Carnitine o-octanoyltransferase is a p53 target that promotes oxidative metabolism and cell survival following nutrient starvation.

Whereas it is known that p53 broadly regulates cell metabolism, the specific activities that mediate this regulation remain partially understood. Here, we identified carnitine o-octanoyltransferase (CROT) as a p53 transactivation target that is upregulated by cellular stresses in a p53-dependent manner. CROT is a peroxisomal enzyme catalyzing very long-chain fatty acids conversion to medium chain fatty acids that can be absorbed by mitochondria during ß-oxidation. p53 induces CROT transcription through binding to consensus response elements in the 5'-UTR of CROT mRNA. Overexpression of WT but not enzymatically inactive mutant CROT promotes mitochondrial oxidative respiration, while downregulation of CROT inhibits mitochondrial oxidative respiration. Nutrient depletion induces p53-dependent CROT expression that facilitates cell growth and survival; in contrast, cells deficient in CROT have blunted cell growth and reduced survival during nutrient depletion. Together, these data are consistent with a model where p53-regulated CROT expression allows cells to be more efficiently utilizing stored very long-chain fatty acids to survive nutrient depletion stresses.

In Silico Analysis of the Structural Dynamics and Substrate Recognition Determinants of the Human Mitochondrial Carnitine/Acylcarnitine SLC25A20 Transporter.

The Carnitine-Acylcarnitine Carrier is a member of the mitochondrial Solute Carrier Family 25 (SLC25), known as SLC25A20, involved in the electroneutral exchange of acylcarnitine and carnitine across the inner mitochondrial membrane. It acts as a master regulator of fatty acids ß-oxidation and is known to be involved in neonatal pathologies and cancer. The transport mechanism, also known as "alternating access", involves a conformational transition in which the binding site is accessible from one side of the membrane or the other. In this study, through a combination of state-of-the-art modelling techniques, molecular dynamics, and molecular docking, the structural dynamics of SLC25A20 and the early substrates recognition step have been analyzed. The results obtained demonstrated a significant asymmetry in the conformational changes leading to the transition from the c- to the m-state, confirming previous observations on other homologous transporters. Moreover, analysis of the MD simulations' trajectories of the apo-protein in the two conformational states allowed for a better understanding of the role of SLC25A20 Asp231His and Ala281Val pathogenic mutations, which are at the basis of Carnitine-Acylcarnitine Translocase Deficiency. Finally, molecular docking coupled to molecular dynamics simulations lend support to the multi-step substrates recognition and translocation mechanism already hypothesized for the ADP/ATP carrier.

Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells.

In eukaryotes, carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for ß-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for posttranslational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did ß-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MG cells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic posttranslational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase is believed to catalyze acetyl group transfer from LAC to coenzyme A, CRAT-/- U87MG cells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.

The Regulators of Peroxisomal Acyl-Carnitine Shuttle CROT and CRAT Promote Metastasis in Melanoma.

Circulating tumor cells are the key link between a primary tumor and distant metastases, but once in the bloodstream, loss of adhesion induces cell death. To identify the mechanisms relevant for melanoma circulating tumor cell survival, we performed RNA sequencing and discovered that detached melanoma cells and isolated melanoma circulating tumor cells rewire lipid metabolism by upregulating fatty acid (FA) transport and FA beta-oxidation‒related genes. In patients with melanoma, high expression of FA transporters and FA beta-oxidation enzymes significantly correlates with reduced progression-free and overall survival. Among the highest expressed regulators in melanoma circulating tumor cells were the carnitine transferases carnitine O-octanoyltransferase and carnitine acetyltransferase, which control the shuttle of peroxisome-derived medium-chain FAs toward mitochondria to fuel mitochondrial FA beta-oxidation. Knockdown of carnitine O-octanoyltransferase or carnitine acetyltransferase and short-term treatment with peroxisomal or mitochondrial FA beta-oxidation inhibitors thioridazine or ranolazine suppressed melanoma metastasis in mice. Carnitine O-octanoyltransferase and carnitine acetyltransferase depletion could be rescued by medium-chain FA supplementation, indicating that the peroxisomal supply of FAs is crucial for the survival of nonadherent melanoma cells. Our study identifies targeting the FA-based cross-talk between peroxisomes and mitochondria as a potential therapeutic opportunity to challenge melanoma progression. Moreover, the discovery of the antimetastatic activity of the Food and Drug Administration‒approved drug ranolazine carries translational potential.

An extensive bioinformatics study on the role of mitochondrial solute carrier family 25 in PC and its mechanism behind affecting immune infiltration and tumor energy metabolism.

BACKGROUND: Several metabolic disorders and malignancies are directly related to abnormal mitochondrial solute carrier family 25 (SLC25A) members activity. However, its biological role in pancreatic cancer (PC) is not entirely understood. METHODS: The lasso method was used to create a novel prognostic risk model for PC based on SLC25A members, and its roles in tumor immunology and energy metabolism were explored. Furthermore, co-expression networks were constructed for SLC25A11, SLC25A29, and SLC25A44. Single-cell RNA sequencing (ScRNA-seq) revealed the distribution of gene expression in PC. Tumor immune infiltration was examined with the TIMER database. Lastly, drug sensitivity was investigated, and co-transcriptional factors were predicted. RESULTS: In the present study, a novel prognostic risk model was established and validated for PC based on SLC25A members. The high-risk group had a lower activation of oxidative phosphorylation and a more abundant immune infiltration phenotype than the low-risk group. According to co-expression network studies, SLC25A11, SLC25A29, and SLC25A44 were involved in the energy metabolism of PC and prevented tumor growth, invasion, and metastasis. ScRNA-seq research also pointed to their contribution to the tumor microenvironment. Moreover, the recruitment of numerous immune cells was positively correlated with SLC25A11 and SLC25A44 but negatively correlated with SLC25A29. Additionally, the sensitivity to 20 Food and Drug Administration-approved antineoplastic medicines was strongly linked to the aforementioned genes, where cisplatin sensitivity increased with the up-regulation of SLC25A29. Finally, the Scleraxis BHLH Transcription Factor (SCX) and other proteins were hypothesized to co-regulate the mRNA transcription of the genes. CONCLUSION: SLC25A members are crucial for tumor immune and energy metabolism in PC, and SLC25A11, SLC25A29, and SLC25A44 can be used as favorable prognostic markers. The use of these markers will provide new directions to unravel their action mechanisms in PC.

Lipid emulsions attenuate the inhibition of carnitine acylcarnitine translocase induced by toxic doses of local anesthetics in rat cardiomyoblasts.

The aim of this study was to examine the effects of lipid emulsions on carnitine palmitoyltransferase I (CPT-I), carnitine acylcarnitine translocase (CACT), carnitine palmitoyltransferase II (CPT-II), and the mitochondrial dysfunctions induced by toxic doses of local anesthetics in H9c2 rat cardiomyoblasts. The effects of local anesthetics and lipid emulsions on the activities of CPT-I, CACT, and CPT-II, and concentrations of local anesthetics were examined. The effects of lipid emulsions, N-acetyl-L-cysteine (NAC), and mitotempo on the bupivacaine-induced changes in cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and intracellular calcium levels were examined. CACT, without significantly altering CPT-I and CPT-II, was inhibited by toxic concentration of local anesthetics. The levobupivacaine- and bupivacaine-induced inhibition of CACT was attenuated by all concentrations of lipid emulsion, whereas the ropivacaine-induced inhibition of CACT was attenuated by medium and high concentrations of lipid emulsion. The concentration of levobupivacaine was slightly attenuated by lipid emulsion. The bupivacaine-induced increase of ROS and calcium and the bupivacaine-induced decrease of MMP were attenuated by ROS scavengers NAC and mitotempo, and the lipid emulsion. Collectively, these results suggested that the lipid emulsion attenuated the levobupivacaine-induced inhibition of CACT, probably through the lipid emulsion-mediated sequestration of levobupivacaine.

Low Molecular Pectin Inhibited the Lipid Accumulation by Upregulation of METTL7B.

Inhibition of lipid accumulation is the key step to prevent nonalcoholic fatty liver (NAFL) progressing to nonalcoholic steatohepatitis. We aimed to study the effect of low-molecular-weight citrus pectin (LCP) against lipid accumulation and the underlying mechanism. Oleic acid (OA)-induced lipid deposition in HepG2 cells was applied to mimic in vitro model of lipid accumulation. Oil Red O (ORO) stain result showed lipid accumulation was significantly reduced, and levels of adipose triglyceride lipase (ATGL) and carnitine palmitoyltransferase-1 (CPT-1), involved in triacylglycerol catabolism and fatty acid ß-oxidation, detected by RT-qPCR were increased after OA-stimulated HepG2 cells treated with LCP. RNA sequencing analysis identified 740 differentially expressed genes (DEGs) in OA-stimulated HepG2 cells treated with the LCP group (OA+LCP group), and bioinformatics analysis indicated that some DEGs were enriched in lipid metabolism-related processes and pathways. The expression of the top 8 known DEGs in the OA+LCP group was then verified by RT-qPCR, which showed that fold change (abs) of METTL7B was the highest among the 8 candidates. In addition, overexpression of METTL7B in HepG2 cells significantly inhibited the lipid accumulation and enhanced levels of ATGL and CPT-1. In conclusion, LCP inhibited lipid accumulation through the upregulation of METTL7B, and further enhancement of ATGL and CPT-1 levels. LCP is expected to develop as a promising agent to ameliorate fat accumulation in NAFL.

Single Cell Gene Co-Expression Network Reveals FECH/CROT Signature as a Prognostic Marker.

Aberrant activation of signaling pathways is frequently observed and reported to be associated with the progression and poor prognosis of prostate cancer (PCa). We aimed to identify key biological processes regulated by androgen receptor (AR) using gene co-expression network from single cell resolution. The bimodal index was used to evaluate whether two subpopulations exist among the single cells. Gene expression among single cells revealed averaging pitfalls and bimodality pattern. Weighted gene co-expression network analysis (WGCNA) was used to identify modules of highly correlated genes. Twenty-nine gene modules were identified and AR-regulated modules were screened by significantly overlapping reported androgen induced differentially expressed genes. The biological function "generation of precursor metabolites and energy" was significantly enriched by AR-regulated modules with bimodality, presenting differential androgen response among subpopulations. Integrating with public ChIP-seq data, two genes FECH, and CROT has AR binding sites. Public in vitro studies also show that androgen regulates FECH and CROT. After receiving androgen deprivation therapy, patients lowly express FECH and CROT. Further survival analysis indicates that FECH/CROT signature can predict PCa recurrence. We reveal the heterogeneous function of "generation of precursor metabolites and energy" upon androgen stimulation from the perspective of single cells. Inhibitors targeting this biological process will facilitate to prevent prostate cancer progression.

Resveratrol shifts energy metabolism to increase lipid oxidation in healthy old mice.

OBJECTIVES: The objective of this work was to determine the specific mechanisms by which resveratrol inhibits lipogenesis and stimulates lipolysis. METHODS: Twelve male mice were individually introduced into a metabolic cage for 24 h to measure basal metabolic rate, prior to intervention. They were randomly divided into two groups, resveratrol (RSV) and control (C), and administered resveratrol intraperitoneally or vehicle, respectively, for two consecutive days. After 24 h, the metabolic energy expenditure was again determined for 24 h, before mice were sacrificed. Protein and gene expression of different enzymes related to metabolism in the hepatic tissue, adipose tissue and gastrocnemius of mice were analyzed by RT-PCR, western blot or ELISA. RESULTS: We report that resveratrol lowers the respiratory quotient in old mice and that this may be due to the activation of fatty acid mobilization from white adipose tissue (because hormone-activated lipase expression is increased) and fatty acid transport into mitochondria and eventual oxidation in muscle and liver (because transport enzymes and beta oxidation enzymes are also increased). Indeed, we have observed that resveratrol in vivo causes an increase in the expression and phosphorylation of AMPKα in liver, muscle and adipose tissue and an increase in the expression of acyl-CoA synthetase, of carnitine palmitoyl transferase 1 and of carnitine acylcarnitine translocase, all enzymes involved in lipid catabolism. On the other hand, the levels of acetyl-CoA carboxylase as well as those its product, i.e. malonyl CoA, are decreased. CONCLUSIONS: We conclude that a controlled dose of resveratrol activates fatty acid mobilization and degradation and inhibits fatty acid synthesis in old mice. This is the first time that these effects of resveratrol in lipid metabolism in healthy old (non-obese) animals are reported.

Clinical and molecular characteristics of carnitine-acylcarnitine translocase deficiency: Experience with six patients in Guangdong China.

Carnitine-acylcarnitine translocase deficiency (CACTD) is a rare autosomal recessive disorder of mitochondrial fatty acid oxidation that occurs due to mutations in the SLC25A20 gene. Severe CACTD results in neonatal or infantile sudden death. Herein, we reported six patients with CACTD diagnosed based on biochemical and molecular findings from 5 unrelated families in Guangdong from 2016 to 2017. Among them, five patients presented with hypotonia, nonketotic hypoglycemia, and arrhythmia 2 days after birth, while the other patient presented with respiratory distress, hypotonia, and arrhythmia. Five of the patients died in the neonatal period. Blood acylcarnitine concentrations determination from dried blood spots (DBS) were measured by tandem mass spectrometry (MS/MS). The SLC25A20 and CPT2 gene sequences were analyzed by direct Sanger sequencing. SLC25A20 gene analysis revealed a c.199-10T>G (IVS2-10T>G) homozygous variants in four unrelated patients and a novel mutation c.199-10T>G/c.719-8_c.719-1dupCCCACAG compound heterozygous variants in twins. This report describes the clinical characteristics, biochemical findings and molecular analysis of SLC25A20 gene of patients with CACTD in Guangdong. And our results show that the c.199-10T>G is likely the most common variant of CACTD in Guangdong population as it accounts for 83% (10/12) of the observed mutant alleles. Individuals with the c.199-10T>G genotype had a severe CACTD phenotype.

Human mitochondrial carnitine acylcarnitine carrier: Molecular target of dietary bioactive polyphenols from sweet cherry (Prunus avium L.).

The effect of polyphenols, recognized as the principal antioxidant and beneficial molecules introduced with the diet, extracted from sweet cherry (Prunus avium L.) on the recombinant human mitochondrial carnitine/acylcarnitine transporter (CACT) has been studied in proteoliposomes. CACT transport activity, which was strongly impaired after oxidation by atmospheric O2 or H2O2, due to the formation of a disulfide bridge between cysteines 136 and 155, was restored by externally added polyphenols. CACT reduction by polyphenols was time dependent. Spectroscopic analysis of polyphenolic extracts revealed eight most represented compounds in four cultivars. Molecular docking of CACT structural omology model with the most either abundant and arguably bio-available phenolic compound (trans 3-O-feruloyl-quinic acid) of the mix, is in agreement with the experimental data since it results located in the active site close to cysteine 136 at the bottom of the translocation aqueous cavity.

Plasma metabonomics investigation reveals involvement of fatty acid oxidation in hematotoxicity in Chinese benzene-exposed workers with low white blood cell count.

Benzene is an environmental and occupational contaminant. Health hazards associated with occupational benzene exposure is a major public health problem in China. In this study, we analyzed metabolite profiles among plasma samples collected from benzene-exposed workers with low white blood cell count (BLWs) and healthy controls using high-performance liquid chromatography-time-of-flight mass spectrometry. To screen potential benzene hematotoxicity biomarkers and metabolic pathways, principal component analysis was used to examine metabolite profile changes in plasma samples. The alterations in fatty acid oxidation (FAO) pathway were consistent with our previous findings in a mouse model; hence, two key genes were selected and verified in WBC samples. A total of nine identified metabolites were significantly changed in BLWs, which were involved in glutathione metabolism, porphyrin metabolism, lipid metabolism pathway, and FAO metabolism. Furthermore, compared with healthy controls, the mRNA expressions of carnitine acyltransferase (CRAT) and ACADVL were significantly increased in BLWs. Particularly, WBC counts was negatively correlated with the expression of AVADVL in BLWs. These aberrant metabolites could act as potential biomarkers for benzene hematotoxicity. In addition, fatty acid oxidation pathway may play a critical role in the development of hematotoxicity caused by benzene.

The carnitine system and cancer metabolic plasticity.

Metabolic flexibility describes the ability of cells to respond or adapt its metabolism to support and enable rapid proliferation, continuous growth, and survival in hostile conditions. This dynamic character of the cellular metabolic network appears enhanced in cancer cells, in order to increase the adaptive phenotype and to maintain both viability and uncontrolled proliferation. Cancer cells can reprogram their metabolism to satisfy the energy as well as the biosynthetic intermediate request and to preserve their integrity from the harsh and hypoxic environment. Although several studies now recognize these reprogrammed activities as hallmarks of cancer, it remains unclear which are the pathways involved in regulating metabolic plasticity. Recent findings have suggested that carnitine system (CS) could be considered as a gridlock to finely trigger the metabolic flexibility of cancer cells. Indeed, the components of this system are involved in the bi-directional transport of acyl moieties from cytosol to mitochondria and vice versa, thus playing a fundamental role in tuning the switch between the glucose and fatty acid metabolism. Therefore, the CS regulation, at both enzymatic and epigenetic levels, plays a pivotal role in tumors, suggesting new druggable pathways for prevention and treatment of human cancer.

Elevated mitochondrial SLC25A29 in cancer modulates metabolic status by increasing mitochondria-derived nitric oxide.

Warburg effect has been recognized as a hallmark of cancer cells for many years, but its modulation mechanism remains a great focus. Our current study found a member of solute carrier family 25 (SLC25A29), the main arginine transporter on mitochondria, significantly elevated in various cancer cells. Knockout of SLC25A29 by CRISPR/Cas9 inhibited proliferation and migration of cancer cells both in vitro and in vivo. SLC25A29-knockout cells also showed an altered metabolic status with enhanced mitochondrial respiration and reduced glycolysis. All of above impacts could be reversed after rescuing SLC25A29 expression in SLC25A29-knockout cells. Arginine is transported into mitochondria partly for nitric oxide (NO) synthesis. Deletion of SLC25A29 resulted in severe decrease of NO production, indicating that the mitochondria is a significant source of NO. SLC25A29-knockout cells dramatically altered the variation of metabolic processes, whereas addition of arginine failed to reverse the effect, highlighting the necessity of transporting arginine into mitochondria by SLC25A29. In conclusion, aberrant elevated SLC25A29 in cancer functioned to transport more arginine into mitochondria, improved mitochondria-derived NO levels, thus modulated metabolic status to facilitate increased cancer progression.

The safety of Lipistart, a medium-chain triglyceride based formula, in the dietary treatment of long-chain fatty acid disorders: a phase I study.

BACKGROUND: Children with long-chain fatty acid ß-oxidation disorders (LCFAOD) presenting with clinical symptoms are treated with a specialist infant formula, with medium chain triglyceride (MCT) mainly replacing long chain triglyceride (LCT). It is essential that the safety and efficacy of any new specialist formula designed for LCFAOD be tested in infants and children. METHODS: In an open-label, 21-day, phase I trial, we studied the safety of a new MCT-based formula (feed 1) in six well-controlled children (three male), aged 7-13 years (median 9 years) with LCFAOD (very long chain acyl CoA dehydrogenase deficiency [VLCADD], n=2; long chain 3-hydroxyacyl CoA dehydrogenase deficiency [LCHADD], n=2; carnitine acyl carnitine translocase deficiency [CACTD], n=2). Feed 1 (Lipistart; Vitaflo) contained 30% energy from MCT, 7.5% LCT and 3% linoleic acid and it was compared with a conventional MCT feed (Monogen; Nutricia) (feed 2) containing 17% energy from MCT, 3% LCT and 1.1% linoleic acid. Subjects consumed feed 2 for 7 days then feed 1 for 7 days and finally resumed feed 2 for 7 days. Vital signs, blood biochemistry, ECG, weight, height, food/feed intake and symptoms were monitored. RESULTS: Five subjects completed the study. Their median daily volume of both feeds was 720 mL (range 500-1900 mL/day). Feed 1 was associated with minimal changes in tolerance, free fatty acids (FFA), acylcarnitines, 3-hydroxybutyrate (3-HB), creatine kinase (CK), blood glucose, liver enzymes and no change in an electrocardiogram (ECG). No child complained of muscle pain or symptoms associated with LCFAOD on either feed. CONCLUSIONS: This is the first safety trial reported of an MCT formula specifically designed for infants and children with LCFAOD. In this short-term study, it appeared safe and well tolerated in this challenging group.

Nitric oxide inhibits the mitochondrial carnitine/acylcarnitine carrier through reversible S-nitrosylation of cysteine 136.

S-nitrosylation of the mitochondrial carnitine/acylcarnitine transporter (CACT) has been investigated on the native and the recombinant proteins reconstituted in proteoliposomes, and on intact mitochondria. The widely-used NO-releasing compound, GSNO, strongly inhibited the antiport measured in proteoliposomes reconstituted with the native CACT from rat liver mitochondria or the recombinant rat CACT over-expressed in E. coli. Inhibition was reversed by the reducing agent dithioerythritol, indicating a reaction mechanism based on nitrosylation of Cys residues of the CACT. The half inhibition constant (IC50) was very similar for the native and recombinant proteins, i.e., 74 and 71µM, respectively. The inhibition resulted to be competitive with respect the substrate, carnitine. NO competed also with NEM, correlating well with previous data showing interference of NEM with the substrate transport path. Using a site-directed mutagenesis approach on Cys residues of the recombinant CACT, the target of NO was identified. C136 plays a major role in the reaction mechanism. The occurrence of S-nitrosylation was demonstrated in intact mitochondria after treatment with GSNO, immunoprecipitation and immunostaining of CACT with a specific anti NO-Cys antibody. In parallel samples, transport activity of CACT measured in intact mitochondria, was strongly inhibited after GSNO treatment. The possible physiological and pathological implications of the post-translational modification of CACT are discussed.

The contribution of the citrate pathway to oxidative stress in Down syndrome.

Inflammatory conditions and oxidative stress have a crucial role in Down syndrome (DS). Emerging studies have also reported an altered lipid profile in the early stages of DS. Our previous works demonstrate that citrate pathway activation is required for oxygen radical production during inflammation. Here, we find up-regulation of the citrate pathway and down-regulation of carnitine/acylcarnitine carrier and carnitine palmitoyl-transferase 1 genes in cells from children with DS. Interestingly, when the citrate pathway is inhibited, we observe a reduction in oxygen radicals as well as in lipid peroxidation levels. Our preliminary findings provide evidence for a citrate pathway dysregulation, which could be related to some phenotypic traits of people with DS.

The mitochondrial carnitine/acylcarnitine carrier is regulated by hydrogen sulfide via interaction with C136 and C155.

BACKGROUND: The carnitine/acylcarnitine carrier (CAC or CACT) mediates transport of acylcarnitines into mitochondria for the ß-oxidation. CAC possesses Cys residues which respond to redox changes undergoing to SH/disulfide interconversion. METHODS: The effect of H2S has been investigated on the [(3)H]carnitine/carnitine antiport catalyzed by recombinant or native CAC reconstituted in proteoliposomes. Site-directed mutagenesis was employed for identifying Cys reacting with H2S. RESULTS: H2S led to transport inhibition, which was dependent on concentration, pH and time of incubation. Best inhibition with IC50 of 0.70 µM was observed at physiological pH after 30-60 min incubation. At longer times of incubation, inhibition was reversed. After oxidation of the carrier by O2, transport activity was rescued by H2S indicating that the inhibition/activation depends on the initial redox state of the protein. The observed effects were more efficient on the native rat liver transporter than on the recombinant protein. Only the protein containing both C136 and C155 responded to the reagent as the WT. While reduced responses were observed in the mutants containing C136 or C155. Multi-alignment of known mitochondrial carriers, highlighted that only the CAC possesses both Cys residues. This correlates well with the absence of effects of H2S on carriers which does not contain the Cys couple. CONCLUSIONS: Altogether, these data demonstrate that H2S regulates the CAC by inhibiting or activating transport on the basis of the redox state of the protein. GENERAL SIGNIFICANCE: CAC represents a specific target of H2S among mitochondrial carriers in agreement with the presence of a reactive Cys couple.

Detection of miR-33 Expression and the Verification of Its Target Genes in the Fatty Liver of Geese.

BACKGROUND: miRNAs are single-stranded, small RNA molecules with a length of 18-25 nucleotides. They bind to the 3' untranslated regions of mRNA transcripts to reduce the translation of these transcripts or to cause their degradation. The roles of these molecules differ in biological processes, such as cell differentiation, proliferation, apoptosis and tumor genesis. miRNA-33 is encoded by the gene introns of proteins that bind sterol-regulatory elements. This molecule cooperates with these proteins to control cholesterol homeostasis, fatty acid levels and the genes that are related to the expression of fat metabolism. The examination of miR-33 expression and its target genes can promote the in-depth study of the miRNA regulation mechanism in the formation process of goose fatty liver and can lay a foundation for research into human fatty liver. METHODOLOGY/PRINCIPAL FINDINGS: (1) Through real-time fluorescent quantitative polymerase chain reaction (TaqMan MicroRNA Assay), we detected the expression of miR-33 during the feeding of Landes geese. The expression level of miR-33 increases significantly in the liver after 19 days in comparison with the control group; (2) By using the bioinformatics software programs TargetScan, miRDB and miRCosm to predict the target genes of miR-33 according to laboratory prophase transcriptome results and references, we screen nine target genes: adenosine triphosphate binding cassette transporters A1, adenosine triphosphate binding cassette transporters G1, Neimann Pick C, carnitine O-octanoyltransferase (CROT), cyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, beta subunit (HADHB), AMP-activated protein kinase, alpha subunit 1 (AMPKα1), insulin receptor substrate 2, glutamic pyruvate transaminase and adipose differentiation-related protein. The dual luciferase reporter gene system in the CHO cell line verifies that CROT, HADHB and NPC1 are the target genes of miR-33 in geese. The inhibition rate of CROT is highest and reaches 70%; (3) The seed sequence (5' 2-8 bases) is the acting site of miR-33. The two predicted target sites of CROT are the target sites of miR-33. Moreover, the predicted target site of HADHB and NPC1 is the target site of miR-33. CONCLUSIONS/SIGNIFICANCE: (1) After 19 days of overfeeding, the expression level of miR-33 increases significantly in the livers of geese; (2) CROT, HADHB and NPC1 are the target genes of miR-33 in geese. These genes determine the combined target site.

Mitochondrial carnitine/acylcarnitine translocase: insights in structure/ function relationships. Basis for drug therapy and side effects prediction.

The mitochondrial carnitine/acylcarnitine translocase has been identified, purified and reconstituted in liposomes in 1990. Since that time it has been object of studies aimed to characterize its function and to define the molecular determinants of the translocation pathway. Thanks to these tenacious studies the molecular map of the amino acids involved in the catalysis has been constructed and the roles of critical residues in the translocation pathway have been elucidated. This has been possible through the combination of transport assay in reconstituted liposomes, site-directed mutagenesis, chemical labeling and bioinformatics. Recently some molecules which modulate CACT activity have been identified, such as glutathione and hydrogen peroxide, constituting some of the few cases of control mechanisms of mitochondrial carriers. The vast knowledge on the carnitine/acylcarnitine translocase is essential both as a progress in basic science and as instrument to foresee therapeutic or toxic effects of xenobiotics and drugs. Such studies have been already started pointing out the inhibitory action of drugs such as K(+)/H(+)-ATPase inhibitors (omeprazole) or antibiotics (ß-lactams) on the carnitine/acylcarnitine translocase, which can explain some of their adverse effects.