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Alterations in mitochondrial function have been linked to a variety of cellular and organismal stress responses including apoptosis, aging, neurodegeneration and tumorigenesis. However, adaptation to mitochondrial dysfunction can occur through the activation of survival pathways, whose mechanisms are still poorly understood. The yeast Saccharomyces cerevisiae is an invaluable model organism for studying how mitochondrial dysfunction can affect stress response and adaptation processes. In this study, we analyzed and compared in the absence and in the presence of osmostress wild-type cells with two models of cells lacking mitochondrial DNA: ethidium bromide-treated cells (ρ 0 ) and cells lacking the mitochondrial pyrimidine nucleotide transporter RIM2 (Δ RIM2 ). Our results revealed that the lack of mitochondrial DNA provides an advantage in the kinetics of stress response. Additionally, wild-type cells exhibited higher osmosensitivity in the presence of respiratory metabolism. Mitochondrial mutants showed increased glycerol levels, required in the short-term response of yeast osmoadaptation, and prolonged oxidative stress. The involvement of the mitochondrial retrograde signaling in osmoadaptation has been previously demonstrated. The expression of CIT2 , encoding the peroxisomal isoform of citrate synthase and whose up-regulation is prototypical of RTG pathway activation, appeared to be increased in the mutants. Interestingly, selected TCA cycle genes, CIT1 and ACO1 , whose expression depends on RTG signaling upon stress, showed a different regulation in ρ 0 and Δ RIM2 cells. These data suggest that osmoadaptation can occur through different mechanisms in the presence of mitochondrial defects and will allow us to gain insight into the relationships among metabolism, mitochondria-mediated stress response, and cell adaptation., Competing Interests: The authors declare no conflicts of interest.

Diffuse intrinsic pontine glioma (DIPG), affecting children aged 4-7 years, is a rare, aggressive tumor that originates in the pons and then spreads to nearby tissue. DIPG is the leading cause of death for pediatric brain tumors due to its infiltrative nature and inoperability. Radiotherapy has only a palliative effect on stabilizing symptoms. In silico and preclinical studies identified ONC201 as a cytotoxic agent against some human cancer cell lines, including DIPG ones. A single-crystal X-ray analysis of the complex of the human mitochondrial caseinolytic serine protease type C ( h ClpP) and ONC201 (PDB ID: 6DL7) allowed h ClpP to be identified as its main target. The hyperactivation of h ClpP causes damage to mitochondrial oxidative phosphorylation and cell death. In some DIPG patients receiving ONC201, an acquired resistance was observed. In this context, a wide program was initiated to discover original scaffolds for new h ClpP activators to treat ONC201-non-responding patients. Harmaline, a small molecule belonging to the chemical class of β-carboline, was identified through Fingerprints for Ligands and Proteins (FLAP), a structure-based virtual screening approach. Molecular dynamics simulations and a deep in vitro investigation showed interesting information on the interaction and activation of h ClpP by harmaline.

Background: Mitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries., Results: In this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica., Conclusions: The Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis., (© 2023. The Author(s).)

Mitochondrial RTG (an acronym for ReTroGrade) signaling plays a cytoprotective role under various intracellular or environmental stresses. We have previously shown its contribution to osmoadaptation and capacity to sustain mitochondrial respiration in yeast. Here, we studied the interplay between RTG2 , the main positive regulator of the RTG pathway, and HAP4 , encoding the catalytic subunit of the Hap2-5 complex required for the expression of many mitochondrial proteins that function in the tricarboxylic acid (TCA) cycle and electron transport, upon osmotic stress. Cell growth features, mitochondrial respiratory competence, retrograde signaling activation, and TCA cycle gene expression were comparatively evaluated in wild type and mutant cells in the presence and in the absence of salt stress. We showed that the inactivation of HAP4 improved the kinetics of osmoadaptation by eliciting both the activation of retrograde signaling and the upregulation of three TCA cycle genes: citrate synthase 1 ( CIT1 ), aconitase 1 ( ACO1 ), and isocitrate dehydrogenase 1 ( IDH1 ). Interestingly, their increased expression was mostly dependent on RTG2 . Impaired respiratory competence in the HAP4 mutant does not affect its faster adaptive response to stress. These findings indicate that the involvement of the RTG pathway in osmostress is fostered in a cellular context of constitutively reduced respiratory capacity. Moreover, it is evident that the RTG pathway mediates peroxisomes-mitochondria communication by modulating the metabolic function of mitochondria in osmoadaptation.

Triclosan (TCS) is a synthetic broad-spectrum antimicrobial agent commonly used world-wide in a range of personal care and sanitizing products detected frequently in aquatic ecosystems. The aim of this study was to examine biochemical markers responses triggered by TCS in Danio rerio and in a native South American fish species ( Corydoras paleatus ). Further, an integrated approach comparing both test fish species was undertaken. These fish organisms were exposed to 100 or 189 µg TCS/L for 48 h. The activities of catalase (CAT), glutathione-s-transferase (GST), superoxide dismutase (SOD), and lipid peroxidation levels (LPO) and total antioxidant capacity against peroxyl radicals (ACAP) were determined in liver, gills, and brain. Acetylcholinesterase activity (AChE) was measured in the brain. Multivariate analysis showed that the most sensitive hepatic parameters were activities of GST and SOD for C. paleatus while LPO levels were for D. rerio . In gills the same parameters were responsive for C. paleatus but CAT in D. rerio . ACAP and GST activity were responsive parameters in brain of both species. Integrated biomarker responses (IBR) index demonstrated similar trends in both species suggesting this parameter might serve as a useful tool for quantification of integrated responses induced by TCS.

Neuromuscular diseases (NMDs) are dysfunctions that involve skeletal muscle and cause incorrect communication between the nerves and muscles. The specific causes of NMDs are not well known, but most of them are caused by genetic mutations. NMDs are generally progressive and entail muscle weakness and fatigue. Muscular impairments can differ in onset, severity, prognosis, and phenotype. A multitude of possible injury sites can make diagnosis of NMDs difficult. Mitochondria are crucial for cellular homeostasis and are involved in various metabolic pathways; for this reason, their dysfunction can lead to the development of different pathologies, including NMDs. Most NMDs due to mitochondrial dysfunction have been associated with mutations of genes involved in mitochondrial biogenesis and metabolism. This review is focused on some mitochondrial routes such as the TCA cycle, OXPHOS, and β-oxidation, recently found to be altered in NMDs. Particular attention is given to the alterations found in some genes encoding mitochondrial carriers, proteins of the inner mitochondrial membrane able to exchange metabolites between mitochondria and the cytosol. Briefly, we discuss possible strategies used to diagnose NMDs and therapies able to promote patient outcome.

SLC25A36 is a pyrimidine nucleotide carrier playing an important role in maintaining mitochondrial biogenesis. Deficiencies in SLC25A36 in mouse embryonic stem cells have been associated with mtDNA depletion as well as mitochondrial dysfunction. In human beings, diseases triggered by SLC25A36 mutations have not been described yet. We report the first known case of SLC25A36 deficiency in a 12-year-old patient with hypothyroidism, hyperinsulinism, hyperammonemia, chronical obstipation, short stature, along with language and general developmental delay. Whole exome analysis identified the homozygous mutation c.803dupT, p.Ser269llefs*35 in the SLC25A36 gene. Functional analysis of mutant SLC25A36 protein in proteoliposomes showed a virtually abolished transport activity. Immunoblotting results suggest that the mutant SLC25A36 protein in the patient undergoes fast degradation. Supplementation with oral uridine led to an improvement of thyroid function and obstipation, increase of growth and developmental progress. Our findings suggest an important role of SLC25A36 in hormonal regulations and oral uridine as a safe and effective treatment.

Mitochondrial RTG -dependent retrograde signaling, whose regulators have been characterized in Saccharomyces cerevisiae , plays a recognized role under various environmental stresses. Of special significance, the activity of the transcriptional complex Rtg1/3 has been shown to be modulated by Hog1, the master regulator of the high osmolarity glycerol pathway, in response to osmotic stress. The present work focuses on the role of RTG signaling in salt-induced osmotic stress and its interaction with HOG1 . Wild-type and mutant cells, lacking HOG1 and/or RTG genes, are compared with respect to cell growth features, retrograde signaling activation and mitochondrial function in the presence and in the absence of high osmostress. We show that RTG2, the main upstream regulator of the RTG pathway, contributes to osmoadaptation in an HOG1 -dependent manner and that, with RTG3 , it is notably involved in a late phase of growth. Our data demonstrate that impairment of RTG signaling causes a decrease in mitochondrial respiratory capacity exclusively under osmostress. Overall, these results suggest that HOG1 and the RTG pathway may interact sequentially in the stress signaling cascade and that the RTG pathway may play a role in inter-organellar metabolic communication for osmoadaptation.

During cultivation under nitrogen starvation, Yarrowia lipolytica produces a mixture of citric acid and isocitric acid whose ratio is mainly determined by the carbon source used. We report that mitochondrial succinate-fumarate carrier YlSfc1 controls isocitric acid efflux from mitochondria. YlSfc1 purified and reconstituted into liposomes transports succinate, fumarate, oxaloacetate, isocitrate and α-ketoglutarate. YlSFC1 overexpression determined the inversion of isocitric acid/citric acid ratio towards isocitric acid, resulting in 33.4 ± 1.9 g/L and 43.3 ± 2.8 g/L of ICA production in test-tube cultivation with glucose and glycerol, respectively. These titers represent a 4.0 and 6.3-fold increase compared to the wild type. YlSFC1 gene expression was repressed in the wild type strain grown in glucose-based medium compared to olive oil medium explaining the reason for the preferred citric acid production during Y. lipolytica growth on carbohydrates. Coexpression of YlSFC1 and adenosine monophosphate deaminase YlAMPD genes together with inactivation of citrate mitochondrial carrier YlYHM2 gene enhanced isocitric acid accumulation up to 41.4 ± 4.1 g/L with an isocitric acid/citric acid ratio of 14.3 in a small-scale cultivation with glucose as a carbon source. During large-scale cultivation with glucose pulse-feeding, the engineered strain produced 136.7 ± 2.5 g/L of ICA with a process selectivity of 88.1%, the highest reported titer and selectivity to date. These results represent the first reported isocitric acid secretion by Y. lipolytica as a main organic acid during cultivation on carbohydrate. Moreover, we demonstrate for the first time that the replacement of one mitochondrial transport system for another can be an efficient tool for switching product accumulation., (Copyright © 2020 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis 1,2 . Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production 2 . The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRAS mut cell lines display decreased glutaminolysis, lower NADPH/NADP + and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRAS WT PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRAS mut PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRAS mut rewired glutamine metabolism 2 , and thus it should be considered a key metabolic target for the treatment of this refractory tumour.

About a thousand genes in the human genome encode for membrane transporters. Among these, several solute carrier proteins (SLCs), representing the largest group of transporters, are still orphan and lack functional characterization. We reasoned that assessing genetic interactions among SLCs may be an efficient way to obtain functional information allowing their deorphanization. Here we describe a network of strong genetic interactions indicating a contribution to mitochondrial respiration and redox metabolism for SLC25A51/MCART1, an uncharacterized member of the SLC25 family of transporters. Through a combination of metabolomics, genomics and genetics approaches, we demonstrate a role for SLC25A51 as enabler of mitochondrial import of NAD, showcasing the potential of genetic interaction-driven functional gene deorphanization.

Heat stress (HS), causing impairment in several physiological processes, is one of the most damaging environmental cues for plants. To counteract the harmful effects of high temperatures, plants activate complex signalling networks, indicated as HS response (HSR). Expression of heat shock proteins (HSPs) and adjustment of redox homeostasis are crucial events of HSR, required for thermotolerance. By pharmacological approaches, the involvement of cAMP in triggering plant HSR has been recently proposed. In this study, to investigate the role of cAMP in HSR signalling, tobacco BY-2 cells overexpressing the 'cAMP-sponge', a genetic tool that reduces intracellular cAMP levels, have been used. in vivo cAMP dampening increased HS susceptibility in a HSPs-independent way. The failure in cAMP elevation during HS caused a high accumulation of reactive oxygen species, due to increased levels of respiratory burst oxidase homolog D, decreased activities of catalase and ascorbate peroxidase, as well as down-accumulation of proteins involved in the control of redox homeostasis. In addition, cAMP deficiency impaired proteasome activity and prevented the accumulation of many proteins of ubiquitin-proteasome system (UPS). By a large-scale proteomic approach together with in silico analyses, these UPS proteins were identified in a specific cAMP-dependent network of HSR., (© 2020 John Wiley & Sons Ltd.)

In the 1980s, after the mitochondrial DNA (mtDNA) had been sequenced, several diseases resulting from mtDNA mutations emerged. Later, numerous disorders caused by mutations in the nuclear genes encoding mitochondrial proteins were found. A group of these diseases are due to defects of mitochondrial carriers, a family of proteins named solute carrier family 25 (SLC25), that transport a variety of solutes such as the reagents of ATP synthase (ATP, ADP, and phosphate), tricarboxylic acid cycle intermediates, cofactors, amino acids, and carnitine esters of fatty acids. The disease-causing mutations disclosed in mitochondrial carriers range from point mutations, which are often localized in the substrate translocation pore of the carrier, to large deletions and insertions. The biochemical consequences of deficient transport are the compartmentalized accumulation of the substrates and dysfunctional mitochondrial and cellular metabolism, which frequently develop into various forms of myopathy, encephalopathy, or neuropathy. Examples of diseases, due to mitochondrial carrier mutations are: combined D-2- and L-2-hydroxyglutaric aciduria, carnitine-acylcarnitine carrier deficiency, hyperornithinemia-hyperammonemia-homocitrillinuria (HHH) syndrome, early infantile epileptic encephalopathy type 3, Amish microcephaly, aspartate/glutamate isoform 1 deficiency, congenital sideroblastic anemia, Fontaine progeroid syndrome, and citrullinemia type II. Here, we review all the mitochondrial carrier-related diseases known until now, focusing on the connections between the molecular basis, altered metabolism, and phenotypes of these inherited disorders.

Itaconic acid (IA) is a naturally occurring dicarboxylic acid with applications in the manufacture of polymers. IA can be produced by fermentation using the fungi Aspergillus terreus or Ustilago maydis as biocatalysts. Indirect evidence has suggested that the mitochondrial carriers U. maydis Um_Mtt1 and A. terreus At_MttA export mitochondrially synthesized cis-aconitate to the cytosol for IA synthesis using malate as a countersubstrate. Here, by assaying the transport features of recombinant Um_Mtt1 and At_MttA in reconstituted liposomes, we find that both proteins efficiently transport cis-aconitate, but malate is well transported only by Um_Mtt1 and 2-oxoglutarate only by At_MttA. Bioinformatic analysis shows that Um_Mtt1 and At_MttA form a distinctive mitochondrial carrier subfamily. Our data show that although fulfilling the same physiological task, Um_Mtt1 and At_MttA have different biochemical features., (© 2019 Federation of European Biochemical Societies.)

Mitochondrial citrate carrier plays a central role in exporting acetyl-CoA in the form of citrate from mitochondria to cytosol thereby connecting carbohydrate catabolism and lipogenesis. In this study, Yarrowia lipolytica mitochondrial citrate carrier was functionally defined and characterized. Firstly, deletion of Y. lipolytica YlCTP1 and YlYHM2 genes coding putative tricarboxylate mitochondrial carriers were performed. ΔYlctp1 strain did not differ significantly from wild type strain in terms of growth rate, organic acids and lipid production. In contrast, ΔYlyhm2 strain did not grow in liquid citrate-containing minimal medium. Moreover, in glucose-containing lipogenic medium YlYHM2 null mutant strain did not produce citric acid; the production of isocitric acid and lipids were decreased. Reintroduction of YlYHM2 gene as well as heterologous expression of Aspergillus niger gene AnYHM2 into ΔYlyhm2 strain restored the growth in minimal citrate medium and even enhanced citric acid production by 45% in both variants compared with wild type strain during test tube cultivation. Mitochondrial extracts isolated from YlYHM2 null mutant and wild type strain were incorporated into liposomes; citrate/citrate and α-ketoglutarate/α-ketoglutarate homoexchange activities were reduced by 87% and 40% in ΔYlyhm2 strain, respectively, compared with the wild type, whereas citrate in /α-ketoglutarate out and α-ketoglutarate in /citrate out heteroexchanges were decreased by 87% and 95%, respectively. YlYhm2p was expressed in Escherichia coli, purified and reconstituted into liposomes. Besides high efficiency to citrate and α-ketoglutarate transport, YlYhm2p also transported oxaloacetate, succinate, fumarate, and to a much lesser extent, aconitate, malate, isocitrate, oxoadipate, and glutamate. The activity of reconstituted YlYhm2p was inhibited strongly by SH-blocking reagents, pyridoxal-5'-phosphate, and partly by N-ethylmaleimide. Co-expression of YlYHM2 and adenosine monophosphate deaminase YlAMPD genes resulted in the production of 49.7 g/L of citric acid during test tube cultivation, whereas wild type strain accumulated 30.1 g/L of citric acid. Large-scale cultivation in bioreactor of the engineered strain resulted in 97.1 g/L of citric acid production with a process selectivity of 94.2% and an overall citric acid yield of 0.5 g/g. The maximal specific rate of citric acid synthesis was 0.93 g/L/h. Therefore, the physiological role of YlYhm2p in glucose-containing medium is to catalyze both import of citrate into mitochondria for catabolic reactions and export of citrate as a source of acetyl-CoA from mitochondria. Possible shuttles for citrate exporting are discussed. Moreover, for the first time evidence has been given for the improvement of TCA cycle intermediate production by manipulation of a gene coding a mitochondrial carrier., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Mitochondria play essential metabolic functions in eukaryotes. Although their major role is the generation of energy in the form of ATP, they are also involved in maintenance of cellular redox state, conversion and biosynthesis of metabolites and signal transduction. Most mitochondrial functions are conserved in eukaryotic systems and mitochondrial dysfunctions trigger several human diseases. By using multi-omics approach, we investigate the effect of methionine supplementation on yeast cellular metabolism, considering its role in the regulation of key cellular processes. Methionine supplementation induces an up-regulation of proteins related to mitochondrial functions such as TCA cycle, electron transport chain and respiration, combined with an enhancement of mitochondrial pyruvate uptake and TCA cycle activity. This metabolic signature is more noticeable in cells lacking Snf1/AMPK, the conserved signalling regulator of energy homeostasis. Remarkably, snf1Δ cells strongly depend on mitochondrial respiration and suppression of pyruvate transport is detrimental for this mutant in methionine condition, indicating that respiration mostly relies on pyruvate flux into mitochondrial pathways. These data provide new insights into the regulation of mitochondrial metabolism and extends our understanding on the role of methionine in regulating energy signalling pathways., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized. Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N 1 -methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N 1 -methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N 1 -methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N 1 -methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N 1 -methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes. In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Combined D-2- and L-2-hydroxyglutaric aciduria (D/L-2-HGA) is a devastating neurometabolic disorder, usually lethal in the first years of life. Autosomal recessive mutations in the SLC25A1 gene, which encodes the mitochondrial citrate carrier (CIC), were previously detected in patients affected with combined D/L-2-HGA. We showed that transfection of deficient fibroblasts with wild-type SLC25A1 restored citrate efflux and decreased intracellular 2-hydroxyglutarate levels, confirming that deficient CIC is the cause of D/L-2-HGA. We developed and implemented a functional assay and applied it to all 17 missense variants detected in a total of 26 CIC-deficient patients, including eight novel cases, showing reduced activities of varying degrees. In addition, we analyzed the importance of residues affected by these missense variants using our existing scoring system. This allowed not only a clinical and biochemical overview of the D/L-2-HGA patients but also phenotype-genotype correlation studies.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability., (© The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The nuclear genes of Saccharomyces cerevisiae YHM2, ODC1 and ODC2 encode three transporters that are localized in the inner mitochondrial membrane. In this study, the roles of YHM2, ODC1 and ODC2 in the assimilation of nitrogen and in the biosynthesis of lysine have been investigated. Both the odc1Δodc2Δ double knockout and the yhm2Δ mutant grew similarly as the YPH499 wild-type strain on synthetic minimal medium (SM) containing 2% glucose and ammonia as the main nitrogen source. In contrast, the yhm2Δodc1Δodc2Δ triple knockout exhibited a marked growth defect under the same conditions. This defect was fully restored by the individual expression of YHM2, ODC1 or ODC2 in the triple deletion strain. Furthermore, the lack of growth of yhm2Δodc1Δodc2Δ on 2% glucose SM was rescued by the addition of glutamate, but not glutamine, to the medium. Using lysine-prototroph YPH499-derived strains, the yhm2Δodc1Δodc2Δ knockout (but not the odc1Δodc2Δ and yhm2Δ mutants) also displayed a growth defect in lysine biosynthesis on 2% glucose SM, which was rescued by the addition of lysine and, to a lesser extent, by the addition of 2-aminoadipate. Additional analysis of the triple mutant showed that it is not respiratory-deficient and does not display mitochondrial DNA instability. These results provide evidence that only the simultaneous absence of YHM2, ODC1 and ODC2 impairs the export from the mitochondrial matrix of i) 2-oxoglutarate which is necessary for the synthesis of glutamate and ammonium fixation in the cytosol and ii) 2-oxoadipate which is required for lysine biosynthesis in the cytosol. Finally, the data presented allow one to suggest that the yhm2Δodc1Δodc2Δ triple knockout is suitable in complementation studies aimed at assessing the pathogenic potential of human SLC25A21 (ODC) mutations., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Punta Lara is located in the Río de la Plata estuary near industrial areas contaminated mainly by organic pollutants. In this work, the responses and status of hepatic biomarkers were studied in juvenile carp (Cyprinus carpio) by means of a 21-day field exposure in cages and collection of juvenile native fish (Leporinus obtusidens) at Punta Lara. The analyzed hepatic biomarkers were: enzymatic activity of glutathione-S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD), lipid peroxidation level using the thiobarbituric acid reaction (TBARS), and CYP1A protein expression, condition factor (CF) and liver somatic (LSI) index. Taking into account oxidative stress responses, SOD activity was increased in both species, while CAT was increased in C. carpio and decreased in L. obtusidens; TBARS levels indicated that oxidative damage was possibly exerted only in L. obtusidens. Biotransformation responses mediated by CYP1A were observed in both species, while GST activity was induced mainly in carps. Considering morphometric indices, CF and LSI were significantly increased in carps while CF decreased in native species. The anthropogenic pollution detected in this study in Punta Lara was associated with differences in biomarkers on both fish species, although a different pattern of response was observed., (Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.)

The Luján River basin, which is located in the northwest area of the province of Buenos Aires, Argentina, receives different anthropogenic inputs before reaching the Río de la Plata estuary. The aim of this study was to assess the adverse impact of the river in the middle part of the basin. To this end, an in situ cage assay was conducted in two sites of the river (S1 and S2) near Luján city, and the responses of hepatic biomarkers of both a standardized (Cyprinus carpio) and a native (Pimelodella laticeps) species were evaluated. The biomarkers studied were the condition factor and liver somatic indices (LSI), the enzymatic activities of catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferase (GST), lipid peroxidation levels (thiobarbituric acid reactive substances, TBARS) and the induction of hepatic cytochrome P450 1A (CYP1A) and vitellogenin (Vtg) proteins. After 14 days, LSI and GST activity increased, and TBARS levels decreased in both species exposed at S1 and S2. In addition, exposure at both sites promoted an increase in SOD activity and CYP1A induction in C. carpio, while Vtg expression was observed only at S1. A shorter exposure period (7 days) caused an initial response only at S2 mediated only by CAT in P. laticeps. Finally, our results demonstrate that a 14-day period of in situ exposure in Luján River could lead to antioxidant and biotransformation processes in C. carpio and to phase II biotransformation responses in P. laticeps., (Copyright © 2012 Wiley Periodicals, Inc.)

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.

Background and Objective: Congenital myasthenic syndromes are rare inherited disorders characterized by fatigable weakness caused by malfunction of the neuromuscular junction. We performed whole exome sequencing to unravel the genetic aetiology in an English sib pair with clinical features suggestive of congenital myasthenia., Methods: We used homozygosity mapping and whole exome sequencing to identify the candidate gene variants. Mutant protein expression and function were assessed in vitro and a knockdown zebrafish model was generated to assess neuromuscular junction development., Results: We identified a novel homozygous missense mutation in the SLC25A1 gene, encoding the mitochondrial citrate carrier. Mutant SLC25A1 showed abnormal carrier function. SLC25A1 has recently been linked to a severe, often lethal clinical phenotype. Our patients had a milder phenotype presenting primarily as a neuromuscular (NMJ) junction defect. Of note, a previously reported patient with different compound heterozygous missense mutations of SLC25A1 has since been shown to suffer from a neuromuscular transmission defect. Using knockdown of SLC25A1 expression in zebrafish, we were able to mirror the human disease in terms of variable brain, eye and cardiac involvement. Importantly, we show clear abnormalities in the neuromuscular junction, regardless of the severity of the phenotype., Conclusions: Based on the axonal outgrowth defects seen in SLC25A1 knockdown zebrafish, we hypothesize that the neuromuscular junction impairment may be related to pre-synaptic nerve terminal abnormalities. Our findings highlight the complex machinery required to ensure efficient neuromuscular function, beyond the proteomes exclusive to the neuromuscular synapse.

Chronic exposure of β-cells to metabolic stresses impairs their function and potentially induces apoptosis. Mitochondria play a central role in coupling glucose metabolism to insulin secretion. However, little is known on mitochondrial responses to specific stresses; i.e. low versus high glucose, saturated versus unsaturated fatty acids, or oxidative stress. INS-1E cells were exposed for 3 days to 5.6 mM glucose, 25 mM glucose, 0.4 mM palmitate, and 0.4 mM oleate. Culture at standard 11.1 mM glucose served as no-stress control and transient oxidative stress (200 µM H2O2 for 10 min at day 0) served as positive stressful condition. Mito-array analyzed transcripts of 60 mitochondrion-associated genes with special focus on members of the Slc25 family. Transcripts of interest were evaluated at the protein level by immunoblotting. Bioinformatics analyzed the expression profiles to delineate comprehensive networks. Chronic exposure to the different metabolic stresses impaired glucose-stimulated insulin secretion; revealing glucotoxicity and lipo-dysfunction. Both saturated and unsaturated fatty acids increased expression of the carnitine/acylcarnitine carrier CAC, whereas the citrate carrier CIC and energy sensor SIRT1 were specifically upregulated by palmitate and oleate, respectively. High glucose upregulated CIC, the dicarboxylate carrier DIC and glutamate carrier GC1. Conversely, it reduced expression of energy sensors (AMPK, SIRT1, SIRT4), metabolic genes, transcription factor PDX1, and anti-apoptotic Bcl2. This was associated with caspase-3 cleavage and cell death. Expression levels of GC1 and SIRT4 exhibited positive and negative glucose dose-response, respectively. Expression profiles of energy sensors and mitochondrial carriers were selectively modified by the different conditions, exhibiting stress-specific signatures.

Background: Agenesis of corpus callosum has been associated with several defects of the mitochondrial respiratory chain and the citric acid cycle. We now report the results of the biochemical and molecular studies of a patient with severe neurodevelopmental disease manifesting by agenesis of corpus callosum and optic nerve hypoplasia., Methods and Results: A mitochondrial disease was suspected in this patient based on the prominent excretion of 2-hydroxyglutaric acid and Krebs cycle intermediates in urine and the finding of increased reactive oxygen species content and decreased mitochondrial membrane potential in her fibroblasts. Whole exome sequencing disclosed compound heterozygosity for two pathogenic variants in the SLC25A1 gene, encoding the mitochondrial citrate transporter. These variants, G130D and R282H, segregated in the family and were extremely rare in controls. The mutated residues were highly conserved throughout evolution and in silico modeling investigations indicated that the mutations would have a deleterious effect on protein function, affecting either substrate binding to the transporter or its translocation mechanism. These predictions were validated by the observation that a yeast strain harbouring the mutations at equivalent positions in the orthologous protein exhibited a growth defect under stress conditions and by the loss of activity of citrate transport by the mutated proteins reconstituted into liposomes., Conclusions: We report for the first time a patient with a mitochondrial citrate carrier deficiency. Our data support a role for citric acid cycle defects in agenesis of corpus callosum as already reported in patients with aconitase or fumarate hydratase deficiency.

The essential cofactors CoA, FAD and NAD+ are synthesized outside the peroxisomes and therefore must be transported into the peroxisomal matrix where they are required for important processes. In the present study we have functionally identified and characterized SLC25A17 (solute carrier family 25 member 17), which is the only member of the mitochondrial carrier family that has previously been shown to be localized in the peroxisomal membrane. Recombinant and purified SLC25A17 was reconstituted into liposomes. Its transport properties and kinetic parameters demonstrate that SLC25A17 is a transporter of CoA, FAD, FMN and AMP, and to a lesser extent of NAD+, PAP (adenosine 3',5'-diphosphate) and ADP. SLC25A17 functioned almost exclusively by a counter-exchange mechanism, was saturable and was inhibited by pyridoxal 5'-phosphate and other mitochondrial carrier inhibitors. It was expressed to various degrees in all of the human tissues examined. Its main function is probably to transport free CoA, FAD and NAD+ into peroxisomes in exchange for intraperoxisomally generated PAP, FMN and AMP. The present paper is the first report describing the identification and characterization of a transporter for multiple free cofactors in peroxisomes.

The transcription factor Sp1 regulates expression of numerous genes involved in many cellular processes. Different post-transcriptional modifications can influence the transcriptional control activity and stability of Sp1. In addition to these modifications, alternative splicing isoforms may also be the basis of its distinct functional activities. In this study, we identified a novel alternative splice isoform of Sp1 named Sp1c. This variant is generated by exclusion of a short domain, which we designate α, through alternative splice acceptor site usage in the exon 3. The existence of this new isoform was confirmed in vivo by Western blotting analysis. Although at very low levels, Sp1c is ubiquitously expressed, as seen in its full-length Sp1. A preliminary characterization of Sp1c shows that: (a) Sp1c works as stronger activator of transcription than full-length Sp1; (b) percentage of HEK293 Sp1c-overexpressing cells is higher in G1 phase and lower in S phase than percentage of HEK293 Sp1-overexpressing cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP(+) and GSH/GSSG ratios in the cytosol of DeltaYHM2 cells as well as an increase in the NADPH/NADP(+) ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the DeltaYHM2 strain and more so by the DeltaYHM2DeltaZWF1 strain upon H(2)O(2) exposure, implying that Yhm2p has an antioxidant function.

Autism is a severe developmental disorder, whose pathogenetic underpinnings are still largely unknown. Temporocortical gray matter from six matched patient-control pairs was used to perform post-mortem biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier (AGC), which participates in the aspartate/malate reduced nicotinamide adenine dinucleotide shuttle and is physiologically activated by calcium (Ca(2+)). AGC transport rates were significantly higher in tissue homogenates from all six patients, including those with no history of seizures and with normal electroencephalograms prior to death. This increase was consistently blunted by the Ca(2+) chelator ethylene glycol tetraacetic acid; neocortical Ca(2+) levels were significantly higher in all six patients; no difference in AGC transport rates was found in isolated mitochondria from patients and controls following removal of the Ca(2+)-containing postmitochondrial supernatant. Expression of AGC1, the predominant AGC isoform in brain, and cytochrome c oxidase activity were both increased in autistic patients, indicating an activation of mitochondrial metabolism. Furthermore, oxidized mitochondrial proteins were markedly increased in four of the six patients. Variants of the AGC1-encoding SLC25A12 gene were neither correlated with AGC activation nor associated with autism-spectrum disorders in 309 simplex and 17 multiplex families, whereas some unaffected siblings may carry a protective gene variant. Therefore, excessive Ca(2+) levels are responsible for boosting AGC activity, mitochondrial metabolism and, to a more variable degree, oxidative stress in autistic brains. AGC and altered Ca(2+) homeostasis play a key interactive role in the cascade of signaling events leading to autism: their modulation could provide new preventive and therapeutic strategies.

In this study, we investigated the effects of statins, fibrates, 9-cis-retinoic acid and forskolin on the transcription of the mitochondrial carnitine/acylcarnitine carrier (CAC) gene. Statins, fibrates, retinoic acid and forskolin activate luciferase gene reporter activity driven by the -334/+3 bp region of the human CAC promoter containing wild-type (but not mutated) PPRE. These four agents also increase CAC transcript and protein levels. The combinations of statins and fibrates, retinoic acid and fibrates and fibrates and forskolin act synergistically. Mevalonate abolishes the activation of CAC gene expression by statins; the inhibitor of the PKA pathway H89 suppresses the stimulation of CAC gene expression by forskolin. Because CAC is essential for fatty acid beta-oxidation, the above results on the regulation of CAC gene expression provide a novel contribution to the understanding of the hypolipidemic action of statins, fibrates and retinoic acid.

Ca2+ concentration in peroxisomal matrix ([Ca2+](perox)) has been monitored dynamically in mammalian cells expressing variants of Ca2+-sensitive aequorin specifically targeted to peroxisomes. Upon stimulation with agonists that induce Ca2+ release from intracellular stores, peroxisomes transiently take up Ca2+ reaching peak values in the lumen as high as 50-100 microm, depending on cell types. Also in resting cells, peroxisomes sustain a Ca2+ gradient, [Ca2+](perox) being approximately 20-fold higher than [Ca2+] in the cytosol ([Ca2+](cyt)). The properties of Ca2+ traffic across the peroxisomal membrane are different from those reported for other subcellular organelles. The sensitivity of peroxisomal Ca2+ uptake to agents dissipating H+ and Na+ gradients unravels the existence of a complex bioenergetic framework including V-ATPase, Ca2+/H+, and Ca2+/Na+ activities whose components are yet to be identified at a molecular level. The different [Ca2+](perox) of resting and stimulated cells suggest that Ca2+ could play an important role in the regulation of peroxisomal metabolism.

The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.

Multiple mitochondrial DNA deletions are associated with clinically heterogeneous disorders transmitted as mendelian traits. Dominant missense mutations were found in the gene encoding the heart and skeletal muscle-specific isoform of the adenine nucleotide translocator (ANT1) in families with autosomal dominant progressive external opthalmoplegia and in a sporadic patient. We herein report on a sporadic patient who presented with hypertrophic cardiomyopathy, mild myopathy with exercise intolerance and lactic acidosis but no ophthalmoplegia. A muscle biopsy showed the presence of numerous ragged-red fibers, and Southern blot analysis disclosed multiple deletions of muscle mitochondrial DNA. Molecular analysis revealed a C to A homozygous mutation at nucleotide 368 of the ANT1 gene. The mutation converted a highly conserved alanine into an aspartic acid at codon 123 and was absent in 500 control individuals. This is the first report of a recessive mutation in the ANT1 gene. The clinical and biochemical features are different from those found in dominant ANT1 mutations, resembling those described in ANT1 knockout mice. No ATP uptake was measured in proteoliposomes reconstituted with protein extracts from the patient's muscle. The equivalent mutation in AAC2, the yeast ortholog of human ANT1, resulted in a complete loss of transport activity and in the inability to rescue the severe Oxidative Phosphorylation phenotype displayed by WB-12, an AAC1/AAC2 defective strain. Interestingly, exposure to reactive oxygen species (ROS) scavengers dramatically increased the viability of the WB-12 transformant, suggesting that increased redox stress is involved in the pathogenesis of the disease and that anti-ROS therapy may be beneficial to patients.

The mitochondrial ADP/ATP carriers (AACs) catalyze the exchange of cytosolic ADP for matrix ATP. We have identified and characterized a novel member of the AAC subfamily of mitochondrial metabolite transport proteins, termed AAC4. The AAC4 gene maps to human chromosome 4q28.1, and its product AAC4 is 66-68% identical to human AAC 1-3 and is localized to mitochondria. AAC4 transcripts are exclusively present in liver, testis and brain unlike those of AAC 1-3. Consistent with its belonging to the AAC subfamily, upon heterologous expression and reconstitution into liposomes AAC4 exchanges ADP for ATP by an electrogenic antiport mechanism with high specificity and high sensitivity to carboxyatractyloside and bongkrekic acid.

The yeast peroxisomal adenine nucleotide carrier, Ant1p, was shown to catalyse unidirectional transport in addition to exchange of substrates. In both transport modes, proton movement occurs. Nucleotide hetero-exchange is H+-compensated and electroneutral. Furthermore, microscopic fluorescence imaging of a pH-sensitive green fluorescent protein targeted to peroxisomes shows that Ant1p is involved in the formation of a DeltapH across the peroxisomal membrane, acidic inside.

Autosomal dominant and recessive forms of progressive external ophthalmoplegia (adPEO and arPEO) are mitochondrial disorders characterized by the presence of multiple deletions of mitochondrial DNA in affected tissues. Four adPEO-associated missense mutations have been identified in the ANT1 gene. In order to investigate their functional consequences on cellular physiology, we introduced three of them at equivalent positions in AAC2, the yeast orthologue of human ANT1. We demonstrate here that expression of the equivalent mutations in aac2-defective haploid strains of Saccharomyces cerevisiae results in (a) a marked growth defect on non-fermentable carbon sources, and (b) a concurrent reduction of the amount of mitochondrial cytochromes, cytochrome c oxidase activity and cellular respiration. The efficiency of ATP and ADP transport was variably affected by the different AAC2 mutations. However, irrespective of the absolute level of activity, the AAC2 pathogenic mutants showed a significant defect in ADP versus ATP transport compared with wild-type AAC2. In order to study whether a dominant phenotype, as in humans, could be observed, the aac2 mutant alleles were also inserted in combination with the endogenous wild-type AAC2 gene. The heteroallelic strains behaved as recessive for oxidative growth and petite-negative phenotype. In contrast, reduction in cytochrome content and increased mtDNA instability appeared to behave as dominant traits in heteroallelic strains. Our results indicate that S. cerevisiae is a suitable in vivo model to study the pathogenicity of the human ANT1 mutations and the pathophysiology leading to impairment of oxidative phosphorylation and damage of mtDNA integrity, as found in adPEO.

The requirement for small molecule transport systems across the peroxisomal membrane has previously been postulated, but not directly proven. Here we report the identification and functional reconstitution of Ant1p (Ypr128cp), a peroxisomal transporter in the yeast Saccharomyces cerevisiae, which has the characteristic sequence features of the mitochondrial carrier family. Ant1p was found to be an integral protein of the peroxisomal membrane and expression of ANT1 was oleic acid inducible. Targeting of Ant1p to peroxisomes was dependent on Pex3p and Pex19p, two peroxins specifically required for peroxisomal membrane protein insertion. Ant1p was essential for growth on medium-chain fatty acids as the sole carbon source. Upon reconstitution of the overexpressed and purified protein into liposomes, specific transport of adenine nucleotides could be demonstrated. Remarkably, both the substrate and inhibitor specificity differed from those of the mitochondrial ADP/ATP transporter. The physiological role of Ant1p in S.cerevisiae is probably to transport cytoplasmic ATP into the peroxisomal lumen in exchange for AMP generated in the activation of fatty acids.