Mitochondrial carriers (MCs) form a large family of nuclear-encoded transporters embedded in the inner mitochondrial membrane and in a few cases in other organelle membranes (Palmieri, 2013). The members of this superfamily are widespread in eukaryotes and involved in numerous metabolic pathways and cell functions. They can be easily recognized by their striking sequence features, i.e., a tripartite structure, six transmembrane α-helices and a 3-fold repeated signature motifs. Members of the family vary greatly in the nature and size of their transported substrates, modes of transport (i.e., uniport, symport or antiport) and driving forces, although the molecular mechanism of substrate translocation may be basically the same. In recent years mutations in the MC genes have been shown to be responsible for 11 diseases (Palmieri, 2013), highlighting the important role of MCs in metabolism. MC impairing mutations affect three main regions crucial for substrate translocation. A first group of mutations affects MC conformational changes and locates at PG levels or at the aromatic belts (Pierri et al., 2013). A second group of mutations affects substrate specificity and locates at the common substrate binding site (Robinson et al., 2008) and at the substrate binding area (Pierri et al., 2013). A further group of mutations locate at residues of the m-/c-gates (Palmieri et al., 2013; Robinson et al., 2008) and at residues of the m-gate area (Pierri et al. 2013). For this last group of mutations, it appears difficult to establish if the impaired function is due to the lack of substrate specificity (or substrate recognition) or to the wrong triggering of conformational changes. Two mutations, one at the PG level 1 and one at the common substrate binding site, impairing citrate translocation within SLC25A1_CTP protein are presented. The two mutations are found to be responsible of agenesis of corpus callosum and optic nerve hypoplasia (Edvardson et al., 2013). References 1. Palmieri F. The mitochondrial transporter family SLC25: identification, properties and physiopathology. Mol Aspects Med. 2013;34:465. 2. Pierri CL, Palmieri F, De Grassi A. Single-nucleotide evolution quantifies the importance of each site along the structure of mitochondrial carriers. Cell Mol Life Sci. 2013. 3. Robinson AJ, Overy C, Kunji ER. The mechanism of transport by mitochondrial carriers based on analysis of symmetry. Proc Natl Acad Sci U S A. 2008;105:17766. 4. Edvardson S, Porcelli V, Jalas C, Soiferman D, Kellner Y, Shaag A, Korman SH, Pierri CL, Scarcia P, Fraenkel ND, Segel R, Schechter A, Frumkin A, Pines O, Saada A, Palmieri L, Elpeleg O. Agenesis of corpus callosum and optic nerve hypoplasia due to mutations in SLC25A1 encoding the mitochondrial citrate transporter. J Med Genet. 2013;50:240.

The mitochondrial citrate-malate exchanger (CIC), a known target of acetylation, is up-regulated in activated immune cells and plays a key role in the production of inflammatory mediators. However, the role of acetylation in CIC activity is elusive. We show that CIC is acetylated in activated primary human macrophages and U937 cells and the level of acetylation is higher in glucose-deprived compared to normal glucose medium. Acetylation enhances CIC transport activity, leading to a higher citrate efflux from mitochondria in exchange with malate. Cytosolic citrate levels do not increase upon activation of cells grown in deprived compared to normal glucose media, indicating that citrate, transported from mitochondria at higher rates from acetylated CIC, is consumed at higher rates. Malate levels in the cytosol are lower in activated cells grown in glucose-deprived compared to normal glucose medium, indicating that this TCA intermediate is rapidly recycled back into the cytosol where it is used by the malic enzyme. Additionally, in activated cells CIC inhibition increases the NADP+/NADPH ratio in glucose-deprived cells; this ratio is unchanged in glucose-rich grown cells due to the activity of the pentose phosphate pathway. Consistently, the NADPH-producing isocitrate dehydrogenase level is higher in activated glucose-deprived as compared to glucose rich cells. These results demonstrate that, in the absence of glucose, activated macrophages increase CIC acetylation to enhance citrate efflux from mitochondria not only to produce inflammatory mediators but also to meet the NADPH demand through the actions of isocitrate dehydrogenase and malic enzyme.

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.

CoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported here and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact.

Grape berries (Vitis vinifera L fruit) exhibit a double-sigmoid pattern of development that results from two successive periods of vacuolar swelling during which the nature of accumulated solutes changes significantly. Throughout the first period, called green or herbaceous stage, berries accumulate high levels of organic acids, mainly malate and tartrate. At the cellular level fruit acidity comprises both metabolism and vacuolar storage. Malic acid compartmentation is critical for optimal functioning of cytosolic enzymes. Therefore, the identification and characterization of the carriers involved in malate transport across sub-cellular compartments is of great importance. The decrease in acid content during grape berry ripening has been mainly associated to mitochondrial malate oxidation. However, no Vitis vinifera mitochondrial carrier involved in malate transport has been reported to date. Here we describe the identification of three V. vinifera mitochondrial dicarboxylate/tricarboxylate carriers (VvDTC1-3) putatively involved in mitochondrial malate, citrate and other di/tricarboxylates transport. The three VvDTCs are very similar, sharing a percentage of identical residues of at least 83 %. Expression analysis of the encoding VvDTC genes in grape berries shows that they are differentially regulated exhibiting a developmental pattern of expression. The simultaneous high expression of both VvDTC2 and VvDTC3 in grape berry mesocarp close to the onset of ripening suggests that these carriers might be involved in the transport of malate into mitochondria.

Successful prediction of protein folding from an amino acid sequence is a challenge in computational biology. In order to reveal the geometric constraints that drive protein folding, highlight those constraints kept or missed by distinct lattices and for establishing which class of intra- and inter-secondary structure element interactions is the most relevant for the correct folding of proteins, we have calculated inter-alpha carbon distances in a set of 42 crystal structures consisting of mainly helix, sheet or mixed conformations. The inter-alpha carbon distances were also calculated in several lattice “hydrophobic-polar” models built from the same protein set. We found that helix structures are more prone to form “hydrophobic–hydrophobic” contacts than beta-sheet structures. At a distance lower than or equal to 3.8 Å (very short-range interactions), “hydrophobic–hydrophobic” contacts are almost absent in the native structures, while they are frequent in all the analyzed lattice models. At distances in-between 3.8 and 9.5 Å (short-/medium-range interactions), the best performing lattice for reproducing mainly helix structures is the body-centered-cubic lattice. If protein structures contain sheet portions, lattice performances get worse, with few exceptions observed for double-tetrahedral and body-centered-cubic lattices. Finally, we can observe that ab initio protein folding algorithms, i.e. those based on the employment of lattices and Monte Carlo simulated annealings, can be improved simply and effectively by preventing the generation of “hydrophobic–hydrophobic” contacts shorter than 3.8 Å, by monitoring the “hydrophobic–hydrophobic/polar–polar” contact ratio in short-/medium distance ranges and by using preferentially a body-centered-cubic lattice.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) initiates a stress response mechanism to clear out the unfolded proteins by either facilitating their re-folding or inducing their degradation. When this fails, an apoptotic cascade is initiated so that the affected cell is eliminated. IRE1α is a critical sensor of the unfolded-protein response, essential for initiating the apoptotic signaling. Here, we report an infantile neurodegenerative disorder associated with enhanced activation of IRE1α and increased apoptosis. Three unrelated affected individuals with congenital microcephaly, infantile epileptic encephalopathy, and profound developmental delay were found to carry heterozygous variants (c.932T>C [p.Leu311Ser] or c.935T>C [p.Leu312Pro]) in RNF13, which codes for an IRE1α-interacting protein. Structural modeling predicted that the variants, located on the surface of the protein, would not alter overall protein folding. Accordingly, the abundance of RNF13 and IRE1α was not altered in affected individuals' cells. However, both IRE1α-mediated stress signaling and stress-induced apoptosis were increased in affected individuals' cells. These results indicate that the RNF13 variants confer gain of function to the encoded protein and thereby lead to altered signaling of the ER stress response associated with severe neurodegeneration in infancy.

Peroxisomes are small organelles found in all eukaryotes, involved in a number of important metabolic pathways, including fatty acid α- and β-oxidation, biosynthesis of ether phospholipids and bile acids, and the degradation of purines, amino acids and polyamines. The functional role of the peroxisomal membrane as a permeability barrier to substrates and cofactors has been controversial for many years. The essential cofactors CoA, FAD and NAD+ are synthesized outside the peroxisomes and must be transported into the peroxisomal matrix where they are required for important processes. SLC25A17 (solute carrier family 25 member 17) is the only member of the mitochondrial carrier family that has 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. Moreover 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 [1]. The plant homologue of SLC25A17 is the peroxisomal protein PXN encoded by the Arabidopsis gene At2g39970 which has recently been found to transport NAD+, NADH, AMP and ADP [2]. Upon heterologous expression of PXN in bacteria followed by purification and reconstitution in liposomes, uptake and efflux experiments revealed that PXN transports coenzyme A (CoA), dephospho-CoA, acetyl-CoA and adenosine 3', 5'-phosphate (PAP), besides NAD+, NADH, AMP and ADP. PXN catalyzed fast counter-exchange of substrates and much slower uniport. Transport was saturable with a submillimolar affinity for NAD+, CoA and other substrates. The physiological role of PXN is probably to provide the peroxisomes with the essential coenzymes NAD+ and CoA [3]. [1] G. Agrimi, A. Russo, P. Scarcia, F. Palmieri, The human gene SLC25A17 encodes a peroxisomal transporter of coenzyme A, FAD and NAD+, Biochem. J., 443 (2012) 241–247.

A pool of twelve cDNA sequences coding for Bowman-Birk inhibitors (BBIs) was identified in the legume grass pea (Lathyrus sativus L.). The corresponding amino acid sequences showed a canonical first anti-trypsin domain, predicted according to the identity of the determinant residue P(1). A more variable second binding loop was observed allowing to identify three groups based on the identity of residue P(1): two groups (Ls_BBI_1 and Ls_BBI_2) carried a second reactive site specific for chymotrypsin, while a third group (Ls_BBI_3) was predicted to inhibit elastase. A fourth variant carrying an Asp in the P(1) position of the second reactive site was identified only from genomic DNA. A phylogenetic tree constructed using grass pea BBIs with their homologs from other legume species revealed grouping based on taxonomy and on specificity of the reactive sites. Five BBI sequences, representing five different second reactive sites, were heterologously expressed in the yeast Pichia pastoris. The recombinant proteins demonstrated to be active against trypsin, while three of them were also active against chymotrypsin, and one against human leukocyte elastase. Comparative modeling and protein docking were used to further investigate interactions between two grass pea BBI isoforms and their target proteases. Thus two reliable 3D models have been proposed, representing two potential ternary complexes, each constituted of an inhibitor and its target enzymes.

The flux of a variety of metabolites, nucleotides and coenzymes across the inner membrane of mitochondria is catalysed by a nuclear-coded superfamily of secondary transport proteins called mitochondrial carriers (MCs) [1]. The importance of MCs is demonstrated by their wide distribution in all eukaryotes, their role in numerous metabolic pathways and cell functions with different tissuespecific expression patterns, and the identification of several diseases caused by alterations of their genes [2]. Until now, 22 MC subfamilies have been functionally characterized, mainly by transport assays upon heterologous gene expression, purification and reconstitution into liposomes [1]. In particular two well characterized MC subfamilies are known to play a crucial role in activating the mitochondrial apoptotic pathway, the first is the subfamily of the ADP/ATP carriers and the second is the subfamily of the citrate carrier. ADP/ATP carriers catalyze the efflux of ATP from the mitochondrial matrix in exchange for cytosolic ADP and their specific inhibition can lead the permeability transition pore opening in case of oxidative stress [3]. Citrate carrier catalyses the efflux of citrate from the mitochondrial matrix in exchange for cytosolic malate and plays a key role in inflammation [4,5]. Our data together with literature data let us suppose that these two MC subfamilies are promising molecular targets for cancer treatment. In particular basing on our knowledge of MC structure, translocation mechanism and substrate specificity [6] we are evaluating neuroendocrine cancer cell resistance to old MC inhibitors and we are screening chemical libraries to develop new specific drugs to be used for viability assays.

Since the end of nineties numerous mitochondrial diseases have been found to be related to mutations in nuclear genes encoding mitochondrial carriers, a family of proteins that shuttle a variety of metabolites across the mitochondrial membrane. To date eleven disorders are known to be caused by defects of mitochondrial carriers. Mutations of mitochondrial carrier genes are responsible for carnitine/acylcarnitine carrier deficiency, ornithine carrier deficiency (HHH syndrome), aspartate/glutamate isoform 1 deficiency (global cerebral hypomyelination), aspartate/glutamate isoform 2 deficiency (CTLN2 and NICCD), amish microcephaly, neonatal myoclonic epilepsy, congenital sideroblastic anemia, PiC deficiency, ADP/ATP carrier isoform 1 deficiency and involved in neuropathy and bilateral striatal necrosis and adPEO (autosomal dominant progressive external ophthalmoplegia). We propose un updated overview of these diseases. We shall also discuss the role of missense mutations in impairing mitochondrial carrier function and the consequent severe damage to the mitochondrial matrix supply with substrates destined to specific metabolic pathways. Despite the substantial progress that has been made in our understanding of the molecular bases of mitochondrial carrier associated diseases, specific pharmacological therapies are not yet available. Current therapies are symptomatic and usually based on specific dietary measures. New therapeutic approaches are under investigation for some of these diseases. For further reading Palmieri F. (2008) Diseases caused by defects of mitochondrial carriers: a review. Biochim Biophys Acta; 1777:564-78. Palmieri F, Pierri CL (2010) Structure and function of mitochondrial carriers - Role of the transmembrane helix P and G residues in the gating and transport mechanism. FEBS Lett. 584:1931-9. Tessa A, Fiermonte G, Dionisi-Vici C, Paradies E, Baumgartner MR, Chien YH,Loguercio C, de Baulny HO, Nassogne MC, Schiff M, Deodato F, Parenti G, Rutledge SL, Vilaseca MA, Melone MA, Scarano G, Aldamiz-Echevarría L, Besley G, Walter J, Martinez-Hernandez E, Hernandez JM, Pierri CL, Palmieri F, Santorelli FM. (2009) Identification of novel mutations in the SLC25A15 gene in hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome: a clinical, molecular, and functional study. Human Mutation; 30:741-8. Wibom R, Lasorsa FM, Töhönen V, Barbaro M, Sterky FH, Kucinski T, Naess K, Jonsson M, Pierri CL, Palmieri F, Wedell A. (2009) AGC1 deficiency associated with global cerebral hypomyelination. N Engl J Med.; 361:489-95. Iacobazzi V, Convertini P, Infantino V, Scarcia P, Todisco S, Palmieri F. (2009) Statins, fibrates and retinoic acid upregulate mitochondrial acylcarinitine carrier gene expression. Biochem Biophys Res Commun.; 388:643-7.

The mitochondrial carriers are members of a family of transport proteins that mediate solute transport across the inner mitochondrial membrane. Two isoforms of the glutamate carriers, GC1 and GC2 (encoded by the SLC25A22 and SLC25A18 genes, respectively), have been identified in humans. Two independent mutations in SLC25A22 are associated with severe epileptic encephalopathy. In the present study we show that two genes (CG18347 and CG12201) phylogenetically related to the human GCs encoding genes are present in the D. melanogaster genome. We have functionally characterized the proteins encoded by CG18347 and CG12201, designated as DmGC1p and DmGC2p respectively, by overexpression in Escherichia coli and reconstitution into liposomes. Their transport properties demonstrate that DmGC1p and DmGC2p both catalyze the transport of glutamate across the inner mitochondrial membrane. Computational approaches have been used in order to highlight residues of DmGC1p and DmGC2p involved in substrate binding. Furthermore, gene expression analysis during development and in various adult tissues reveals that CG18347 is ubiquitously expressed in all examined D. melanogaster tissues, while the expression of CG12201 is strongly testis-biased. Finally, we identified mitochondrial glutamate carrier orthologs in 49 eukariotic species in order to attempt the reconstruction of the evolutionary history of the glutamate carrier function. Comparison of the exon/intron structure and other key features of the analyzed orthologs suggest that eukaryotic glutamate carrier genes descend from an intron-rich ancestral gene already present in the common ancestor of lineages that diverged as early as bilateria and radiata.

Therapeutic monoclonal antibodies (mAbs) have high efficacy in treating TNF α-related immunological diseases. Other than neutralizing TNF α, these IgG1 antibodies exert Fc receptor-mediated effector functions such as the complement-dependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC). The crystallizable fragment (Fc) of these IgG1 contains a single glycosylation site at Asn 297/300 that is essential for the CDC and ADCC. Glycosylated antibodies lacking core fucosylation showed an improved ADCC. However, no structural data are available concerning the ligand-binding interaction of these mAbs used in TNF α-related diseases and the role of the fucosylation. We therefore used comparative modeling for generating complete 3D mAb models that include the antigen-binding fragment (Fab) portions of infliximab, complexed with TNF α (4G3Y.pdb), the Fc region of the human IGHG1 fucosylated (3SGJ) and afucosylated (3SGK) complexed with the Fc receptor subtype Fcγ RIIIA, and the Fc region of a murine immunoglobulin (1IGT). After few thousand steps of energy minimization on the resulting 3D mAb models, minimized final models were used to quantify interactions occurring between Fcγ RIIIA and the fucosylated/afucosylated Fc fragments. While fucosylation does not affect Fab-TNF α interactions, we found that in the absence of fucosylation the Fc-mAb domain and Fcγ RIIIA are closer and new strong interactions are established between G129 of the receptor and S301 of the Chimera 2 Fc mAb; new polar interactions are also established between the Chimera 2 Fc residues Y299, N300, and S301 and the Fcγ RIIIA residues K128, G129, R130, and R155. These data help to explain the reduced ADCC observed in the fucosylated mAbs suggesting the specific AA residues involved in binding interactions.

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.

L’agenesi del corpo calloso (ACC) è stata associata a diversi difetti della catena respiratoria mitocondriale e degli enzimi del ciclo dell’acido citrico. In questo studio sono riportati i dati relativi ad una paziente che mostrava un severo difetto del neurosviluppo, caratterizzato da ACC e ipoplasia del nervo ottico. Essa presentava elevati valori di 2-idrossiglutarato e degli intermedi del ciclo di Krebs nelle urine e un incremento delle specie reattive dell’ossigeno (ROS) ed una diminuzione del potenziale di membrana mitocondriale nei fibroblasti. Questi dati suggerivano un alterazione del metabolismo mitocondriale. Mediante il sequenziamento dell’intero esoma della paziente sono state individuate due varanti alleliche del gene SLC25A1, che codifica per il trasportatore mitocondriale del citrato. Tali varianti determinano le mutazioni amminoacidiche G130D e R282H. Le due varianti segregano nella famiglia ma risultano essere estremamente rare nei soggetti controllo, dove sono presenti sempre in eterozigosi con l’allele wild-type. Le analisi in silico della struttura del trasportatore mitocondriale hanno evidenziato che i residui amminoacidici mutati sono molto conservati all’inetrno della famiglia dei carrier mitocondriali (MCF) e potrebbero alterare la funzionalità della proteina. Questo dato predittivo è stato validato studiando la proteina ortologa di lievito sia da un punto di vista fenotipico che funzionale. Infatti il ceppo di lievito che presentava la proteina con le due mutazioni ha mostrato un difetto di crescita in condizioni di stress. Inoltre le proteine mutate ricostituite in vescicole fosfolipidiche mostravano una scarsa capacità di catalizzare il trasporto di citrato. In conclusione, i nostri dati hanno evidenziato un collegamento tra un difetto del gene del carrier mitocondriale del citrato e l’agenesi del corpo calloso.

Haem–copper oxidases are the terminal enzymes in both prokaryotic and eukaryotic respiratory chains. They catalyse the reduction of dioxygen to water and convert redox energy into a transmembrane electrochemical proton gradient during their catalytic activity. Haem–copper oxidases show substantial structure similarity, but spectroscopic and biochemical analyses indicate that these enzymes contain diverse prosthetic groups and use different substrates (i.e. cytochrome c or quinol). Owing to difficulties in membrane protein crystallization, there are no definitive structural data about the quinol oxidase physiological substrate-binding site(s). In the present paper, we propose an atomic structure model for the menaquinol:O2 oxidoreductase of Bacillus subtilis (QOx.aa3). Furthermore, a multistep computational approach is used to predict residues involved in the menaquinol/menaquinone binding within B. subtilis QOx.aa3 as well as those involved in quinol/quinone binding within Escherichia coli QOx.bo3. Two specific sequence motifs, R70GGXDX4RXQX3PX3FX[D/N/E/Q]X2HYNE97 and G159GSPX2GWX2Y169 (B. subtilis numbering), were highlighted within QOx from Bacillales. Specific residues within the first and the second sequencemotif participate in the high- and low-affinity substrate-binding sites respectively. Using comparative analysis, two analogous motifs, R71GFXDX4RXQX8[Y/F]XPPHHYDQ101 and G163EFX3GWX2Y173 (E. coli numbering) were proposed to be involved in Enterobacteriales/Rhodobacterales/Rhodospirillales QOx high- and low-affinity quinol-derivative-binding sites. Results and models are discussed in the context of the literature.

Dihydrolipoamide dehydrogenase (DLD, E3) is a flavoprotein common to pyruvate, α-ketoglutarate and branched-chain α-keto acid dehydrogenases. We found two novel DLD mutations (p.I40Lfs*4; p.G461E) in a 19year-old patient with lactic acidosis and a complex amino- and organic aciduria consistent with DLD deficiency, manifesting progressive exertional fatigue. Muscle biopsy showed mitochondrial proliferation and lack of DLD cross-reacting material. Riboflavin supplementation determined the complete resolution of exercise intolerance with the partial restoration of the DLD protein and disappearance of mitochondrial proliferation in the muscle. Morphological and functional studies support the riboflavin chaperon-like role in stabilizing DLD protein with rescue of its expression in the muscle.

Mitochondrial carriers are membrane-embedded proteins consisting of a tripartite structure, a three-fold pseudo-symmetry, related sequences, and similar folding whose main function is to catalyze the transport of various metabolites, nucleotides, and coenzymes across the inner mitochondrial membrane. In this study, the evolutionary rate in vertebrates was screened at each of the approximately 50,000 nucleotides corresponding to the amino acids of the 53 human mitochondrial carriers. Using this information as a starting point, a scoring system was developed to quantify the evolutionary pressure acting on each site of the common mitochondrial carrier structure and estimate its functional or structural relevance. The degree of evolutionary selection varied greatly among all sites, but it was highly similar among the three symmetric positions in the tripartite structure, known as symmetry-related sites or triplets, suggesting that each triplet constitutes an evolutionary unit. Based on evolutionary selection, 111 structural sites (37 triplets) were found to be important. These sites play a key role in structure/function of mitochondrial carriers and are involved in either conformational changes (sites of the gates, proline-glycine levels, and aromatic belts) or in binding and specificity of the transported substrates (sites of the substrate-binding area in between the two gates). Furthermore, the evolutionary pressure analysis revealed that the matrix short helix sites underwent different degrees of selection with high inter-paralog variability. Evidence is presented that these sites form a new sequence motif in a subset of mitochondrial carriers, including the ADP/ATP translocator, and play a regulatory function by interacting with ligands and/or proteins of the mitochondrial matrix.

Lon is a mitochondrial protease that degrades oxidized damaged proteins, assists protein folding and participates in maintaining mitochondrial DNA levels. Changes in Lon mRNA levels, protein levels and activity are not always directly correlated, suggesting that Lon could be regulated at post translational level. We found that Lon and SIRT3, the most important mitochondrial sirtuin, colocalize and coimmunoprecipitate in breast cancer cells, and silencing or inhibition of Lon did not alter SIRT3 levels. Silencing of SIRT3 increased the levels of Lon protein and of its acetylation, suggesting that Lon is a target of SIRT3, likely at K917.

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 to 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 to 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 peroxisomal protein PXN encoded by the Arabidopsis gene At2g39970 has very recently been found to transport NAD(+), NADH, AMP and ADP. In this work we have reinvestigated the substrate specificity and the transport properties of PXN by using a wide range of potential substrates. Heterologous expression in bacteria followed by purification, reconstitution in liposomes, and uptake and efflux experiments revealed that PNX transports coenzyme A (CoA), dephospho-CoA, acetyl-CoA and adenosine 3', 5'-phosphate (PAP), besides NAD(+), NADH, AMP and ADP. PXN catalyzed fast counter-exchange of substrates and much slower uniport and was strongly inhibited by pyridoxal 5'-phosphate, bathophenanthroline and tannic acid. Transport was saturable with a submillimolar affinity for NAD(+), CoA and other substrates. The physiological role of PXN is probably to provide the peroxisomes with the essential coenzymes NAD(+) and CoA.

The inner mitochondrial membrane contains a superfamily of proteins, called mitochondrial carriers (MCs), which transport several metabolites into and out of the mitochondrial matrix. As observed in the ADP/ATP carrier structure, crystallized in complex with its powerful inhibitor carboxyatractyloside, the main structural fold of the MCs consists of a barrel of six transmembrane α-helices whose charged surfaces form the wall of a water-filled cavity. Multiple sequence alignment and 3D comparative models of mitochondrial carriers of known function have recently allowed the identification of i) a similarly located binding site located in the carrier cavity, ii) two ion pair networks or gates that are on the matrix or the cytosolic side of the carrier molecules, and iii) two Pro-Gly levels above and below the substrate binding site. As a result of the substrate–protein interactions, ‘hinged helix movements’ consisting of a tilt of the entire helical segments and a kink/swivel of the helical termini at the level of their Pro and Gly have been proposed to be fundamental for the alternative opening and closure of the gates on the matrix or the cytosolic side and thus for the translocation mechanism. The key role of residues of the binding site, gates and Pro-Gly levels in substrate translocation is supported by the localization of most missense mutations found in patients affected by diseases associated to mitochondrial carriers. References Klingenberg M (2007 ) Transport viewed as a catalytic process. Biochimie. 89:1042-8. Palmieri F (2008) Diseases caused by defects of mitochondrial carriers: a review. Biochim Biophys Acta 1777: 564-57 Palmieri F, Pierri CL (2010) Structure and function of mitochondrial carriers - Role of the transmembrane helix P and G residues in the gating and transport mechanism. FEBS Lett. 584:1931-9 Pebay-Peyroula E, Dahout-Gonzalez C, Kahn R, Trézéguet V, Lauquin GJ, Brandolin G. (2003) Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature. 426:39-44 Robinson AJ, Kunji ER. (2006) Mitochondrial carriers in the cytoplasmic state have a common substrate binding site. Proc Natl Acad Sci U S A. 103:2617-22 Robinson AJ, Overy C, Kunji ER. (2008) The mechanism of transport by mitochondrial carriers based on analysis of symmetry. Proc Natl Acad Sci U S A. 105:17766-71 Wibom R, Lasorsa F, Töhönen V, Barbaro M, Sterky F, Kucinski T, Naess K, Jonsson M, Pierri CL, Palmieri F, Wedell A (2009) AGC1 deficiency associated with global cerebral hypomyelination. N Engl J Med 361: 489-495

Nitric oxide (NO) is thought to have a role in the pathogenesis of achalasia. We performed a genetic analysis of 2 siblings with infant-onset achalasia. Exome analysis revealed that they were homozygous for a premature stop codon in the gene encoding nitric oxide synthase 1 (NOS1). Kinetic analyses and molecular modeling showed that the truncated protein product has defects in folding, NO production, and binding of cofactors. Heller myotomy had no effect in these patients, but sildenafil therapy increased their ability to drink. The finding recapitulates the previously reported phenotype of NOS1-deficient mice, which have achalasia. NO signaling appears to be involved in the pathogenesis of achalasia in humans.