Ornithine Carrier Research Articles

Overexpression of SLC25A15 is involved in the proliferation of cutaneous melanoma and leads to poor prognosis.

Melanoma is a skin tumor with a high degree of malignancy, poor prognosis and few effective therapies. Deprivation of the arginine from cancer cells through transport inhibition and arginine depletion is a novel strategy for cancer therapy. In this study, we have investigated the effect of SLC25A15, which encodes the mitochondrial ornithine carrier 1, on melanoma progression. Using bioinformatics methods to screen the data from TCGA and GEO, we found that SLC25A15 is overexpressed in patients with melanoma and negatively related with the overall and disease-free survival rates. Knockdown the expression of SLC25A15 by siRNA could effectively inhibit the proliferation of A375 melanoma cells, as detected by CCK8 and colony formation. Furthermore, SLC25A15 siRNA was able to promote apoptosis of A375 cells, which exhibited decreased expression levels of the anti-apoptotic protein Bcl-2 while showing increased pro-apoptotic protein Bax and cleaved caspase-3. All these results suggest that the overexpression of SLC25A15 is involved in the progression of melanoma and may predict the prognosis of melanoma. This may shed new lights on the diagnosis and therapy of melanoma in the future.

The hyperornithinemia-hyperammonemia-homocitrullinuria syndrome.

BackgroundHyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is a rare autosomal recessive disorder of the urea cycle. HHH has a panethnic distribution, with a major prevalence in Canada, Italy and Japan. Acute clinical signs include intermittent episodes of vomiting, confusion or coma and hepatitis-like attacks. Alternatively, patients show a chronic course with aversion for protein rich foods, developmental delay/intellectual disability, myoclonic seizures, ataxia and pyramidal dysfunction. HHH syndrome is caused by impaired ornithine transport across the inner mitochondrial membrane due to mutations in SLC25A15 gene, which encodes for the mitochondrial ornithine carrier ORC1. The diagnosis relies on clinical signs and the peculiar metabolic triad of hyperammonemia, hyperornithinemia, and urinary excretion of homocitrulline. HHH syndrome enters in the differential diagnosis with other inherited or acquired conditions presenting with hyperammonemia.MethodsA systematic review of publications reporting patients with HHH syndrome was performed.ResultsWe retrospectively evaluated the clinical, biochemical and genetic profile of 111 HHH syndrome patients, 109 reported in 61 published articles, and two unpublished cases. Lethargy and coma are frequent at disease onset, whereas pyramidal dysfunction and cognitive/behavioural abnormalities represent the most common clinical features in late-onset cases or during the disease course. Two common mutations, F188del and R179* account respectively for about 30% and 15% of patients with the HHH syndrome. Interestingly, the majority of mutations are located in residues that have side chains protruding into the internal pore of ORC1, suggesting their possible interference with substrate translocation. Acute and chronic management consists in the control of hyperammonemia with protein-restricted diet supplemented with citrulline/arginine and ammonia scavengers. Prognosis of HHH syndrome is variable, ranging from a severe course with disabling manifestations to milder variants compatible with an almost normal life.ConclusionsThis paper provides detailed information on the clinical, metabolic and genetic profiles of all HHH syndrome patients published to date. The clinical phenotype is extremely variable and its severity does not correlate with the genotype or with recorded ammonium/ornithine plasma levels. Early intervention allows almost normal life span but the prognosis is variable, suggesting the need for a better understanding of the still unsolved pathophysiology of the disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-015-0242-9) contains supplementary material, which is available to authorized users.

Substrate Specificity of the Two Mitochondrial Ornithine Carriers Can Be Swapped by Single Mutation in Substrate Binding Site

Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. 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. Altogether the substrate specificity changes demonstrate that Arg-179 and Glu-180 of contact point II bind the C(α) carboxylate and amino group of the substrates, respectively. Residue Glu-77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because Arg-179 is probably connected with Arg-275 of contact point III through Trp-224 by cation-π interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that Arg-179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side.

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.

Identification of a mitochondrial transporter for basic amino acids in Arabidopsis thaliana by functional reconstitution into liposomes and complementation in yeast.

We describe the identification and functional characterization of two Arabidopsis mitochondrial basic amino acid carriers (BAC), AtmBAC1 and AtmBAC2, which are related to the yeast ornithine (Orn) carrier Ort1p, also known as Arg11p. The arg11 mutant requires arginine (Arg) supplementation because it fails to export sufficient ornithine from the mitochondrion to the cytosol where it is converted to arginine. AtmBAC1 and, to a lesser extent, AtmBAC2 partially replaced the function of Ort1p in yeast arg11. The more efficient putative carrier, AtmBAC1, was expressed in E. coli, purified, and reconstituted into phospholipid vesicles, where it transported the basic l-amino acids arginine, lysine, ornithine and histidine (in order of decreasing affinity). AtmBAC1 recognized l-histidine whereas both yeast Ort1p and the mammalian ortholog ORNT1p do not. Also different from ORNT1p, AtmBAC1 did not transport citrulline. AtmBAC1 appeared to be more stereospecific than the yeast and mammalian ornithine carriers, exhibiting greater preference for the l-forms of arginine, lysine and ornithine. By RT-PCR, both AtmBAC1 and AtmBAC2 transcripts were detected in stems, leaves, flowers, siliques, and seedlings. Expression of AtmBAC1 in seedlings is consistent with its involvement in Arg breakdown in early seedling development, i.e. delivery of Arg to mitochondrial arginase. The Km (0.19 mm) for Arg uptake by AtmBAC1 was close to the value we previously determined for the saturable component of Arg uptake into intact mitochondria from soybean seedling cotyledons.

The purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria catalyses a second transport mode: ornithine+/H+ exchange

The mechanism of unidirectional transport of ornithine (i.e. in the absence of a counter-metabolite) has been investigated in proteoliposomes reconstituted with the ornithine carrier purified from rat liver mitochondria. The efflux of [3H]ornithine from proteoliposomes was stimulated by the addition of H+ (but not of other cations) to the incubation medium . On keeping the pH in the compartment containing ornithine constant at 8.0, the flux of ornithine into or out of the proteoliposomes increased on decreasing the pH in the opposite compartment from 8.0 to 6.0. Ornithine influx was also stimulated when a higher H+ concentration was generated inside the vesicles relative to the outside by the K+/H+ exchanger nigericin in the presence of an outwardly directed K+ gradient. A valinomycin-induced electrogenic flux of K+ did not affect ornithine transport in the absence of a counter-metabolite. Furthermore, changes in fluorescence of the pH indicator pyranine, included inside the proteoliposomes, showed that the flux of ornithine is accompanied by translocation of H+ in the opposite direction. It is concluded that the mitochondrial ornithine carrier catalyses an electroneutral exchange of ornithine+ for H+, in addition to the well-known 1:1 exchange of metabolites. Lysine+, but not citrulline, can also be exchanged for H+ by the ornithine carrier. The ornithine+/H+ transport mode of the exchanger is an essential step in the catabolism of excess arginine.

The purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria catalyses a second transport mode: ornithine+/H+ exchange

The mechanism of unidirectional transport of ornithine (i.e. in the absence of a counter-metabolite) has been investigated in proteoliposomes reconstituted with the ornithine carrier purified from rat liver mitochondria. The efflux of [(3)H]ornithine from proteoliposomes was stimulated by the addition of H(+) (but not of other cations) to the incubation medium. On keeping the pH in the compartment containing ornithine constant at 8.0, the flux of ornithine into or out of the proteoliposomes increased on decreasing the pH in the opposite compartment from 8.0 to 6.0. Ornithine influx was also stimulated when a higher H(+) concentration was generated inside the vesicles relative to the outside by the K(+)/H(+) exchanger nigericin in the presence of an outwardly directed K(+) gradient. A valinomycin-induced electrogenic flux of K(+) did not affect ornithine transport in the absence of a counter-metabolite. Furthermore, changes in fluorescence of the pH indicator pyranine, included inside the proteoliposomes, showed that the flux of ornithine is accompanied by translocation of H(+) in the opposite direction. It is concluded that the mitochondrial ornithine carrier catalyses an electroneutral exchange of ornithine(+) for H(+), in addition to the well-known 1:1 exchange of metabolites. Lysine(+), but not citrulline, can also be exchanged for H(+) by the ornithine carrier. The ornithine(+)/H(+) transport mode of the exchanger is an essential step in the catabolism of excess arginine.

Identification of the yeast ARG-11 gene as a mitochondrial ornithine carrier involved in arginine biosynthesis

The ARG-11 gene in Saccharomyces cerevisiae encodes a protein with the characteristic features of a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 29 members of the family are unknown. The yeast ARG-11 protein has been over-produced as inclusion bodies in Escherichia coli. It has been solubilized in the presence of sarkosyl, re-constituted into liposomes and shown to transport ornithine in exchange for protons. Its main physiological role is probably to take ornithine synthesized from glutamate in the mitochondrial matrix to the cytosol where it is converted to arginine.

Kinetic characterization of the reconstituted ornithine carrier from rat liver mitochondria

The ornithine carrier was purified from rat liver mitochondria and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-2. The efficiency of reconstitution was optimized with respect to the concentration of protein and phospholipid, the Triton X-100/phospholipid ratio, the Amberlite/detergent ratio and the number of passages through a single Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, increasing in the presence of acidic phospholipids and decreasing in the presence of dioleoylphosphatidylcholine. In the reconstituted system the incorporated ornithine carrier catalyzed a first-order reaction of ornithine/ornithine or ornithine/citrulline exchange. The maximum transport rate of external [ 14C]ornithine was 3.2 mmol/min per g protein at 25°C. This value was independent of the type of substrate present at the external or internal space of the liposomes (ornithine, citrulline and lysine). The half-saturation constant ( K m) was 0.16 mM for ornithine, 1.2 mM for lysine and 3.6 mM for citrulline. The activation energy of the ornithine/ornithine exchange reaction was 89 kJ/mol. The rate of exchange had a pH optimum at 8 and was inhibited by cations.

Mitochondrial substrate carriers

The inner mitochondrial membrane contains several transport systems for metabolites, which are necessary for oxidative phosphorylation, for the transfer of reducing equivalents and for important metabolic pathways such as fatty acid oxidation, gluconeogenesis and the urea cycle. So far nine mitochondrial carriers have been purified and functionally reconstituted into liposomes (for a review see Ref. 1). With the exception of the AGC, all the other carriers have been found to have a very similar molecular mass of 28-34 kDa. The primary structure of the ADP/ATP carrier (AAC), the phosphate carrier (PiC), the uncoupling protein (UNC) and the oxoglutarate carrier (OGC) has been determined by amino acid analysis and/or by DNA sequencing. These proteins have a tripartite structure made of related sequences about 100 amino acids in length. Furthermore, the repetitive elements of each protein are related to those of the others, suggesting that these proteins belong to a carrier protein family evolved from a common ancestor gene. In this paper we report on several structural and functional properties of previously purified carriers, as well as on the first identification and purification of the aspartate/ glutamate carrier (AGC) and the ornithine carrier (ORC).

Identification and purification of the ornithine/citrulline carrier from rat liver mitochondria.

The ornithine/citrulline carrier from rat liver mitochondria, solubilized with Triton X-100 and partially purified on hydroxyapatite, was identified and completely purified by PD-10, DEAE-Sephacel and celite chromatography. On SDS/polyacrylamide gel electrophoresis, the purified ornithine/citrulline carrier consisted of a single protein band with an apparent molecular mass of 33.5 kDa. When reconstituted into liposomes the ornithine carrier protein catalyzed an active mersalyl sensitive ornithine/ornithine exchange. It was purified 438-fold with a recovery of 38% and a protein yield of 0.09% with respect to the extract derived from mitoplasts. The purified and reconstituted protein did not catalyze a significant unidirectional transport of ornithine. Citrulline was found to be the best countersubstrate for the transport of ornithine, followed by lysine and arginine. The exchange activity was inhibited by several sulphydryl reagents.

Metabolite transport in rat kidney mitochondria: Ornithine/phosphate translocator

Ornithine uptake by rat kidney mitochondria is here first shown by monitoring the reduction of the intramitochondrial pyridine nucleotides which occurs as a result of metabolism of imported ornithine via ornithine aminotransferase and 1-pyrroline-carboxylate dehydrogenase. Ornithine uptake shows saturation features (Km and Vmax values, measured at 20°C and at pH 7.20, were found to be about 0.85 mM and 23 nmoles/min × mg protein, respectively) and proves to be inhibited by D-ornithine, inorganic phosphate, praseodimium chloride and mersalyl. Neither malate nor glutamate, but phosphate was found to exchange with ornithine. Phosphate efflux caused by externally added ornithine was shown both as revealed by a colorimetric assay and as continuously monitored by measuring extramitochondrial reduction of NAD + in the presence of glyceraldehyde-3-phosphate, glyceraldehyde-3-phosphate dehydrogenase, ADP and 3-phosphoglycerate kinase. The role of ornithine carrier in kidney metabolism will also be discussed.