Evidence for Non-Essential Salt Bridges in the M-Gates of Mitochondrial Carrier Proteins

Mitochondrial carriers, which transport metabolites, nucleotides, and cofactors across the mitochondrial inner membrane, have six transmembrane α-helices enclosing a translocation pore with a central substrate binding site whose access is controlled by a cytoplasmic and a matrix gate (M-gate). The salt bridges formed by the three PX[DE]XX[RK] motifs located on the odd-numbered transmembrane α-helices greatly contribute to closing the M-gate. We have measured the transport rates of cysteine mutants of the charged residue positions in the PX[DE]XX[RK] motifs of the bovine oxoglutarate carrier, the yeast GTP/GDP carrier, and the yeast NAD+ transporter, which all lack one of these charged residues. Most single substitutions, including those of the non-charged and unpaired charged residues, completely inactivated transport. Double mutations of charged pairs showed that all three carriers contain salt bridges non-essential for activity. Two double substitutions of these non-essential charge pairs exhibited higher transport rates than their corre-sponding single mutants, whereas swapping the charged residues in these positions did not increase activity. The results demonstrate that some of the residues in the charged residue positions of the PX[DE]XX[KR] motifs are important for reasons other than forming salt bridges, probably for playing specific roles related to the substrate interaction-mediated conformational changes leading to the M-gate opening/closing

Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome.

iskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS

The mitochondrial oxoglutarate carrier: from identification to mechanism

The 2-oxoglutarate carrier (OGC) belongs to the mitochondrial carrier protein family whose members are responsible for the exchange of metabolites, cofactors and nucleotides between the cytoplasm and mitochondrial matrix. Initially, OGC was characterized by determining substrate specificity, kinetic parameters of transport, inhibitors and molecular probes that form covalent bonds with specific residues. It was shown that OGC specifically transports oxoglutarate and certain carboxylic acids. The substrate specificity combination of OGC is unique, although many of its substrates are also transported by other mitochondrial carriers. The abundant recombinant expression of bovine OGC in Escherichia coli and its ability to functionally reconstitute into proteoliposomes made it possible to deduce the individual contribution of each and every residue of OGC to the transport activity by a complete set of cys-scanning mutants. These studies give experimental support for a substrate binding site constituted by three major contact points on the even-numbered α-helices and identifies other residues as important for transport function through their crucial positions in the structure for conserved interactions and the conformational changes of the carrier during the transport cycle. The results of these investigations have led to utilize OGC as a model protein for understanding the transport mechanism of mitochondrial carriers

Inactivation of the reconstituted oxoglutarate carrier from bovine heart mitochondria by pyridoxal 5'-phosphate

The effect of pyridoxal 5'-phosphate and some other lysine reagents on the purified, reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition of oxoglutarate/oxoglutarate exchange by pyridoxal 5'-phosphate can be reversed by passing the proteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodium borohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal 5'-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutarate transport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutarate carrier (Ki = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal 5'-phosphate indicated that modification of a single amino acid residue/carrier molecule was sufficient for complete inhibition of oxoglutarate transport. After reduction with sodium borohydride [3H]pyridoxal 5'-phosphate bound covalently to the oxoglutarate carrier. Incubation of the proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivation and no radioactivity was found associated with the carrier protein. In contrast, glutarate and substrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl, which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier against inhibition by pyridoxal 5'-phosphate. These results indicate that pyridoxal 5'-phosphate interacts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminal glycine residue) which is essential for substrate translocation and may be localized at or near the substrate-binding site.The effect of pyridoxal 5'-phosphate and some other lysine reagents on the purified, reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition of oxoglutarate/oxoglutarate exchange by pyridoxal 5'-phosphate can be reversed by passing the proteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodium borohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal 5'-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutarate transport with an IC50 Of 0.5 mM, competed with the substrate for binding to the oxoglutarate carrier (K-i = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal 5'-phosphate indicated that modification of a single amino acid residue/carrier molecule was sufficient for complete inhibition of oxoglutarate transport. After reduction with sodium borohydride [H-3]pyridoxal 5'-phosphate bound covalently to the oxoglutarate carrier. Incubation of the proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivation and no radioactivity was found associated with the carrier protein. In contrast, glutarate and substrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl, which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier against inhibition by pyridoxal 5'-phosphate. These results indicate that pyridoxal 5'-phosphate interacts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminal glycine residue) which is essential for substrate translocation and may be localized at or near the substrate-binding site

The mitochondrial oxoglutarate carrier: Cysteine-scanning mutagenesis of transmembrane domain IV and sensitivity of Cys mutants to sulfhydryl reagents

Using a functional mitochondrial oxoglutarate carrier mutant devoid of Cys residues (C-less carrier), each amino acid residue in transmembrane domain IV and flanking hydrophilic loops (from T179 to S205) was replaced individually with Cys. The great majority of the 27 mutants exhibited significant oxoglutarate transport in reconstituted liposomes as compared to the activity of the C-less carrier. In contrast, Cys substitution for G183, R190, Q198, and Y202, in either C-less or wild-type carriers, yielded molecules with complete loss of oxoglutarate transport activity. G183 and R190 could be partially replaced only by Ala and Lys, respectively, whereas Q198 and Y202 were irreplaceable with respect to oxoglutarate transport. Of the single-Cys mutants tested, only T187C, A191C, V194C, and N195C were strongly inactivated by N-ethylmaleimide and by low concentrations of methanethiosulfonate derivatives. Oxoglutarate protects Cys residues at positions 187, 191, and 194 against reaction with N-ethylmaleimide. These positions as well as the residues found to be essential for the carrier activity, except Y202 which is located in the extramembrane loop IV-V, reside on the same face of transmembrane helix IV, probably lining part of a water-accessible crevice or channel between helices of the oxoglutarate carrier

Identification of the residues determining the substrate specificities of the two human mitochondrial ornithine carrier isoforms

The mitochondrial carrier family consists of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has suggested that these transporters have a common substrate binding site consisting of three major contact points in the central carrier cavity. Here we have probed the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 with site-directed mutagenesis and a set of different substrates in transport assays. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Together with further mutations we demonstrate that the residues in position 179 and E180 of contact point II selects the stereospecificity and side chain length of the amino acid substrates, suggesting that these residues bind the substrate carboxylate and alpha-amino group, respectively. Residue E77 of contact point I probably interacts with the terminal amino group of the substrate side chain. For the substrate-induced conformational changes required for substrate translocation, it is likely that all three contact points are involved by R179 connecting to R275 of contact point III with cation-pi interactions through W224. Mutations at position 179 also severely affect the turnover number, implying that substrate binding to residue 179 is a rate-limiting step in the catalytic transport cycle. Since R179 is located in vicinity of the matrix gate of the cavity, it may be a key residue in the opening of the carrier to the matrix side

Editorial: Understanding membrane transporters: from structure to function

Membrane transporters are critical to the passage of molecules across biological membranes and compartments, including the extracellular and intracellular environments. For this reason, these proteins play a crucial role in a plethora of pathophysiological processes in all organisms and as such are of great interest from the biomedical and biotechnological viewpoints. They include primary and secondary active and passive transporters which exert their function through conformational changes induced by ligand binding or other mechanisms including, among others, allosteric gating, transmembrane potential variation, and ATP hydrolysis. This Research Topic collects scientific contributions concerning the study of membrane transporters, with a specific focus on the characterization of the relationships between their structure, dynamics, and mechanism of action through a combination of experimental and computational techniques. They also include studies on the interactions between transporters and membrane lipids and how these affect the transport mechanism

The interaction of hemin, a porphyrin derivative, with the purified rat brain 2-oxoglutarate carrier

The mitochondrial 2-oxoglutarate carrier (OGC), isolated and purified from rat brain mito-chondria, was reconstituted into proteoliposomes to study the interaction with hemin, a porphyrin derivative, which may result from the breakdown of heme-containing proteins and plays a key role in several metabolic pathways. By kinetic approaches, on the basis of the single binding centre gated pore mechanism, we analyzed the effect of hemin on the transport rate of OGC in uptake and efflux experiments in proteoliposomes reconstituted in the presence of the substrate 2-oxoglutarate. Overall, our experimental data fit the hypothesis that hemin operates a competitive inhibition in the 0.5–10 µM concentration range. As a consequence of the OGC inhibition, the malate/aspartate shuttle might be impaired, causing an alteration of mitochondrial function. Hence, considering that the metabolism of porphyrins implies both cytoplasmic and mitochondrial processes, OGC may participate in the regulation of porphyrin derivatives availability and the related metabolic pathways that depend on them (such as oxidative phosphorylation and apoptosis). For the sake of clarity, a simplified model based on induced-fit molecular docking supported the in vitro transport assays findings that hemin was as good as 2-oxoglutarate to bind the carrier by engaging specific ionic hydrogen bond interactions with a number of key residues known for participating in the similarly located mitochondrial carrier substrate binding site

Chronic idiopathic thrombocytopenia treated with immunoglobulin

Twenty five children with chronic idiopathic thrombocytopenic purpura followed from 6-96 months in 7 Italian paediatric departments were treated with high dose immunoglobulin according to a multicentre protocol. Positive responses were observed in 20 of 25 patients (80%) and negative responses in 5 of 25 (20%). On previous steroid treatment 7 of 10 positive responders were steroid resistant and 13 of 15 were steroid dependent. Within four weeks of beginning treatment 16 of 20 patients (80%) relapsed, while 4 of 20 (20%) maintained normal platelet values after 4-12 months' follow up. Statistical analysis of the platelet count on day five of treatment enabled the authors to divide positive responders into three groups: good, intermediate, and poor. The possible mode of action and clinical application of high dose immunoglobulin are discussed