding was found specifically at accessible chromatin regions characterized by p300 binding and hypoacetylated histones. Identifying regions with a similar epigenetic make up in t(8;21) acute myeloid leukemia (AML) cells, another subtype of AMLs, revealed that these regions are occupied by the oncofusion protein AML1-ETO. Together, our results suggest that oncofusion proteins localize to accessible regions and that chromatin accessibility together with p300 binding and histone acetylation characterize AML1-ETO and PML-RAR? binding sites.
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Loss, replacement and gain of proteins at the origin of the mitochondria.
Biochim. Biophys. Acta
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We review what has been inferred about the changes at the level of the proteome that accompanied the evolution of the mitochondrion from an alphaproteobacterium. We regard these changes from an alphaproteobacterial perspective: which proteins were lost during mitochondrial evolution? And, of the proteins that were lost, which ones have been replaced by other, non-orthologous proteins with a similar function? Combining literature-supported replacements with quantitative analyses of mitochondrial proteomics data we infer that most of the loss and replacements that separate current day mitochondria in mammals from alphaproteobacteria took place before the radiation of the eukaryotes. Recent analyses show that also the acquisition of new proteins to the large protein complexes of the oxidative phosphorylation and the mitochondrial ribosome occurred mainly before the divergence of the eukaryotes. These results indicate a significant number of pivotal evolutionary events between the acquisition of the endosymbiont and the radiation of the eukaryotes and therewith support an early acquisition of mitochondria in eukaryotic evolution. Technically, advancements in the reconstruction of the evolutionary trajectories of loss, replacement and gain of mitochondrial proteins depend on using profile-based homology detection methods for sequence analysis. We highlight the mitochondrial Holliday junction resolvase endonuclease, for which such methods have detected new "family members" and in which function differentiation is accompanied by the loss of catalytic residues for the original enzymatic function and the gain of a protein domain for the new function. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.
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A three-dimensional topology of complex I inferred from evolutionary correlations.
BMC Struct. Biol.
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The quaternary structure of eukaryotic NADH:ubiquinone oxidoreductase (complex I), the largest complex of the oxidative phosphorylation, is still mostly unresolved. Furthermore, it is unknown where transiently bound assembly factors interact with complex I. We therefore asked whether the evolution of complex I contains information about its 3D topology and the binding positions of its assembly factors. We approached these questions by correlating the evolutionary rates of eukaryotic complex I subunits using the mirror-tree method and mapping the results into a 3D representation by multidimensional scaling.
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BOLA1 is an aerobic protein that prevents mitochondrial morphology changes induced by glutathione depletion.
Antioxid. Redox Signal.
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The BolA protein family is widespread among eukaryotes and bacteria. In Escherichia coli, BolA causes a spherical cell shape and is overexpressed during oxidative stress. Here we aim to elucidate the possible role of its human homolog BOLA1 in mitochondrial morphology and thiol redox potential regulation.
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Evolution and diversification of the organellar release factor family.
Mol. Biol. Evol.
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Translation termination is accomplished by proteins of the Class I release factor family (RF) that recognize stop codons and catalyze the ribosomal release of the newly synthesized peptide. Bacteria have two canonical RFs: RF1 recognizes UAA and UAG, RF2 recognizes UAA and UGA. Despite that these two release factor proteins are sufficient for de facto translation termination, the eukaryotic organellar RF protein family, which has evolved from bacterial release factors, has expanded considerably, comprising multiple subfamilies, most of which have not been functionally characterized or formally classified. Here, we integrate multiple sources of information to analyze the remarkable differentiation of the RF family among organelles. We document the origin, phylogenetic distribution and sequence structure features of the mitochondrial and plastidial release factors: mtRF1a, mtRF1, mtRF2a, mtRF2b, mtRF2c, ICT1, C12orf65, pRF1, and pRF2, and review published relevant experimental data. The canonical release factors (mtRF1a, mtRF2a, pRF1, and pRF2) and ICT1 are derived from bacterial ancestors, whereas the others have resulted from gene duplications of another release factor. These new RF family members have all lost one or more specific motifs relevant for bona fide release factor function but are mostly targeted to the same organelle as their ancestor. We also characterize the subset of canonical release factor proteins that bear nonclassical PxT/SPF tripeptide motifs and provide a molecular-model-based rationale for their retained ability to recognize stop codons. Finally, we analyze the coevolution of canonical RFs with the organellar genetic code. Although the RF presence in an organelle and its stop codon usage tend to coevolve, we find three taxa that encode an RF2 without using UGA stop codons, and one reverse scenario, where mamiellales green algae use UGA stop codons in their mitochondria without having a mitochondrial type RF2. For the latter, we put forward a "stop-codon reinvention" hypothesis that involves the retargeting of the plastid release factor to the mitochondrion.
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Structure based hypothesis of a mitochondrial ribosome rescue mechanism.
Biol. Direct
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mtRF1 is a vertebrate mitochondrial protein with an unknown function that arose from a duplication of the mitochondrial release factor mtRF1a. To elucidate the function of mtRF1, we determined the positions that are conserved among mtRF1 sequences but that are different in their mtRF1a paralogs. We subsequently modeled the 3D structure of mtRF1a and mtRF1 bound to the ribosome, highlighting the structural implications of these differences to derive a hypothesis for the function of mtRF1.
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Iterative orthology prediction uncovers new mitochondrial proteins and identifies C12orf62 as the human ortholog of COX14, a protein involved in the assembly of cytochrome c oxidase.
Genome Biol.
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Orthology is a central tenet of comparative genomics and ortholog identification is instrumental to protein function prediction. Major advances have been made to determine orthology relations among a set of homologous proteins. However, they depend on the comparison of individual sequences and do not take into account divergent orthologs.
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CATCHprofiles: clustering and alignment tool for ChIP profiles.
PLoS ONE
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Chromatin Immuno Precipitation (ChIP) profiling detects in vivo protein-DNA binding, and has revealed a large combinatorial complexity in the binding of chromatin associated proteins and their post-translational modifications. To fully explore the spatial and combinatorial patterns in ChIP-profiling data and detect potentially meaningful patterns, the areas of enrichment must be aligned and clustered, which is an algorithmically and computationally challenging task. We have developed CATCHprofiles, a novel tool for exhaustive pattern detection in ChIP profiling data. CATCHprofiles is built upon a computationally efficient implementation for the exhaustive alignment and hierarchical clustering of ChIP profiling data. The tool features a graphical interface for examination and browsing of the clustering results. CATCHprofiles requires no prior knowledge about functional sites, detects known binding patterns "ab initio", and enables the detection of new patterns from ChIP data at a high resolution, exemplified by the detection of asymmetric histone and histone modification patterns around H2A.Z-enriched sites. CATCHprofiles capability for exhaustive analysis combined with its ease-of-use makes it an invaluable tool for explorative research based on ChIP profiling data. CATCHprofiles and the CATCH algorithm run on all platforms and is available for free through the CATCH website: http://catch.cmbi.ru.nl/. User support is available by subscribing to the mailing list catch-users@bioinformatics.org.
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C7orf30 specifically associates with the large subunit of the mitochondrial ribosome and is involved in translation.
Nucleic Acids Res.
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In a comparative genomics study for mitochondrial ribosome-associated proteins, we identified C7orf30, the human homolog of the plant protein iojap. Gene order conservation among bacteria and the observation that iojap orthologs cannot be transferred between bacterial species predict this protein to be associated with the mitochondrial ribosome. Here, we show colocalization of C7orf30 with the large subunit of the mitochondrial ribosome using isokinetic sucrose gradient and 2D Blue Native polyacrylamide gel electrophoresis (BN-PAGE) analysis. We co-purified C7orf30 with proteins of the large subunit, and not with proteins of the small subunit, supporting interaction that is specific to the large mitoribosomal complex. Consistent with this physical association, a mitochondrial translation assay reveals negative effects of C7orf30 siRNA knock-down on mitochondrial gene expression. Based on our data we propose that C7orf30 is involved in ribosomal large subunit function. Sequencing the gene in 35 patients with impaired mitochondrial translation did not reveal disease-causing mutations in C7orf30.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.