A mitochondrial carrier gene, <Emphasis Type="Italic">CG32103</Emphasis>, is highly expressed in the corpora allata in the fruit fly <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> (Diptera: Drosophilidae)

Here we describe a novel gene that is highly expressed in the corpora allata, an endocrine organ responsible for synthesizing juvenile hormones (JHs), in the fruit fly, Drosophila melanogaster Meigen. We isolated an enhancer-trap line in which the transgene was inserted at the locus CG32103, which encodes a mitochondrial carrier family protein with calcium-binding motifs. RNA in situ hybridization revealed that CG32103 is predominantly expressed in the corpora allata in D. melanogaster larvae. Putative orthologs of CG32103 are conserved in many insect species. Mitochondrial carriers are responsible for transporting metabolites across the inner mitochondrial membrane. Given that both mitochondrial membrane transport and cytoplasmic calcium signaling are important for JH biosynthesis regulation, we speculated that CG32103 represents a new member of the family of JH biosynthesis regulators in insects.     

The human uncoupling proteins 5 and 6 (UCP5/SLC25A14 and UCP6/SLC25A30) transport sulfur oxyanions,phosphate and dicarboxylates

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family. In this work, two members of this family, UCP5 (BMCP1, brain mitochondrial carrier protein 1 encoded by SLC25A14) and UCP6 (KMCP1, kidney mitochondrial carrier protein 1 encoded by SLC25A30) have been thoroughly characterized biochemically. They were overexpressed in bacteria, purified and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that UCP5 and UCP6 transport inorganic anions (sulfate, sulfite, thiosulfate and phosphate) and, to a lesser extent, a variety of dicarboxylates (e.g. malonate, malate and citramalate) and, even more so, aspartate and (only UCP5) glutamate and tricarboxylates. Both carriers catalyzed a fast counter-exchange transport and a very low uniport of substrates. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors at various degrees. The transport affinities of UCP5 and UCP6 were higher for sulfate and thiosulfate than for any other substrate, whereas the specific activity of UCP5 was much higher than that of UCP6. It is proposed that a main physiological role of UCP5 and UCP6 is to catalyze the export of sulfite and thiosulfate (the H₂S degradation products) from the mitochondria, thereby modulating the level of the important signal molecule H₂S.     

Mitochondrial Glutamate Carrier GC1 as a Newly Identified Player in the Control of Glucose-stimulated Insulin Secretion

The SLC25 carrier family mediates solute transport across the inner mitochondrial membrane, a process that is still poorly characterized regarding both the mechanisms and proteins implicated. This study investigated mitochondrial glutamate carrier GC1 in insulin-secreting β-cells. GC1 was cloned from insulin-secreting cells, and sequence analysis revealed hydropathy profile of a six-transmembrane protein, characteristic of mitochondrial solute carriers. GC1 was found to be expressed at the mRNA and protein levels in INS-1E β-cells and pancreatic rat islets. Immunohistochemistry showed that GC1 was present in mitochondria, and ultrastructural analysis by electron microscopy revealed inner mitochondrial membrane localization of the transporter. Silencing of GC1 in INS-1E β-cells, mediated by adenoviral delivery of short hairpin RNA, reduced mitochondrial glutamate transport by 48% (p < 0.001). Insulin secretion at basal 2.5 mm glucose and stimulated either by intermediate 7.5 mm glucose or non-nutrient 30 mm KCl was not modified by GC1 silencing. Conversely, insulin secretion stimulated with optimal 15 mm glucose was reduced by 23% (p < 0.005) in GC1 knocked down cells compared with controls. Adjunct of cell-permeant glutamate (5 mm dimethyl glutamate) fully restored the secretory response at 15 mm glucose (p < 0.005). Kinetics of insulin secretion were investigated in perifused isolated rat islets. GC1 silencing in islets inhibited the secretory response induced by 16.7 mm glucose, both during first (−25%, p < 0.05) and second (−33%, p < 0.05) phases. This study demonstrates that insulin-secreting cells depend on GC1 for maximal glucose response, thereby assigning a physiological function to this newly identified mitochondrial glutamate carrier.Functions of mitochondria require regulated flux of molecules across the two membranes surrounding the matrix. Mitochondrial solute carriers (SLC25) are a large family of nuclearly encoded membrane-embedded proteins that promote solute transport across the inner mitochondrial membrane (1–4). The human genome contains 48 members of the SLC25 family, among them about 30 have been identified and characterized biochemically (1, 5–8). In particular, very little is known on solute carrier proteins transporting metabolites, such as glutamate. The two isoforms of the glutamate carrier GC1 and GC2 (encoded by SLC25A22 and SLC25A18, respectively) catalyze the transport of glutamate across the inner mitochondrial membrane, either by proton co-transport or in exchange for hydroxyl ions. To date, one human pathology has been associated with GC1, exhibiting a correlation between GC1 mutation and neonatal myoclonic epilepsy (9). Of interest, the high K_m isoform GC1 was shown to be expressed in different tissues, especially in the brain, liver, and pancreas (10). Despite the importance of these studies, we still lack subcellular localization and demonstration of the physiological function of glutamate carriers. The elevated expression levels in the pancreas triggered our interest, given that the glutamate pathway has been highlighted over the last years in the endocrine pancreas in general and the β-cell in particular (11). Still, the putative mechanisms responsible for mitochondrial glutamate transport have not yet been characterized in specialized tissues such as insulin-secreting cells. Only two carriers involved in mitochondrial shuttles have been shown to play an important role in the control of insulin secretion, i.e. the aspartate/glutamate carrier (AGC1 or Aralar1) (12) and the citrate/isocitrate carrier (13).It is well founded that mitochondrial metabolism is crucial in pancreatic β-cells by generating signals involved in metabolism-secretion coupling (14). Upon glucose stimulation, generation of ATP through mitochondrial activation leads to the closure of ATP-sensitive K⁺ channels and depolarization of the plasma membrane (15). This, in turn, induces the opening of voltage-dependent calcium channels resulting in elevation of cytosolic Ca²⁺ (16). Ca²⁺ is necessary but not sufficient for the full development of the insulin secretory response (17). Other messengers have been proposed to contribute to stimulation of insulin exocytosis, such as protein kinases A and C, long chain acyl-CoAs, nucleotides, and glutamate (18). The involvement of the latter amino acid was deduced from experiments performed under conditions of intracellular [Ca²⁺] clamped at permissive concentrations, during which intracellular provision of glutamate directly stimulated insulin exocytosis (19–21). Based on these results, it was proposed that glutamate could act downstream of mitochondrial function, participating in the coupling of glucose metabolism to insulin secretion (21). The importance of the glutamate pathway for β-cell function is illustrated in transgenic mice (named βGlud1^−/−) with conditional β-cell-specific deletion of the mitochondrial enzyme glutamate dehydrogenase, resulting in about 40% reduction of glucose-stimulated insulin secretion (22). The exact role of glutamate in β-cell function is still debated as the glutamate pathway might raise insulin release by participating in the amplifying pathway (19–21) and/or by relaying signals of protein abundance to mitochondria (23–25). In both models, glutamate should be transported in and out of the mitochondria by some putative mitochondrial carrier that remains to be identified in β-cells. Overall, better characterization of mitochondrial glutamate handling will contribute to our comprehension of mechanisms implicated in the control of insulin secretion.In this study, we identified glutamate carrier GC1 as being expressed in the inner mitochondrial membrane of insulinoma INS-1E cells as well as in primary rat islets. Adenovirus-mediated knockdown of GC1 by shRNA² demonstrated physiological functionality of GC1 in insulin secretion.     

Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number

Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.     

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown.     

The Human Gene SLC25A29, of Solute Carrier Family 25, Encodes a Mitochondrial Transporter of Basic Amino Acids

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.     

A Novel Member of Solute Carrier Family 25 (SLC25A42) Is a Transporter of Coenzyme A and Adenosine 3��,5��-Diphosphate in Human Mitochondria

Mitochondrial carriers are a family of proteins that transport metabolites, nucleotides, and cofactors across the inner mitochondrial membrane thereby connecting cytosolic and matrix functions. The essential cofactor coenzyme A (CoA) is synthesized outside the mitochondrial matrix and therefore must be transported into mitochondria where it is required for a number of fundamental processes. In this work we have functionally identified and characterized SLC25A42, a novel human member of the mitochondrial carrier family. The SLC25A42 gene (Haitina, T., Lindblom, J., Renström, T., and Fredriksson, R., 2006, Genomics 88, 779–790) was overexpressed in Escherichia coli, purified, and reconstituted into phospholipid vesicles. Its transport properties, kinetic parameters, and targeting to mitochondria demonstrate that SLC25A42 protein is a mitochondrial transporter for CoA and adenosine 3′,5′-diphosphate. SLC25A42 catalyzed only a counter-exchange transport, exhibited a high transport affinity for CoA, dephospho-CoA, ADP, and adenosine 3′,5′-diphosphate, was saturable and inhibited by bongkrekic acid and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A42 is to import CoA into mitochondria in exchange for intramitochondrial (deoxy)adenine nucleotides and adenosine 3′,5′-diphosphate. This is the first time that a mitochondrial carrier for CoA and adenosine 3′,5′-diphosphate has been characterized biochemically.The mitochondrial carrier family, or the solute carrier family 25 (SLC25),³ comprises a large group of proteins that transport a variety of substrates across the inner mitochondrial membrane and, in a few cases, across other membranes (1, 2). Common structural features of the mitochondrial carrier family members consist in a tripartite structure (three repeats of ∼100 amino acids), the presence of two transmembrane α-helices separated by hydrophilic loops in each repeat, and the presence of a signature motif at the C terminus of the first helix in each repeat (Ref. 3 and references therein). The SLC25 family is by far the largest of the currently known 43 SLC families. The Saccharomyces cerevisiae genome contains 35 members, that of Arabidopsis thaliana 58, and the human genome at least 48 SLC25 members. Until now, nearly 30 members and isoforms of this family have been identified in humans. These include the uncoupling protein and the carriers for ADP/ATP, phosphate, 2-oxoglutarate/malate, citrate, carnitine/acylcarnitine, dicarboxylates, ornithine and other basic amino acids, oxodicarboxylates, deoxynucleotides and thiamine pyrophosphate, aspartate-glutamate, glutamate, S-adenosylmethionine, ATP-Mg/Pi, pyrimidine nucleotides, and adenine nucleotides in peroxisomes (see Ref. 1 for a review and Refs. 4–8). The present investigation was undertaken to identify the function of SLC25A42, a novel member of the SLC25 family recently found in the human genome (9). SLC25A42 is 318 amino acids long and is highly expressed in virtually all tissues, in most at higher levels than many other SLC25 family members (9).In this study we provide direct evidence that SLC25A42 is a mitochondrial transporter for CoA and PAP. SLC25A42 was overexpressed in Escherichia coli, purified, reconstituted in phospholipid vesicles, and shown to transport CoA, dephospho-CoA, PAP, and (deoxy)adenine nucleotides with high specificity and by a counter-exchange mechanism. The main function of SLC25A42 is probably to catalyze the entry of CoA into the mitochondria in exchange for adenine nucleotides and PAP.     

Comparison of the oxidative phosphorylation (OXPHOS) nuclear genes in the genomes of Drosophila melanogaster, Drosophila pseudoobscura and Anopheles gambiae

Background

In eukaryotic cells, oxidative phosphorylation (OXPHOS) uses the products of both nuclear and mitochondrial genes to generate cellular ATP. Interspecies comparative analysis of these genes, which appear to be under strong functional constraints, may shed light on the evolutionary mechanisms that act on a set of genes correlated by function and subcellular localization of their products.

Results

We have identified and annotated the Drosophila melanogaster, D. pseudoobscura and Anopheles gambiae orthologs of 78 nuclear genes encoding mitochondrial proteins involved in oxidative phosphorylation by a comparative analysis of their genomic sequences and organization. We have also identified 47 genes in these three dipteran species each of which shares significant sequence homology with one of the above-mentioned OXPHOS orthologs, and which are likely to have originated by duplication during evolution. Gene structure and intron length are essentially conserved in the three species, although gain or loss of introns is common in A. gambiae. In most tissues of D. melanogaster and A. gambiae the expression level of the duplicate gene is much lower than that of the original gene, and in D. melanogaster at least, its expression is almost always strongly testis-biased, in contrast to the soma-biased expression of the parent gene.

Conclusions

Quickly achieving an expression pattern different from the parent genes may be required for new OXPHOS gene duplicates to be maintained in the genome. This may be a general evolutionary mechanism for originating phenotypic changes that could lead to species differentiation.     

Molecular identification and functional characterization of a novel glutamate transporter in yeast and plant mitochondria

The genome of Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family (MCF) and 58 MCF members are coded by the genome of Arabidopsis thaliana, most of which have been functionally characterized. Here two members of this family, Ymc2p from S. cerevisiae and BOU from Arabidopsis, have been thoroughly characterized. These proteins were overproduced in bacteria and reconstituted into liposomes. Their transport properties and kinetic parameters demonstrate that Ymc2p and BOU transport glutamate, and to a much lesser extent L-homocysteinesulfinate, but not other amino acids and many other tested metabolites. Transport catalyzed by both carriers was saturable, inhibited by mercuric chloride and dependent on the proton gradient across the proteoliposomal membrane. The growth phenotype of S. cerevisiae cells lacking the genes ymc2 and agc1, which encodes the only other S. cerevisiae carrier capable to transport glutamate besides aspartate, was fully complemented by expressing Ymc2p, Agc1p or BOU. Mitochondrial extracts derived from ymc2Δagc1Δ cells, reconstituted into liposomes, exhibited no glutamate transport at variance with wild-type, ymc2Δ and agc1Δ cells, showing that S. cerevisiae cells grown in the presence of acetate do not contain additional mitochondrial transporters for glutamate besides Ymc2p and Agc1p. Furthermore, mitochondria isolated from wild-type, ymc2Δ and agc1Δ strains, but not from the double mutant ymc2Δagc1Δ strain, swell in isosmotic ammonium glutamate showing that glutamate is transported by Ymc2p and Agc1p together with a H⁺. It is proposed that the function of Ymc2p and BOU is to transport glutamate across the mitochondrial inner membrane and thereby play a role in intermediary metabolism, C1 metabolism and mitochondrial protein synthesis.     

The circadian protein CLOCK regulates cell metabolism via the mitochondrial carrier SLC25A10

Physiological function and metabolic regulation are the most important outputs of circadian clock controls in mammals. Mitochondrial respiration and ROS production show rhythmic activity. Mitochondrial carriers, which are responsible for mitochondrial substance transfer, are vital for mitochondrial metabolism. Clock (Circadian Locomotor Output Cycles Kaput) is the first core circadian gene identified in mammalian animals. However, whether CLOCK protein can regulate mitochondrial functions via mitochondrial carriers is unclear. Here, we showed that CLOCK can bind to the mitochondrial carrier SLC25A10. For further analysis, we established a Slc25a10^−/−-Hepa1-6 cell line using CRISPR/Cas9 gene-editing technology. Slc25a10^−/−-Hepa1-6 cells showed disordered glucose homeostasis, increased oxidative stress levels, and damaged electron transport chains. Next, using an immunoprecipitation assay, we found that amino acids 43–84 and 169–210 in SLC25A10 are key sites that respond to CLOCK binding. Finally, forced expression of wild-type SLC25A10 in Slc25a10^−/−-Hepa1-6 cells could compensate for the loss of SLC25A10; the decreased glucose metabolism, severe oxidative stress and damaged electron transport chain were recovered. In addition, a mutant Slc25a10 with changes in two key sites did not show a rescue effect. In conclusion, we identified a new protein-protein interaction mechanism in which CLOCK can directly regulate cell metabolism via the mitochondrial membrane transporter SLC25A10. Our study might provide some new insights into the relationship between circadian clock and mitochondrial metabolism.     

SLC25A23 augments mitochondrial Ca2+ uptake,interacts with MCU,and induces oxidative stress–mediated cell death

Emerging findings suggest that two lineages of mitochondrial Ca²⁺ uptake participate during active and resting states: 1) the major eukaryotic membrane potential–dependent mitochondrial Ca²⁺ uniporter and 2) the evolutionarily conserved exchangers and solute carriers, which are also involved in ion transport. Although the influx of Ca²⁺ across the inner mitochondrial membrane maintains metabolic functions and cell death signal transduction, the mechanisms that regulate mitochondrial Ca²⁺ accumulation are unclear. Solute carriers—solute carrier 25A23 (SLC25A23), SLC25A24, and SLC25A25—represent a family of EF-hand–containing mitochondrial proteins that transport Mg-ATP/Pi across the inner membrane. RNA interference–mediated knockdown of SLC25A23 but not SLC25A24 and SLC25A25 decreases mitochondrial Ca²⁺ uptake and reduces cytosolic Ca²⁺ clearance after histamine stimulation. Ectopic expression of SLC25A23 EF-hand–domain mutants exhibits a dominant-negative phenotype of reduced mitochondrial Ca²⁺ uptake. In addition, SLC25A23 interacts with mitochondrial Ca²⁺ uniporter (MCU; CCDC109A) and MICU1 (CBARA1) while also increasing I_MCU. In addition, SLC25A23 knockdown lowers basal mROS accumulation, attenuates oxidant-induced ATP decline, and reduces cell death. Further, reconstitution with short hairpin RNA–insensitive SLC25A23 cDNA restores mitochondrial Ca²⁺ uptake and superoxide production. These findings indicate that SLC25A23 plays an important role in mitochondrial matrix Ca²⁺ influx.     

Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Mitochondrial Thymidine Kinase 2 Deficiency

A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK^+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK^+/−TK2^−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK^+/−TK2^−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency.     

Two Rapidly Evolving Genes Contribute to Male Fitness in Drosophila

Purifying selection often results in conservation of gene sequence and function. The most functionally conserved genes are also thought to be among the most biologically essential. These observations have led to the use of sequence conservation as a proxy for functional conservation. Here we describe two genes that are exceptions to this pattern. We show that lack of sequence conservation among orthologs of CG15460 and CG15323—herein named jean-baptiste (jb) and karr, respectively—does not necessarily predict lack of functional conservation. These two Drosophila melanogaster genes are among the most rapidly evolving protein-coding genes in this species, being nearly as diverged from their D. yakuba orthologs as random sequences are. jb and karr are both expressed at an elevated level in larval males and adult testes, but they are not accessory gland proteins and their loss does not affect male fertility. Instead, knockdown of these genes in D. melanogaster via RNA interference caused male-biased viability defects. These viability effects occur prior to the third instar for jb and during late pupation for karr. We show that putative orthologs to jb and karr are also expressed strongly in the testes of other Drosophila species and have similar gene structure across species despite low levels of sequence conservation. While standard molecular evolution tests could not reject neutrality, other data hint at a role for natural selection. Together these data provide a clear case where a lack of sequence conservation does not imply a lack of conservation of expression or function.     

Down-regulation of SLC25A20 promotes hepatocellular carcinoma growth and metastasis through suppression of fatty-acid oxidation

Solute carrier family 25 member 20 (SLC25A20) is a mitochondrial-membrane–carrier protein involved in the transport of acylcarnitines into mitochondrial matrix for oxidation. A previous-integrated-proteogenomic study had identified SLC25A20 as one of the top-three prognostic biomarkers in HCC. However, the expression and the biological function of SLC25A20 have not yet been investigated in HCC. In the present study, we found that SLC25A20 expression is frequently down-regulated in HCC cells mainly due to the up-regulation of miR-132-3p. Down-regulation of SLC25A20 is associated with a poor prognosis in patients with HCC. SLC25A20 suppressed HCC growth and metastasis, both in vitro and in vivo, by suppression of G1–S cell transition, epithelial-to-mesenchymal transition (EMT), and induction of cell apoptosis. Mechanistically, SLC25A20 down-regulation promoted HCC growth and metastasis through suppression of fatty-acid oxidation. Altogether, SLC25A20 plays a critical tumor-suppressive role in carcinogenesis of HCC; SLC25A20 may serve as a novel prognostic factor and therapeutic target for patients with HCC.Subject terms: Liver cancer, Liver cancer     

TRF4 is involved in polyadenylation of snRNAs in Drosophila melanogaster

The Saccharomyces cerevisiae poly(A) polymerases Trf4 and Trf5 are involved in an RNA quality control mechanism, where polyadenylated RNAs are degraded by the nuclear exosome. Although Trf4/5 homologue genes are distributed throughout multicellular organisms, their biological roles remain to be elucidated. We isolated here the two homologues of Trf4/5 in Drosophila melanogaster, named DmTRF4-1 and DmTRF4-2, and investigated their biological function. DmTRF4-1 displayed poly(A) polymerase activity in vitro, whereas DmTRF4-2 did not. Gene knockdown of DmTRF4-1 by RNA interference is lethal in flies, as is the case for the trf4 trf5 double mutants. In contrast, disruption of DmTRF4-2 results in viable flies. Cellular localization analysis suggested that DmTRF4-1 localizes in the nucleolus. Abnormal polyadenylation of snRNAs was observed in transgenic flies overexpressing DmTRF4-1 and was slightly increased by the suppression of DmRrp6, the 3′-5′ exonuclease of the nuclear exosome. These results suggest that DmTRF4-1 and DmRrp6 are involved in the polyadenylation-mediated degradation of snRNAs in vivo.     

The mitochondrial solute carrier SLC25A5 at Xq24 is a novel candidate gene for non-syndromic intellectual disability

Loss-of-function mutations in several different neuronal pathways have been related to intellectual disability (ID). Such mutations often are found on the X chromosome in males since they result in functional null alleles. So far, microdeletions at Xq24 reported in males always have been associated with a syndromic form of ID due to the loss of UBE2A. Here, we report on overlapping microdeletions at Xq24 that do not include UBE2A or affect its expression, in patients with non-syndromic ID plus some additional features from three unrelated families. The smallest region of overlap, confirmed by junction sequencing, harbors two members of the mitochondrial solute carrier family 25, SLC25A5 and SLC25A43. However, identification of an intragenic microdeletion including SLC25A43 but not SLC25A5 in a healthy boy excluded a role for SLC25A43 in cognition. Therefore, our findings point to SLC25A5 as a novel gene for non-syndromic ID. This highly conserved gene is expressed ubiquitously with high levels in cortex and hippocampus, and a presumed role in mitochondrial exchange of ADP/ATP. Our data indicate that SLC25A5 is involved in memory formation or establishment, which could add mitochondrial processes to the wide array of pathways that regulate normal cognitive functions.     

The phosphoCTD-interacting domain of Topoisomerase I

The N-terminal domain (NTD) of Drosophila melanogaster (Dm) Topoisomerase I has been shown to bind to RNA polymerase II, but the domain of RNAPII with which it interacts is not known. Using bacterially-expressed fusion proteins carrying all or half of the NTDs of Dm and human (Homo sapiens, Hs) Topo I, we demonstrate that the N-terminal half of each NTD binds directly to the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of the largest RNAPII subunit, Rpb1. Thus, the amino terminal segment of metazoan Topo I (1-157 for Dm and 1-114 for Hs) contains a novel phosphoCTD-interacting domain that we designate the Topo I-Rpb1 interacting (TRI) domain. The long-known in vivo association of Topo I with active genes presumably can be attributed, wholly or in part, to the TRI domain-mediated binding of Topo I to the phosphoCTD of transcribing RNAPII.     

The cys-loop ligand-gated ion channel superfamily of the honeybee, Apis mellifera

Members of the cys-loop ligand-gated ion channel (cys-loop LGIC) superfamily mediate neurotransmission in insects and are targets of successful insecticides. We have described the cys-loop LGIC superfamily of the honeybee, Apis mellifera, which is an important crop pollinator and a key model for social interaction. The honeybee superfamily consists of 21 genes, which is slightly smaller than that of Drosophila melanogaster comprising 23 genes. As with Drosophila, the honeybee possesses ion channels gated by acetylcholine, γ-amino butyric acid, glutamate and histamine as well as orthologs of the Drosophila pH-sensitive chloride channel (pHCl), CG8916, CG12344 and CG6927. Similar to Drosophila, honeybee cys-loop LGIC diversity is broadened by differential splicing which may also serve to generate species-specific receptor isoforms. These findings on Apis mellifera enhance our understanding of cys-loop LGIC functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. DQ667181–DQ667195.     

Deregulation of Genes Related to Iron and Mitochondrial Metabolism in Refractory Anemia with Ring Sideroblasts

The presence of SF3B1 gene mutations is a hallmark of refractory anemia with ring sideroblasts (RARS). However, the mechanisms responsible for iron accumulation that characterize the Myelodysplastic Syndrome with ring sideroblasts (MDS-RS) are not completely understood. In order to gain insight in the molecular basis of MDS-RS, an integrative study of the expression and mutational status of genes related to iron and mitochondrial metabolism was carried out. A total of 231 low-risk MDS patients and 81 controls were studied. Gene expression analysis revealed that iron metabolism and mitochondrial function had the highest number of genes deregulated in RARS patients compared to controls and the refractory cytopenias with unilineage dysplasia (RCUD). Thus mitochondrial transporters SLC25 (SLC25A37 and SLC25A38) and ALAD genes were over-expressed in RARS. Moreover, significant differences were observed between patients with SF3B1 mutations and patients without the mutations. The deregulation of genes involved in iron and mitochondrial metabolism provides new insights in our knowledge of MDS-RS. New variants that could be involved in the pathogenesis of these diseases have been identified.