Seven novel mutations in the ORNT1 gene (SLC25A15) in patients with hyperornithinemia, hyperammonemia, and homocitrullinuria syndrome

Salvi, Sergio; Dionisi‐Vici, Carlo; Bertini, Enrico; Verardo, Margherita; Santorelli, Filippo M.

doi:10.1002/humu.1221

Cited by 29 publications

(22 citation statements)

References 8 publications

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“…All of the nonconservative substitutions of Arg-275 abolish transport activity, which is consistent with the previous observation that R275Q causes HHH syndrome (26) and renders the ORC1 protein inactive (23). It should be noted that arginine at position 275 is highly conserved in almost all subfamilies of mitochondrial carriers (8,13).…”

Section: Discussionsupporting

confidence: 92%

“…The tissue distribution of the isoforms is overlapping; they are found in most tissues with ORC1 expressed more than ORC2 especially in liver, pancreas, lung, and testis (23). Mutations of the ORC1 gene that inactivate the carrier protein and cause the autosomal recessive hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome (OMIM238970) have provided clues for the functional importance of certain residues (23)(24)(25)(26)(27). Some of the missense mutations causing HHH syndrome, such as E180K (24), G220R (25), R275Q (23,26), G27R, A70L, F188L, G216S, T272I, and L283F (27), affect residues protruding into the substrate translocation pathway of ORC1, suggesting that they might affect substrate binding (8,13,28).…”

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confidence: 99%

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

Monné

Miniero

Daddabbo

et al. 2012

Journal of Biological Chemistry

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Background: Substrate binding and transport mechanisms of mitochondrial carriers are inadequately understood. Results: The effect of mutations on substrate specificity and transport activity was assessed in two human ornithine carrier isoforms. Conclusion:The substrate specificity and transport rate of the two isoforms are defined by a few residues and can be swapped. Significance: The results show how small substrates can trigger transport in carriers.

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Section: Discussionsupporting

confidence: 92%

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confidence: 99%

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

Monné

Miniero

Daddabbo

et al. 2012

Journal of Biological Chemistry

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“…1). Mutagenesis of residue 279 BtAAC1 (contact point III) affects carrier activity in Aac2p (10), and in the human ORNT1 is associated with hyperornithineamiahyperammonia-homocitrullinuria syndrome (23). Although it does not confer substrate specificity, it could constitute a third important contact point of the substrate-binding site.…”

Section: Resultsmentioning

confidence: 99%

Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

Robinson

Kunji

2006

Proc. Natl. Acad. Sci. U.S.A.

234

289

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Mitochondrial carriers link biochemical pathways in the cytosol and mitochondrial matrix by transporting substrates across the inner mitochondrial membrane. Substrate recognition is specific for each carrier, but sequence similarities suggest the carriers have similar structures and mechanisms of substrate translocation. By considering conservation of amino acids, distance and chemical constraints, and by modeling family members on the known structure of the ADP͞ATP translocase, we have identified a common substrate binding site. It explains substrate selectivity and proton coupling and provides a mechanistic link to carrier opening by substrate-induced perturbation of the salt bridges that seal the pathway to and from the mitochondrial matrix. It enables the substrate specificity of uncharacterized mitochondrial carriers to be predicted.comparative modeling ͉ membrane protein ͉ sequence alignment M embers of the mitochondrial carrier family are located in the inner mitochondrial membrane and allow exchange of substrates between the cytosol and the mitochondrial matrix (1). The family is exclusive to eukaryotes, and its members are unrelated to other transporter families, including the major facilitator superfamily and the ABC transporters. Their substrates include nucleotides, amino acids, cofactors, carboxylic acids, and inorganic anions required by the organelle for oxidative phosphorylation, gluconeogenesis, synthesis and degradation of amino and fatty acids, macromolecular synthesis of proteins and nucleic acids, sterol metabolism, and thermogenesis (1). Mutations of the carriers are associated with diseases, including progressive external opthalmoplegia, adult-and neonatal-onset type II citrullinaemia, Amish-type microcephaly, hyperornithinemia-hyperammonia-homocitrullinuria, carnitineacylcarnitine translocase deficiency, neonatal myoclonic epilepsy, severe obesity, and type II diabetes (1).The mitochondrial carriers have three tandem repeats of Ϸ100 aa, each containing two transmembrane ␣-helices linked by a large loop (2) and a conserved signature motif P-X-[DE]-X-X-[RK] (3). The carriers exist in two distinct conformational states: the cytoplasmic state (c-state) in which the carrier accepts its substrate from the cytoplasm, and the matrix state in which it accepts a substrate from the mitochondrial matrix. For the ADP͞ATP carrier, the inhibitor carboxyatractyloside (CATR) is known to bind to the carrier in the c-state only and prevent the binding of ADP. In the atomic model of the ADP͞ATP carrier 1 from Bos taurus (BtAAC1) in complex with CATR (4), the six-transmembrane ␣-helices form a barrel that has pseudo-3-fold symmetry. CATR is bound in the internal aqueous cavity (see Fig. 4, which is published as supporting information on the PNAS web site), which is open to the cytosol and closed to the mitochondrial matrix, consistent with the structure representing the c-state. The prolines of the signature motif kink the transmembrane helices, bringing their C-terminal ends together at the base of the cavit...

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“…The cDNA for ORC2 was amplified from the same six samples. The mutant forms of ORC1 corresponded to 7 alleles from 6 HHH patients (10,11). Five of them were homozygous for the mutations G27R, R275Q, R179X, C861insG, and IVS5ϩ1g3a, and the other one was heterozygous for G190D and F188⌬.…”

Section: Methodsmentioning

confidence: 99%

The Mitochondrial Ornithine Transporter

Fiermonte

Dolce

David

et al. 2003

Journal of Biological Chemistry

117

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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.Rat liver mitochondria contain an ornithine-citrulline transport protein, often known as the ornithine carrier (ORC) 1 (1-5). The reconstituted purified protein is highly active in ornithinecitrulline exchange and is somewhat less active in unidirectional transport of ornithine. It transports lysine and arginine also but does not transport histidine. The positive charges of ornithine and lysine are compensated by cotransport of a proton with citrulline in exchange. Its affinity for ornithine is lower than for other substrates, and under saturating internal concentrations it is not dependent on the nature of the countersubstrate. The exchange reaction operates by a simultaneous (sequential) mechanism, and the unidirectional transport of ornithine or lysine (but not of citrulline) is compensated by a proton. The exchange of cytosolic ornithine for matrix citrulline is part of the urea cycle (1, 3, 6), and the ornithine-H ϩ exchange also has an important role in the catabolism of excess arginine and the biosynthesis of polyamines (4).In Saccharomyces cerevisiae, the ornithine carrier is encoded by ARG11/ORT1, and its identity has been established by overexpression in bacteria, reconstitution, and study of its transport properties (7). Subsequently, the gene for the human mitochondrial ornithine carrier, which is defective in the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome (8), was identified by homology with the yeast sequence, and its function as an ORC was inferred from its ability to complement the defect of HHH fibroblasts by incorporating radioactive ornithine into cellular protein. The HHH phenotype is milder than those associated with a deficiency of an enzyme in the urea cycle, and it has been suggested that carriers with other functions can compensate for the defective protein by transporting ornithine (8).As described below, the human and mouse genomes contain a spliced pseudogene that in man encodes a second isoform, ORC2 (9). The isoforms are expressed...

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Seven novel mutations in the ORNT1 gene (SLC25A15) in patients with hyperornithinemia, hyperammonemia, and homocitrullinuria syndrome

Cited by 29 publications

References 8 publications

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

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

Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

The Mitochondrial Ornithine Transporter

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