Identification of the Human Mitochondrial Oxodicarboxylate Carrier BACTERIAL EXPRESSION, RECONSTITUTION, FUNCTIONAL CHARACTERIZATION, TISSUE DISTRIBUTION, AND CHROMOSOMAL LOCATION

In Saccharomyces cerevisiae, the genes ODC1 and ODC2 encode isoforms of the oxodicarboxylate carrier. They both transport C5-C7 oxodicarboxylates across the inner membranes of mitochondria and are members of the family of mitochondrial carrier proteins. Orthologs are encoded in the genomes of Caenorhabditis elegans and Drosophila melanogaster, and a human expressed sequence tag (EST) encodes part of a closely related protein. Information from the EST has been used to complete the human cDNA sequence. This sequence has been used to map the gene to chromosome 14q11.2 and to show that the gene is expressed in all tissues that were examined. The human protein was produced by overexpression in Escherichia coli, purified, and reconstituted into phospholipid vesicles. It has similar transport characteristics to the yeast oxodicarboxylate carrier proteins (ODCs). Both the human and yeast ODCs catalyzed the transport of the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a counter-exchange mechanism. Adipate, glutarate, and to a lesser extent, pimelate, 2-oxopimelate, 2-aminoadipate, oxaloacetate, and citrate were also transported by the human ODC. The main differences between the human and yeast ODCs are that 2-aminoadipate is transported by the former but not by the latter, whereas malate is transported by the yeast ODCs but not by the human ortholog. In mammals, 2-oxoadipate is a common intermediate in the catabolism of lysine, tryptophan, and hydroxylysine. It is transported from the cytoplasm into mitochondria where it is converted into acetyl-CoA. Defects in human ODC are likely to be a cause of 2-oxoadipate acidemia, an inborn error of metabolism of lysine, tryptophan, and hydroxylysine

The Mitochondrial Ornithine Transporter BACTERIAL EXPRESSION, RECONSTITUTION, FUNCTIONAL CHARACTERIZATION, AND TISSUE DISTRIBUTION OF TWO HUMAN ISOFORMS

Two isoforms of the human ornithine carrier, ORC1 and ORC2, have been identified by overexpression of the proteins in bacteria and by study of the transport properties of the purified proteins reconstituted into liposomes. Both transport L-isomers of ornithine, lysine, arginine, and citrulline by exchange and by unidirectional mechanisms, and they are inactivated by the same inhibitors. ORC2 has a broader specificity than ORC1, and L- and D-histidine, L-homoarginine, and D-isomers of ornithine, lysine, and ornithine are all substrates. Both proteins are expressed in a wide range of human tissues, but ORC1 is the predominant form. The highest levels of expression of both isoforms are in the liver. Five mutant forms of ORC1 associated with the human disease hyperornithinemia-hyperammonemia-homocitrullinuria were also made. The mutations abolish the transport properties of the protein. In patients with hyperornithinemia-hyperammonemia-homocitrullinuria, isoform ORC2 is unmodified, and its presence compensates partially for defective ORC1

Calorimetry and FTIR reveal the ability of URG7 protein to modify the aggregation state of both cell lysate and amylogenic α-synuclein

Differential scanning calorimetry and FITR analyses allowed to investigate the role of URG7 (up-regulated gene clone 7) protein involved in the development of hepatocellular carcinoma induced by hepatitis B virus infection, on the physical structure both of lysates of human hepatoblastoma cells (HepG2) stressed with tunicamycin and α-synuclein, one of the proteins associated with neurogenerative diseases. The protein-water interfacial region was identified and correlated with protein structure. DSC results confirm through the interfacial water behavior that URG7 is able to act in two ways: it maintains the interfacial water stability and controls the mobile fraction level, thereby the flexibility and the protein folding. The mobile water phase increases strongly for cells exposed to α-synuclein, demonstrating an important influence on water hydration. FTIR results evidenced an increase of about 30% of cross β structures in cells exposed to α-synuclein, associated with aggregated proteins. In stress conditions, URG7 was able to maintain the same fraction of mobile water as untreated cells. URG7 was able to restore the water reorientation ability around the complex lysate system and reduced abnormal protein folding

Biochemical characterization of a new mitochondrial transporter of dephosphocoenzyme A in Drosophila melanogaster

none13noCoA 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.Vozza, Angelo; Leonardis, Francesco De; Paradies, Eleonora; Grassi, Anna De; Pierri, Ciro Leonardo; Parisi, Giovanni; Marobbio, Carlo Marya Thomas; Lasorsa, Francesco Massimo; Muto, Luigina; Capobianco, Loredana; Dolce, Vincenza; Raho, Susanna; Fiermonte, GiuseppeVozza, Angelo; Leonardis, Francesco De; Paradies, Eleonora; Grassi, Anna De; Pierri, Ciro Leonardo; Parisi, Giovanni; Marobbio, Carlo Marya Thomas; Lasorsa, Francesco Massimo; Muto, Luigina; Capobianco, Loredana; Dolce, Vincenza; Raho, Susanna; Fiermonte, Giusepp

How Detergent Impacts Membrane Proteins: Atomic-Level Views of Mitochondrial Carriers in Dodecylphosphocholine.

Characterizing the structure of membrane proteins (MPs) generally requires extraction from their native environment, most commonly with detergents. Yet, the physicochemical properties of detergent micelles and lipid bilayers differ markedly and could alter the structural organization of MPs, albeit without general rules. Dodecylphosphocholine (DPC) is the most widely used detergent for MP structure determination by NMR, but the physiological relevance of several prominent structures has been questioned, though indirectly, by other biophysical techniques, e.g., functional/thermostability assay (TSA) and molecular dynamics (MD) simulations. Here, we resolve unambiguously this controversy by probing the functional relevance of three different mitochondrial carriers (MCs) in DPC at the atomic level, using an exhaustive set of solution-NMR experiments, complemented by functional/TSA and MD data. Our results provide atomic-level insight into the structure, substrate interaction and dynamics of the detergent-membrane protein complexes and demonstrates cogently that, while high-resolution NMR signals can be obtained for MCs in DPC, they systematically correspond to nonfunctional states

Identification and biochemical-molecular characterization of mitochondrial carrier proteins in human andmodel organisms and associated diseases

Dottorato di Ricercain Medicina Traslazionale. Ciclo XXIXThe mitochondrial carriers (MCs) are transmembrane proteins found in the mitochondrial inner membrane, which catalyze the translocation of solutes through the membrane. These belong to a family of carrier proteins, the SLC25 or Mitochondrial Carrier Family (MCF). Their function is to create a connection between mitochondria and cytosol, facilitating the flow of a large variety of solutes across the permeability barrier of the inner mitochondrial membrane, which is necessary for many physiological processes. The functional information obtained from the study of mitochondrial carrier was fundamental in correlating MCs physiological and pathological roles in cellular metabolism. It was possible to identify genes, and their possible defects, responsible for the onset of certain diseases such as the Stanley syndrome, Amish microcephaly, HHH syndrome (hyperornithinemia, hyperammonemia and homocitrullinuria) and type II citrullinemia, their molecular basis and their symptoms. Further studies on the functional characterization of the gene family SLC25 will clarify other diseases caused by a mitochondrial carrier deficiency. This work was focused in particular on the study of some carriers belonging to the MCF: - the mitochondrial glycine carrier, important in heme synthesis and congenital sideroblastic anemia; - the mitochondrial dephosphocoenzyme A carrier, important in regulating the compartmentalization of the CoA, the study of which is crucial for a better understanding of some neurodegenerative diseases that depend on the biosynthesis of CoA; - the mitochondrial oxoglutarate carrier, of which the functional and structural rearrangements required for substrate transport were analyzed The studies were focused on the biochemical and molecular characterization of human glycine carrier protein (GlyC) and its yeast homolog (Hem25p) providing evidence that they are mitochondrial carriers for glycine. Glycine carrier is required for the uptake of glycine in the mitochondrial matrix, where this amino acid is condensed with succinyl coenzyme A to yield δ-aminolevulinic acid, necessary for heme biosynthesis. A detailed knowledge of this transporter could be helpful to clearly understand congenital sideroblastic anemia (CSA), caused by defects of heme biosynthesis in developing erythroblasts. In particular, Hem25p was cloned into a bacterial expression system (Escherichia coli BL21), overexpressed at high levels as inclusion bodies, and purified by Ni2+-NTAagarose affinity chromatography. The protein was then reconstituted in liposomes and its transport activity of glycine was observed. The kinetic constants, Km and Vmax, were calculated. Subsequently, other evidences of glycine uptake were obtained carrying out experiments on mitochondrial proteins from the yeast wild-type strain, the hem25Δ strain and the hem25Δ HEM25-pYES2. The protein subcellular localization was found to be mitochondrial. Furthermore, the hem25Δ mutant manifested a defect in the biosynthesis of δ-aminolevulinic acid and displayed reduced levels of downstream heme and mitochondrial cytochromes. The observed defects were rescued by complementation with yeast HEM25 or human SLC25A38 genes. This work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia. In human, the transport of CoA across the inner mitochondrial membrane has been attributed to two different genes, SLC25A16 and SLC25A42. Presumed orthologs of both genes are present in many eukaryotic genomes, but not in that of D. melanogaster, which contains only one gene, CG4241, phylogenetically close to SLC25A42. CG4241 encodes a long and a short isoform of the dPCoA carrier, respectively dPCoAC1 and dPCoAC2, which arise from an alternative translational start site. dPCoAC1 and dPCoAC2 were expressed as inclusion bodies in E. coli C0214, and reconstituted in proteoliposomes to observe the transport activity in order to characterize them functionally.The functional characterization of the D. melanogaster dPCoA carrier is of particular interest as it is the first mitochondrial carrier showing a particular substrate specificity for dPCoA and ADP. The expression of both isoforms in a S. 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. This work will be useful in the near future to better understand the deficiency of enzymes involved in the CoA biosynthesis associated with a neurodegenerative disorder known as neurodegeneration with brain iron accumulation (NBIA). The oxoglutarate carrier (OGC) plays a key role in important metabolic pathways. Its transport activity has been extensively studied, and, to investigate new structural rearrangements required for substrate translocation, site-directed mutagenesis was used to conservatively replace lysine 122 by arginine. K122R mutant was kinetically characterized, exhibiting a significant Vmax reduction with respect to the wild-type (WT) OGC, whereas Km value was unaffected, implying that this substitution does not interfere with 2-oxoglutarate binding site. Moreover, K122R mutant was more inhibited by several sulfhydryl reagents with respect to the WT OGC, suggesting that the reactivity of some cysteine residues towards these Cys-specific reagents is increased in this mutant. Different sulfhydryl reagents were employed in transport assays to test the effect of the cysteine modifications on single-cysteine OGC mutants named C184, C221, C224 (constructed in the WT background) and K122R/C184, K122R/C221, K122R/C224 (constructed in the K122R background). Cysteines 221 and 224 were more deeply influenced by some sulfhydryl reagents in the K122R background. Furthermore, the presence of 2- oxoglutarate significantly enhanced the degree of inhibition of K122R/C221, K122R/C224 and C224 activity by the sulfhydryl reagent 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), suggesting that cysteines 221 and 224, together with K122, take part to structural rearrangements required for the transition from the c- to the m-state during substrate translocationUniversità della Calabri