The cardiac troponin T variations have often been used as an example of the application of clinical genotyping for prognostication and risk stratification measures for the management of patients with a family history of sudden cardiac death or familial cardiomyopathy. Given the disparity in patient outcomes and therapy options, we investigated the impact of variations on the intermolecular interactions across the thin filament complex as an example of an unbiased systems biology method to better define clinical prognosis to aid future management options. We present a novel unbiased dynamic model to define and analyse the functional, structural and physico-chemical consequences of genetic variations among the troponins. This was subsequently integrated with clinical data from accessible global multi-centre systematic reviews of familial cardiomyopathy cases from 106 articles of the literature: 136 disease-causing variations pertaining to 981 global clinical cases. Troponin T variations showed distinct pathogenic hotspots for dilated and hypertrophic cardiomyopathies; considering the causes of cardiovascular death separately, there was a worse survival in terms of sudden cardiac death for patients with a variation at regions 90–129 and 130–179 when compared to amino acids 1–89 and 200–288. Our data support variations among 90–130 as being a hotspot for sudden cardiac death and the region 131–179 for heart failure death/transplantation outcomes wherein the most common phenotype was dilated cardiomyopathy. Survival analysis into regions of high risk (regions 90–129 and 130–180) and low risk (regions 1–89 and 200–288) was significant for sudden cardiac death (p = 0.011) and for heart failure death/transplant (p = 0.028). Our integrative genomic, structural, model from genotype to clinical data integration has implications for enhancing clinical genomics methodologies to improve risk stratification.

Mitochondrial complex I (CI) deficiency is the most common oxidative phosphorylation disorder described. It shows a wide range of phenotypes with poor correlation within genotypes. Herein we expand the clinics and genetics of CI deficiency in the brazilian population by reporting three patients with pathogenic (c.640G>A, c.1268C>T, c.1207dupG) and likely pathogenic (c.766C>T) variants in the NDUFV1 gene. We show the mutation c.766C>T associated with a childhood onset phenotype of hypotonia, muscle weakness, psychomotor regression, lethargy, dysphagia, and strabismus. Additionally, this mutation was found to be associated with headaches and exercise intolerance in adulthood. We also review reported pathogenic variants in NDUFV1 highlighting the wide phenotypic heterogeneity in CI deficiency.

Objective To expand the clinical phenotype of POLR3A mutations by assessing the functional consequences of a missense and a splicing acceptor mutation. Methods We performed whole-exome sequencing for identification of likely pathogenic mutations in a 9-year-old female patient with severe generalized dystonia, metabolic acidosis, leukocytosis, hypotonia, and dysphagia. Brain MRI showed basal ganglia atrophy and presence of lactate and lipid peaks by [H-1]-magnetic resonance spectroscopy. Expression levels of Pol III target genes were measured by quantitative real-time (qRT)-PCR to study the pathogenicity of the biallelic mutations in patient fibroblasts. Results The patient is a compound heterozygous for a novel missense c.3721G>A (p.Val1241Met) and the splicing region c.1771-6C>G mutation in POLR3A, the gene coding for the catalytic subunit of RNA polymerase III (Pol III). Aberrant splicing was observed for the c.1771-6C>G mutation. Decreased RNA expression levels of Pol III targets (HNRNPH2, ubiquitin B, lactotransferrin, and HSP90AA1) were observed in patient fibroblasts with rescue to normal levels by overexpression of the wild-type protein but not by the p.Val1241Met variant. Conclusions Mutations in the POLR3A gene cause POLR3A-related hypomyelinating leukodystrophy with or without oligodontia or hypogonadotropic hypogonadism (HLD7, OMIM: 607694) and neonatal progeroid syndrome (OMIM: 264090), both with high phenotypic variability. We demonstrated the pathogenicity of c.1771-6C>G and c.3721G>A mutations causing an early-onset disorder. The phenotype of our patient expands the clinical presentation of POLR3A-related mutations and suggests a new classification that we propose designating as Neurodevelopmental Disorder with Regression, Abnormal Movements, and Increased Lactate.

Acyl-CoAs are reactive metabolites that can non-enzymatically S-acylate and N-acylate protein cysteine and lysine residues, respectively. N-acylation is irreversible and enhanced if a nearby cysteine residue undergoes an initial reversible S-acylation, as proximity leads to rapid S → N-transfer of the acyl moiety. We reasoned that protein-bound acyl-CoA could also facilitate S → N-transfer of acyl groups to proximal lysine residues. Furthermore, as CoA contains an ADP backbone this may extend beyond CoA-binding sites and include abundant Rossmann-fold motifs that bind the ADP moiety of NADH, NADPH, FADH and ATP. Here, we show that excess nucleotides decrease protein lysine N-acetylation in vitro. Furthermore, by generating modelled structures of proteins N-acetylated in mouse liver, we show that proximity to a nucleotide-binding site increases the risk of N-acetylation and identify where nucleotide binding could enhance N-acylation in vivo. Finally, using glutamate dehydrogenase as a case study, we observe increased in vitro lysine N-malonylation by malonyl-CoA near nucleotide-binding sites which overlaps with in vivo N-acetylation and N-succinylation. Furthermore, excess NADPH, GTP and ADP greatly diminish N-malonylation near their nucleotide-binding sites, but not at distant lysine residues. Thus, lysine N-acylation by acyl-CoAs is enhanced by nucleotide-binding sites and may contribute to higher stoichiometry protein N-acylation in vivo.

Mitochondrial disorders affect 1/5,000 and have no cure. Inducing mitochondrial biogenesis with bezafibrate improves mitochondrial function in animal models, but there are no comparable human studies. We performed an open-label observational experimental medicine study of six patients with mitochondrial myopathy caused by the m.3243A>G MTTL1 mutation. Our primary aim was to determine the effects of bezafibrate on mitochondrial metabolism, whilst providing preliminary evidence of safety and efficacy using biomarkers. The participants received 600-1,200 mg bezafibrate daily for 12 weeks. There were no clinically significant adverse events, and liver function was not affected. We detected a reduction in the number of complex IV-immunodeficient muscle fibres and improved cardiac function. However, this was accompanied by an increase in serum biomarkers of mitochondrial disease, including fibroblast growth factor 21 (FGF-21), growth and differentiation factor 15 (GDF-15), plus dysregulation of fatty acid and amino acid metabolism. Thus, although potentially beneficial in short term, inducing mitochondrial biogenesis with bezafibrate altered the metabolomic signature of mitochondrial disease, raising concerns about long-term sequelae.

Increasing numbers of diseases are associated with mitochondrial dysfunction. This is unsurprising given mitochondria have major roles in bioenergy generation, signalling, detoxification, apoptosis and biosynthesis. However, fundamental questions of mitochondrial biology remain, including: which nuclear genes encode mitochondrial proteins; how their expression varies with tissue; and which are associated with disease. But experiments to catalogue the mitochondrial proteome are incomplete and sometimes contradictory. This arises because the mitochondrial proteome has tissue- and stage-specific variability, plus differences among experimental techniques and localization evidence types used. This leads to limitations in each technique's coverage and inevitably conflicting results. To support identification of mitochondrial proteins, we developed MitoMiner (http://mitominer.mrc-mbu.cam.ac.uk/), a database combining evidence of mitochondrial localization with information from public resources. Here we report upgrades to MitoMiner, including its re-engineering to be gene-centric to enable easier sharing of evidence among orthologues and support next generation sequencing, plus new data sources, including expression in different tissues, information on phenotypes and diseases of genetic mutations and a new mitochondrial proteome catalogue. MitoMiner is a powerful platform to investigate mitochondrial localization by providing a unique combination of experimental sub-cellular localization datasets, tissue expression, predictions of mitochondrial targeting sequences, gene annotation and links to phenotype and disease.

NUBPL (Nucleotide-binding protein like) protein encodes a member of the Mrp/NBP35 ATP-binding proteins family, deemed to be involved in mammalian complex I (CI) assembly process. Exome sequencing of a patient presenting with infantile-onset hepatopathy, renal tubular acidosis, developmental delay, short stature, leukoencephalopathy with minimal cerebellar involvement and multiple OXPHOS deficiencies revealed the presence of two novel pathogenic compound heterozygous variants in NUBPL (p.Phe242Leu/p.Leu104Pro). We investigated patient's and control immortalised fibroblasts and demonstrated that both the peripheral and the membrane arms of complex I are undetectable in mutant NUBPL cells, resulting in virtually absent CI holocomplex and loss of enzyme activity. In addition, complex III stability was moderately affected as well. Lentiviral-mediated expression of the wild-type NUBPL cDNA rescued both CI and CIII assembly defects, confirming the pathogenicity of the variants. In conclusion, this is the first report describing a complex multisystemic disorder due to NUBPL defect. In addition, we confirmed the role of NUBPL in Complex I assembly associated with secondary effect on Complex III stability and we demonstrated a defect of mtDNA-related translation which suggests a potential additional role of NUBPL in mtDNA expression.

During heart transplantation, storage in cold preservation solution is thought to protect the organ by slowing metabolism; by providing osmotic support; and by minimising ischaemia-reperfusion (IR) injury upon transplantation into the recipient1,2. Despite its widespread use our understanding of the metabolic changes prevented by cold storage and how warm ischaemia leads to damage is surprisingly poor. Here, we compare the metabolic changes during warm ischaemia (WI) and cold ischaemia (CI) in hearts from mouse, pig, and human. We identify common metabolic alterations during WI and those affected by CI, thereby elucidating mechanisms underlying the benefits of CI, and how WI causes damage. Succinate accumulation is a major feature within ischaemic hearts across species, and CI slows succinate generation, thereby reducing tissue damage upon reperfusion caused by the production of mitochondrial reactive oxygen species (ROS)3,4. Importantly, the inevitable periods of WI during organ procurement lead to the accumulation of damaging levels of succinate during transplantation, despite cooling organs as rapidly as possible. This damage is ameliorated by metabolic inhibitors that prevent succinate accumulation and oxidation. Our findings suggest how WI and CI contribute to transplant outcome and indicate new therapies for improving the quality of transplanted organs.

This Article was originally published under Nature Research's License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the Article have been modified accordingly.

Cardiolipin in eukaryotes is found in the mitochondrial inner membrane, where it interacts with membrane proteins and, although not essential, is necessary for the optimal activity of a number of proteins. One of them is the mitochondrial ADP/ATP carrier, which imports ADP into the mitochondrion and exports ATP. In the crystal structures, cardiolipin is bound to three equivalent sites of the ADP/ATP carrier, but its role is unresolved. Conservation of residues at these cardiolipin binding sites across other members of the mitochondrial carrier superfamily indicates cardiolipin binding is likely to be important for the function of all mitochondrial carriers. Multiscale simulations were performed in a cardiolipin-containing membrane to investigate the dynamics of cardiolipin around the yeast and bovine ADP/ATP carriers in a lipid bilayer and the properties of the cardiolipin-binding sites. In coarse-grain simulations, cardiolipin molecules bound to the carriers for longer periods of time than phosphatidylcholine and phosphatidylethanolamine lipids—with timescales in the tens of microseconds. Three long-lived cardiolipin binding sites overlapped with those in the crystal structures of the carriers. Other shorter-lived cardiolipin interaction sites were identified in both membrane leaflets. However, the timescales of the interactions were of the same order as phosphatidylcholine and phosphatidylethanolamine, suggesting that these sites are not specific for cardiolipin binding. The calculation of lipid binding times and the overlap of the cardiolipin binding sites between the structures and simulations demonstrate the potential of multiscale simulations to investigate the dynamics and behavior of lipids interacting with membrane proteins.

TIMM50 is an essential component of the TIM23 complex, the mitochondrial inner membrane machinery that imports cytosolic proteins containing a mitochondrial targeting presequence into the mitochondrial inner compartment. Whole exome sequencing (WES) identified compound heterozygous pathogenic mutations in TIMM50 in an infant patient with rapidly progressive, severe encephalopathy. Patient fibroblasts presented low levels of TIMM50 and other components of the TIM23 complex, lower mitochondrial membrane potential, and impaired TIM23-dependent protein import. As a consequence, steady-state levels of several components of mitochondrial respiratory chain were decreased, resulting in decreased respiration and increased ROS production. Growth of patient fibroblasts in galactose shifted energy production metabolism toward oxidative phosphorylation (OxPhos), producing an apparent improvement in most of the above features but also increased apoptosis. Complementation of patient fibroblasts with TIMM50 improved or restored these features to control levels. Moreover, RNASEH1 and ISCU mutant fibroblasts only shared a few of these features with TIMM50 mutant fibroblasts. Our results indicate that mutations in TIMM50 cause multiple mitochondrial bioenergetic dysfunction and that functional TIMM50 is essential for cell survival in OxPhos-dependent conditions.

Thousands of protein acyl modification sites have now been identified in vivo. However, at most sites the acylation stoichiometry is low, making functional enzyme-driven regulation in the majority of cases unlikely. As unmediated acylation can occur on the surface of proteins when acyl-CoA thioesters react with nucleophilic cysteine and lysine residues, slower nonenzymatic processes likely underlie most protein acylation. Here, we review how nonenzymatic acylation of nucleophilic lysine and cysteine residues occurs; the factors that enhance acylation at particular sites; and the strategies that have evolved to limit protein acylation. We conclude that protein acylation is an unavoidable consequence of the central role of reactive thioesters in metabolism. Finally, we propose a hypothesis for why low-stoichiometry protein acylation is selected against by evolution and how it might contribute to degenerative processes such as aging.

Biallelic mutations in NDUFAF6 have been identified as responsible for cases of autosomal recessive Leigh syndrome associated with mitochondrial complex I deficiency. Here we report two siblings and two unrelated subjects with Leigh syndrome, in which we found the same compound heterozygous missense (c.532G>C:p.A178P) and deep intronic (c.420+784C>T) variants in NDUFAF6. We demonstrated that the identified intronic variant creates an alternative splice site, leading to the production of an aberrant transcript. A detailed analysis of whole-exome sequencing data together with the functional validation based on mRNA analysis may reveal pathogenic variants even in non-exonic regions.

Acetyl-coenzyme A (CoA) is an abundant metabolite that can also alter protein function through non-enzymatic N-acetylation of protein lysines. This N-acetylation is greatly enhanced in vitro if an adjacent cysteine undergoes initial S-acetylation, as this can lead to S→N transfer of the acetyl moiety. Here, using modeled mouse structures of 619 proteins N-acetylated in mouse liver, we show lysine N-acetylation is greater in vivo if a cysteine is within ∼10 Å. Extension to the genomes of 52 other mammalian and bird species shows pairs of proximal cysteine and N-acetylated lysines are less conserved, implying most N-acetylation is detrimental. Supporting this, there is less conservation of cytosolic pairs of proximal cysteine and N-acetylated lysines in species with longer lifespans. As acetyl-CoA levels are linked to nutrient supply, these findings suggest how dietary restriction could extend lifespan and how pathologies resulting from dietary excess may occur.

Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an early onset, fatal mitochondrial encephalopathy, leading to multiple neurological failure and eventually death, usually in the first decade of life. Mutations in SURF1, a nuclear gene encoding a mitochondrial protein involved in COX assembly, are among the most common causes of LS. LSSURF1 patients display severe, isolated COX deficiency in all tissues, including cultured fibroblasts and skeletal muscle. Recombinant, constitutive SURF1−/− mice show diffuse COX deficiency, but fail to recapitulate the severity of the human clinical phenotype. Pigs are an attractive alternative model for human diseases, because of their size, as well as metabolic, physiological and genetic similarity to humans. Here, we determined the complete sequence of the swine SURF1 gene, disrupted it in pig primary fibroblast cell lines using both TALENs and CRISPR/Cas9 genome editing systems, before finally generating SURF1−/− and SURF1−/+ pigs by Somatic Cell Nuclear Transfer (SCNT). SURF1−/− pigs were characterized by failure to thrive, muscle weakness and highly reduced life span with elevated perinatal mortality, compared to heterozygous SURF1−/+ and wild type littermates. Surprisingly, no obvious COX deficiency was detected in SURF1−/− tissues, although histochemical analysis revealed the presence of COX deficiency in jejunum villi and total mRNA sequencing (RNAseq) showed that several COX subunit-encoding genes were significantly down-regulated in SURF1−/− skeletal muscles. In addition, neuropathological findings, indicated a delay in central nervous system development of newborn SURF1−/− piglets. Our results suggest a broader role of sSURF1 in mitochondrial bioenergetics.

105 new or altered reactions in the MitoCore model that differ from those in the Virtual Metabolic Human database (and used by the Recon 2.2 model). (XLSX 82Â kb)

Background: The complexity of metabolic networks can make the origin and impact of changes in central metabolism occurring during diseases difficult to understand. Computer simulations can help unravel this complexity, and progress has advanced in genome-scale metabolic models. However, many models produce unrealistic results when challenged to simulate abnormal metabolism as they include incorrect specification and localisation of reactions and transport steps, incorrect reaction parameters, and confounding of prosthetic groups and free metabolites in reactions. Other common drawbacks are due to their scale, making them difficult to parameterise and simulation results hard to interpret. Therefore, it remains important to develop smaller, manually curated models. Results: We present MitoCore, a manually curated constraint-based computer model of human metabolism that incorporates the complexity of central metabolism and simulates this metabolism successfully under normal and abnormal physiological conditions, including hypoxia and mitochondrial diseases. MitoCore describes 324 metabolic reactions, 83 transport steps between mitochondrion and cytosol, and 74 metabolite inputs and outputs through the plasma membrane, to produce a model of manageable scale for easy interpretation of results. Its key innovations include a more accurate partitioning of metabolism between cytosol and mitochondrial matrix; better modelling of connecting transport steps; differentiation of prosthetic groups and free co-factors in reactions; and a new representation of the respiratory chain and the proton motive force. MitoCore's default parameters simulate normal cardiomyocyte metabolism, and to improve usability and allow comparison with other models and types of analysis, its reactions and metabolites have extensive annotation, and cross-reference identifiers from Virtual Metabolic Human database and KEGG. These innovations-including over 100 reactions absent or modified from Recon 2-are necessary to model central metabolism more accurately. Conclusion: We anticipate MitoCore as a research tool for scientists, from experimentalists looking to interpret their data and test hypotheses, to experienced modellers predicting the consequences of disease or using computationally intensive methods that are infeasible with larger models, as well as a teaching tool for those new to modelling and needing a small, manageable model on which to learn and experiment.