Ciro Leonardo Pierri

Evolution, structure and function of mitochondrial carriers: a review with new insights

The mitochondrial carriers (MC) constitute a large family (MCF) of inner membrane transporters displaying different substrate specificities, patterns of gene expression and even non-mitochondrial organelle localization. In Arabidopsis thaliana 58 genes encode these six trans-membrane domain proteins. The number in other sequenced plant genomes varies from 37 to 125, thus being larger than that of Saccharomyces cerevisiae and comparable with that of Homo sapiens. In addition to displaying highly similar secondary structures, the proteins of the MCF can be subdivided into subfamilies on the basis of substrate specificity and the presence of specific symmetry-related amino acid triplets. We assessed the predictive power of these triplets by comparing predictions with experimentally determined data for Arabidopsis MCs, and applied these predictions to the not yet functionally characterized mitochondrial carriers of the grass, Brachypodium distachyon, and the alga, Ostreococcus lucimarinus. We additionally studied evolutionary aspects of the plant MCF by comparing sequence data of the Arabidopsis MCF with those of Saccharomyces cerevisiae and Homo sapiens, then with those of Brachypodium distachyon and Ostreococcus lucimarinus, employing intra- and inter-genome comparisons. Finally, we discussed the importance of the approaches of global gene expression analysis and in vivo characterizations in order to address the relevance of these vital carrier proteins.

Mitochondrial metabolite transport

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 MCs (mitochondrial carriers). The importance of MCs is demonstrated by their wide distribution in all eukaryotes, their role in numerous metabolic pathways and cell functions, and the identification of several diseases caused by alterations of their genes. MCs can easily be recognized in databases thanks to their striking sequence features. Until now, 22 MC subfamilies, which are well conserved throughout evolution, have been functionally characterized, mainly by transport assays upon heterologous gene expression, purification and reconstitution into liposomes. Given the significant sequence conservation, it is thought that all MCs use the same basic transport mechanism, although they exhibit different modes of transport and driving forces and their substrates vary in nature and size. Based on substrate specificity, sequence conservation and carrier homology models, progress has recently been made in understanding the transport mechanism of MCs by new insights concerning the existence of a substrate-binding site in the carrier cavity, of cytosolic and matrix gates and conserved proline and glycine residues in each of the six transmembrane alpha-helices. These structural properties are believed to play an important role in the conformational changes required for substrate translocation.

AGC1 Deficiency Associated with Global Cerebral Hypomyelination

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), specific to neurons and muscle, supplies aspartate to the cytosol and, as a component of the malate-aspartate shuttle, enables mitochondrial oxidation of cytosolic NADH, thought to be important in providing energy for neurons in the central nervous system. We describe AGC1 deficiency, a novel syndrome characterized by arrested psychomotor development, hypotonia, and seizures in a child with a homozygous missense mutation in the solute carrier family 25, member 12, gene SLC25A12, which encodes the AGC1 protein. Functional analysis of the mutant AGC1 protein showed abolished activity. The child had global hypomyelination in the cerebral hemispheres, suggesting that impaired efflux of aspartate from neuronal mitochondria prevents normal myelin formation.

Structure and function of mitochondrial carriers - Role of the transmembrane helix P and G residues in the gating and transport mechanism

To date, 22 mitochondrial carrier subfamilies have been functionally identified based on substrate specificity. Structural, functional and bioinformatics studies have pointed to the existence in the mitochondrial carrier superfamily of a substrate‐binding site in the internal carrier cavity, of two salt‐bridge networks or gates that close the cavity alternatively on the matrix or the cytosolic side of the membrane, and of conserved prolines and glycines in the transmembrane α‐helices. The significance of these properties in the structural changes occurring during the catalytic substrate translocation cycle are discussed within the context of a transport mechanism model. Most experimentally produced and disease‐causing missense mutations concern carrier regions corresponding to the substrate‐binding site, the two gates and the conserved prolines and glycines.

Novel Hydroxycinnamoyl-Coenzyme A Quinate Transferase Genes from Artichoke Are Involved in the Synthesis of Chlorogenic Acid

Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes.

Computational approaches for protein function prediction: A combined strategy from multiple sequence alignment to molecular docking-based virtual screening

The functional characterization of proteins represents a daily challenge for biochemical, medical and computational sciences. Although finally proved on the bench, the function of a protein can be successfully predicted by computational approaches that drive the further experimental assays. Current methods for comparative modeling allow the construction of accurate 3D models for proteins of unknown structure, provided that a crystal structure of a homologous protein is available. Binding regions can be proposed by using binding site predictors, data inferred from homologous crystal structures, and data provided from a careful interpretation of the multiple sequence alignment of the investigated protein and its homologs. Once the location of a binding site has been proposed, chemical ligands that have a high likelihood of binding can be identified by using ligand docking and structure-based virtual screening of chemical libraries. Most docking algorithms allow building a list sorted by energy of the lowest energy docking configuration for each ligand of the library. In this review the state-of-the-art of computational approaches in 3D protein comparative modeling and in the study of protein-ligand interactions is provided. Furthermore a possible combined/concerted multistep strategy for protein function prediction, based on multiple sequence alignment, comparative modeling, binding region prediction, and structure-based virtual screening of chemical libraries, is described by using suitable examples. As practical examples, Abl-kinase molecular modeling studies, HPV-E6 protein multiple sequence alignment analysis, and some other model docking-based characterization reports are briefly described to highlight the importance of computational approaches in protein function prediction.

Identification of novel mutations in the SLC25A15 gene in hyperornithinemia‐hyperammonemia‐homocitrullinuria (HHH) syndrome: A clinical, molecular, and functional study

Hyperornithinemia‐hyperammonemia‐homocitrullinuria (HHH) syndrome is an autosomal recessive disorder of the urea cycle. With the exception of the French‐Canadian founder effect, no common mutation has been detected in other populations. In this study, we collected 16 additional HHH cases and expanded the spectrum of SLC25A15/ORC1 mutations. Eleven novel mutations were identified including six new missense and one microrearrangement. We also measured the transport properties of the recombinant purified proteins in reconstituted liposomes for four new and two previously reported missense mutations and proved that the transport activities of these mutant forms of ORC1 were reduced as compared with the wild‐type protein; residual activity ranged between 4% and 19%. Furthermore, we designed three‐dimensional (3D)‐modeling of mutant ORC1 proteins. While modeling the changes in silico allowed us to obtain new information on the pathomechanisms underlying HHH syndrome, we found no clear‐cut genotype–phenotype correlations. Although patient metabolic alterations responded well to low‐protein therapy, predictions concerning the long‐term evolution of HHH syndrome remain uncertain. The preference for a hepatic rather than a neurological presentation at onset also continues, largely, to elude us. Neither modifications in oxidative metabolism‐related energy, such as those expected in different mtDNA haplogroups, nor sequence variants in SLC25A2/ORC2 seem to be crucial. Other factors, including protein stability and function, and ORC1‐ORC2 structural interactions should be further investigated. Hum Mutat 0, 1–8, 2009.

α-Isopropylmalate, a Leucine Biosynthesis Intermediate in Yeast, Is Transported by the Mitochondrial Oxalacetate Carrier

In Saccharomyces cerevisiae, α-isopropylmalate (α-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported α-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di- and tricarboxylate transporters as well as the previously proposed α-IPM transporter. Transport was saturable with a half-saturation constant of 75 ± 4 μm for α-IPM and 0.31 ± 0.04 mm for β-IPM and was inhibited by the substrates of Oac1p. Though not transported, α-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, α-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an α-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with α-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant ΔOAC1ΔLEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of ΔOAC1ΔLEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates α-ketoisocaproate and α-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of α-IPM, probably in exchange for oxalacetate.

Acetylation of human mitochondrial citrate carrier modulates mitochondrial citrate/malate exchange activity to sustain NADPH production during macrophage activation

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.

The peroxisomal NAD+ carrier of Arabidopsis thaliana transports coenzyme A and its derivatives

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.