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

Abstract

The 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 translocation