RESUMEN
Gamma-butyrobetaine dioxygenase (BBOX1) is a catalyst for the conversion of gamma-butyrobetaine to l-carnitine, which is detected in normal renal tubules. The purpose of this study was to analyze the prognosis, immune response, and genetic alterations associated with low BBOX1 expression in patients with clear cell renal cell carcinoma (RCC). We analyzed the relative influence of BBOX1 on survival using machine learning and investigated drugs that can inhibit renal cancer cells with low BBOX1 expression. We analyzed clinicopathologic factors, survival rates, immune profiles, and gene sets according to BBOX1 expression in a total of 857 patients with kidney cancer from the Hanyang University Hospital cohort (247 cases) and The Cancer Genome Atlas (610 cases). We employed immunohistochemical staining, gene set enrichment analysis, in silico cytometry, pathway network analyses, in vitro drug screening, and gradient boosting machines. BBOX1 expression in RCC was decreased compared with that in normal tissues. Low BBOX1 expression was associated with poor prognosis, decreased CD8+ T cells, and increased neutrophils. In gene set enrichment analyses, low BBOX1 expression was related to gene sets with oncogenic activity and a weak immune response. In pathway network analysis, BBOX1 was linked to regulation of various T cells and programmed death-ligand 1. In vitro drug screening showed that midostaurin, BAY-61-3606, GSK690693, and linifanib inhibited the growth of RCC cells with low BBOX1 expression. Low BBOX1 expression in patients with RCC is related to short survival time and reduced CD8+ T cells; midostaurin, among other drugs, may have enhanced therapeutic effects in this context.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , gamma-Butirobetaína Dioxigenasa/genética , Pronóstico , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , BiomarcadoresRESUMEN
OBJECTIVE: Cervical cancer (CC) ranks fourth most diagnosed cancer and cancer mortality in women. Long non-coding RNAs (lncRNAs) take important roles in CC development. This study aimed to identify more and novel competing endogenous RNA (ceRNA) mechanisms of lncRNAs in CC. MATERIALS AND METHODS: The miRNA expression dataset GSE20592 and lncRNA/mRNA expression dataset GSE63514 were downloaded from Gene Expression Omnibus. The differentially expressed genes (DEGs), differentially expressed lncRNAs (DElncRNAs), and differentially expressed miRNAs (DEmiRNAs) between CC tumor and normal samples were identified with the criteria of adj.P.Value < 0.05 (Benjamini & Hochberg) and |log2(fold change)|>2. Functional enrichment analysis was performed for DEGs. The interaction pairs among lncRNAs, miRNAs and mRNAs were predicted and the ceRNA network was then constructed. Survival analysis was performed based on the TCGA dataset. RESULTS: Totally, 42 DEmiRNAs, 25 DElncRNAs, and 518 DEGs were identified in CC tumor samples versus normal tissues. The DEGs were associated with 'GO:0006260: DNA replication', 'GO:0051301: cell division', and 'hsa01100:Metabolic pathways'. The ceRNA network consisted of 878 lncRNA-miRNA-mRNA pairs. Of the miRNAs, lncRNAs, and genes with the top 10 interaction degrees in the ceRNA network, the upregulated cyclin dependent kinase inhibitor 2A gene (CDKN2A) was targeted by the downregulated DEmiRNAs including hsa-miR-125b-5p and hsa-miR-125a-5p, which were targeted by the upregulated DElncRNA BBOX1-AS1. The high expression level of CDKN2A contributed to the poor overall survival of patients with CC. CONCLUSIONS: The BBOX1-AS1-hsa-miR-125b-5p/hsa-miR-125a-5p-CDKN2A ceRNA network is of great value in CC development.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Redes Reguladoras de Genes/genética , MicroARNs/genética , Neoplasias del Cuello Uterino/genética , gamma-Butirobetaína Dioxigenasa/genética , Biomarcadores de Tumor/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Pronóstico , ARN Largo no Codificante/genética , ARN Mensajero/genética , Neoplasias del Cuello Uterino/mortalidadRESUMEN
BACKGROUND: Gastric cancer (GC) is one type of the most general malignancies in the globe. Research increasingly suggests long non-coding RNAs (lncRNAs) exert crucial roles in GC. However, the function of BBOX1-AS1 in GC has not been reported yet, it needs more explorations. AIMS: The aim of the study is to figure out the role and related regulation mechanism of BBOX1-AS1 in GC. METHODS: RT-qPCR assay was applied to detect genes expression. The role of BBOX1-AS1 in GC was investigated by cell counting kit-8, colony formation, tunel detection, and western blot assays. The binding ability between miR-3940-3p and BBOX1-AS1 (or BIRC5) by RIP, RNA pull-down and luciferase reporter assays. RESULTS: The expression of BBOX1-AS1 presented significantly upregulation in GC tissues and cells. Moreover, upregulation of BBOX1-AS1 promoted GC cell proliferation, and inhibited GC cell apoptosis. However, downregulation of BBOX1-AS1 led to opposite results. Furtherly, we discovered that BBOX1-AS1 bound with miR-3940-3p and also negatively regulated miR-3940-3p. Besides, it proved that miR-3940-3p interplayed with BIRC5 and negatively regulated BIRC5. Through rescue experiments, we proved that BIRC5 reversed miR-3940-3p-mediated cell proliferation or apoptosis in BBOX1-AS1-dysregulated GC cells. CONCLUSIONS: BBOX1-AS1 accelerates GC proliferation by sponging miR-3940-3p to upregulate BIRC5 expression, which may guide a new direction into the therapeutic strategies of GC.
Asunto(s)
MicroARNs/genética , Neoplasias Gástricas , Survivin/genética , gamma-Butirobetaína Dioxigenasa/genética , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Factores de Transcripción , Activación Transcripcional , Regulación hacia ArribaRESUMEN
OBJECTIVES: Over the past years, growing attention has been paid to deciphering the pivotal role of long non-coding RNAs (lncRNAs) in regulating the occurrence and development of human malignancies, cervical cancer (CC) included. Nonetheless, the regulatory role of lncRNA BBOX1 antisense RNA 1 (BBOX1-AS1) has not been explored as yet. MATERIAL AND METHODS: The expression of BBOX1-AS1 was detected by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8 (CCK-8), colony formation, TUNEL, Western blot, transwell and immunofluorescence assays testified the critical role of BBOX1-AS1 in CC. The relationship between RNAs (BBOX1-AS1, miR-361-3p, HOXC6 and HuR) was analysed by luciferase reporter, RNA Immunoprecipitation (RIP) and RNA pull-down assays. RESULTS: BBOX1 antisense RNA 1 antisense RNA 1 was revealed to be highly expressed in CC. Decreased expression of BBOX1-AS1 had suppressive effects on CC cell growth and migration. Molecular mechanism assays verified that BBOX1-AS1 had negative interaction with miR-361-3p in CC. Additionally, homeobox C6 (HOXC6) was validated to be a downstream target of miR-361-3p in CC. Furthermore, ELAV-like RNA-binding protein 1, also known as HuR, was uncovered to be capable of regulating the mRNA stability of HOXC6 in CC. More importantly, rescue assays delineated that knockdown of HuR after overexpressing miR-361-3p could reverse BBOX1-AS1 upregulation-mediated effect on CC progression. Similarly, the function induced by BBOX1-AS1 upregulation on CC progression could be countervailed by HOXC6 depletion. CONCLUSIONS: BBOX1 antisense RNA 1 facilitates CC progression by upregulating HOXC6 expression via miR-361-3p and HuR.
Asunto(s)
Proteína 1 Similar a ELAV/genética , Proteínas de Homeodominio/genética , MicroARNs/genética , ARN Largo no Codificante/genética , Regulación hacia Arriba/genética , Neoplasias del Cuello Uterino/genética , gamma-Butirobetaína Dioxigenasa/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Células HeLa , Humanos , Activación Transcripcional/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
BACKGROUND & AIMS: Nonalcoholic fatty liver disease is characterized by excessive hepatic accumulation of triglycerides. We aimed to identify metabolites that differ in plasma of patients with liver steatosis vs healthy individuals (controls) and investigate the mechanisms by which these might contribute to fatty liver in mice. METHODS: We obtained blood samples from 15 patients with liver steatosis and 15 controls from a single center in China (discovery cohort). We performed untargeted liquid chromatography with mass spectrometry analysis of plasma to identify analytes associated with liver steatosis. We then performed targeted metabolomic analysis of blood samples from 2 independent cohorts of individuals who underwent annual health examinations in China (1157 subjects with or without diabetes and 767 subjects with or without liver steatosis; replication cohorts). We performed mass spectrometry analysis of plasma from C57BL/6J mice, germ-free, and mice given antibiotics. C57BL/6J mice were given 0.325% (m/v) N,N,N-trimethyl-5-aminovaleric acid (TMAVA) in their drinking water and placed on a 45% high-fat diet (HFD) for 2 months. Plasma, liver tissues, and fecal samples were collected; fecal samples were analyzed by 16S ribosomal RNA gene sequencing. C57BL/6J mice with CRISPR-mediated disruption of the gene encoding γ-butyrobetaine hydroxylase (BBOX-knockout mice) were also placed on a 45% HFD for 2 months. Hepatic fatty acid oxidation (FAO) in liver tissues was determined by measuring liberation of 3H2O from [3H] palmitic acid. Liver tissues were analyzed by electron microscopy, to view mitochondria, and proteomic analyses. We used surface plasmon resonance analysis to quantify the affinity of TMAVA for BBOX. RESULTS: Levels of TMAVA, believed to be a metabolite of intestinal microbes, were increased in plasma from subjects with liver steatosis compared with controls, in the discovery and replication cohorts. In 1 replication cohort, the odds ratio for fatty liver in subjects with increased liver plasma levels of TMAVA was 1.82 (95% confidence interval [CI], 1.14-2.90; P = .012). Plasma from mice given antibiotics or germ-free mice had significant reductions in TMAVA compared with control mice. We found the intestinal bacteria Enterococcus faecalis and Pseudomonas aeruginosa to metabolize trimethyllysine to TMAVA; levels of trimethyllysine were significantly higher in plasma from patients with steatosis than controls. We found TMAVA to bind and inhibit BBOX, reducing synthesis of carnitine. Mice given TMAVA had alterations in their fecal microbiomes and reduced cold tolerance; their plasma and liver tissue had significant reductions in levels of carnitine and acyl-carnitine and their hepatocytes had reduced mitochondrial FAO compared with mice given only an HFD. Mice given TMAVA on an HFD developed liver steatosis, which was reduced by carnitine supplementation. BBOX-knockout mice had carnitine deficiency and decreased FAO, increasing uptake and liver accumulation of free fatty acids and exacerbating HFD-induced fatty liver. CONCLUSIONS: Levels of TMAVA are increased in plasma from subjects with liver steatosis. In mice, intestinal microbes metabolize trimethyllysine to TMAVA, which reduces carnitine synthesis and FAO to promote steatosis.
Asunto(s)
Bacterias/metabolismo , Microbioma Gastrointestinal , Intestinos/microbiología , Hígado/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Valeratos/metabolismo , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/sangre , Estudios de Casos y Controles , Estudios Transversales , Dieta Alta en Grasa , Disbiosis , Ácidos Grasos no Esterificados/metabolismo , Heces/microbiología , Femenino , Humanos , Lipólisis/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/microbiología , Enfermedad del Hígado Graso no Alcohólico/patología , Oxidación-Reducción , Regulación hacia Arriba , Valeratos/sangre , Valeratos/toxicidad , Adulto Joven , gamma-Butirobetaína Dioxigenasa/genética , gamma-Butirobetaína Dioxigenasa/metabolismoRESUMEN
Carnitine plays essential roles in intermediary metabolism. In non-vegetarians, most of carnitine sources (~75%) are obtained from diet whereas endogenous synthesis accounts for around 25%. Renal carnitine reabsorption along with dietary intake and endogenous production maintain carnitine homeostasis. The precursors for carnitine biosynthesis are lysine and methionine. The biosynthetic pathway involves four enzymes: 6-N-trimethyllysine dioxygenase (TMLD), 3-hydroxy-6-N-trimethyllysine aldolase (HTMLA), 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABADH), and γ-butyrobetaine dioxygenase (BBD). OCTN2 (organic cation/carnitine transporter novel type 2) transports carnitine into the cells. One of the major functions of carnitine is shuttling long-chain fatty acids across the mitochondrial membrane from the cytosol into the mitochondrial matrix for ß-oxidation. This transport is achieved by mitochondrial carnitine-acylcarnitine cycle, which consists of three enzymes: carnitine palmitoyltransferase I (CPT I), carnitine-acylcarnitine translocase (CACT), and carnitine palmitoyltransferase II (CPT II). Carnitine inborn errors of metabolism could result from defects in carnitine biosynthesis, carnitine transport, or mitochondrial carnitine-acylcarnitine cycle. The presentation of these disorders is variable but common findings include hypoketotic hypoglycemia, cardio(myopathy), and liver disease. In this review, the metabolism and homeostasis of carnitine are discussed. Then we present details of different inborn errors of carnitine metabolism, including clinical presentation, diagnosis, and treatment options. At the end, we discuss some of the causes of secondary carnitine deficiency.
Asunto(s)
Cardiomiopatías/genética , Carnitina/deficiencia , Carnitina/genética , Hiperamonemia/genética , Errores Innatos del Metabolismo/genética , Mitocondrias/enzimología , Enfermedades Musculares/genética , Aldehído Oxidorreductasas/genética , Cardiomiopatías/metabolismo , Carnitina/biosíntesis , Carnitina/metabolismo , Carnitina Aciltransferasas/genética , Carnitina O-Palmitoiltransferasa/genética , Humanos , Hiperamonemia/metabolismo , Mitocondrias/genética , Oxigenasas de Función Mixta/genética , Enfermedades Musculares/metabolismo , Oxidación-Reducción , Miembro 5 de la Familia 22 de Transportadores de Solutos/genética , gamma-Butirobetaína Dioxigenasa/genéticaRESUMEN
The biologically important carnitine biosynthesis pathway in humans proceeds via four enzymatic steps. The first step in carnitine biosynthesis is catalyzed by trimethyllysine hydroxylase (TMLH), a non-heme Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase, which catalyzes the stereospecific hydroxylation of (2S)-Nε-trimethyllysine to (2S,3S)-3-hydroxy-Nε-trimethyllysine. Here, we report biocatalytic studies on human TMLH and its 19 variants introduced through site-directed mutagenesis. Amino acid substitutions at the sites involved in binding of the Fe(II) cofactor, 2OG cosubstrate and (2S)-Nε-trimethyllysine substrate provide a basic insight into the binding requirements that determine an efficient TMLH-catalyzed conversion of (2S)-Nε-trimethyllysine to (2S,3S)-3-hydroxy-Nε-trimethyllysine. This work demonstrates the importance of the recognition sites that contribute to the enzymatic activity of TMLH: the Fe(II)-binding H242-D244-H389 residues, R391-R398 involved in 2OG binding and several residues (D231, N334 and the aromatic cage comprised of W221, Y217 and Y234) associated with binding of (2S)-Nε-trimethyllysine.
Asunto(s)
Oxigenasas de Función Mixta/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Biocatálisis , Carnitina/biosíntesis , Dominio Catalítico/genética , Humanos , Cinética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , gamma-Butirobetaína Dioxigenasa/química , gamma-Butirobetaína Dioxigenasa/genética , gamma-Butirobetaína Dioxigenasa/metabolismoRESUMEN
BACKGROUND: Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease (ESKD) in the world. Emerging evidence has shown that urinary mRNAs may serve as early diagnostic and prognostic biomarkers of DKD. In this article, we aimed to first establish a novel bioinformatics-based methodology for analyzing the "urinary kidney-specific mRNAs" and verify their potential clinical utility in DKD. METHODS: To select candidate mRNAs, a total of 127 Affymetrix microarray datasets of diabetic kidney tissues and other tissues from humans were compiled and analyzed using an integrative bioinformatics approach. Then, the urinary expression of candidate mRNAs in stage 1 study (n = 82) was verified, and the one with best performance moved on to stage 2 study (n = 80) for validation. To avoid potential detection bias, a one-step Taqman PCR assay was developed for quantification of the interested mRNA in stage 2 study. Lastly, the in situ expression of the selected mRNA was further confirmed using fluorescent in situ hybridization (FISH) assay and bioinformatics analysis. RESULTS: Our bioinformatics analysis identified sixteen mRNAs as candidates, of which urinary BBOX1 (uBBOX1) levels were significantly upregulated in the urine of patients with DKD. The expression of uBBOX1 was also increased in normoalbuminuric diabetes subjects, while remained unchanged in patients with urinary tract infection or bladder cancer. Besides, uBBOX1 levels correlated with glycemic control, albuminuria and urinary tubular injury marker levels. Similar results were obtained in stage 2 study. FISH assay further demonstrated that BBOX1 mRNA was predominantly located in renal tubular epithelial cells, while its expression in podocytes and urothelium was weak. Further bioinformatics analysis also suggested that tubular BBOX1 mRNA expression was quite stable in various types of kidney diseases. CONCLUSIONS: Our study provided a novel methodology to identify and analyze urinary kidney-specific mRNAs. uBBOX1 might serve as a promising biomarker of DKD. The performance of the selected urinary mRNAs in monitoring disease progression needs further validation.
Asunto(s)
Biología Computacional , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/orina , gamma-Butirobetaína Dioxigenasa/genética , gamma-Butirobetaína Dioxigenasa/orina , Biomarcadores/orina , Bases de Datos Genéticas , Femenino , Humanos , Riñón/metabolismo , Riñón/patología , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/orina , Reproducibilidad de los Resultados , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Carnitine is a key molecule in energy metabolism that helps transport activated fatty acids into the mitochondria. Its homeostasis is achieved through oral intake, renal reabsorption and de novo biosynthesis. Unlike dietary intake and renal reabsorption, the importance of de novo biosynthesis pathway in carnitine homeostasis remains unclear, due to lack of animal models and description of a single patient defective in this pathway. CASE PRESENTATION: We identified by array comparative genomic hybridization a 42 months-old girl homozygote for a 221 Kb interstitial deletions at 11p14.2, that overlaps the genes encoding Fibin and butyrobetaine-gamma 2-oxoglutarate dioxygenase 1 (BBOX1), an enzyme essential for the biosynthesis of carnitine de novo. She presented microcephaly, speech delay, growth retardation and minor facial anomalies. The levels of almost all evaluated metabolites were normal. Her serum level of free carnitine was at the lower limit of the reference range, while her acylcarnitine to free carnitine ratio was normal. CONCLUSIONS: We present an individual with a completely defective carnitine de novo biosynthesis. This condition results in mildly decreased free carnitine level, but not in clinical manifestations characteristic of carnitine deficiency disorders, suggesting that dietary carnitine intake and renal reabsorption are sufficient to carnitine homeostasis. Our results also demonstrate that haploinsufficiency of BBOX1 and/or Fibin is not associated with Primrose syndrome as previously suggested.
Asunto(s)
Carnitina/deficiencia , Proteínas Portadoras/genética , Deleción Cromosómica , Cromosomas Humanos Par 11 , Proteínas de la Membrana/genética , gamma-Butirobetaína Dioxigenasa/genética , Anomalías Múltiples/patología , Calcinosis/patología , Carnitina/metabolismo , Preescolar , Hibridación Genómica Comparativa , Enfermedades del Oído/patología , Femenino , Genoma Humano , Homocigoto , Humanos , Discapacidad Intelectual/patología , Atrofia Muscular/patologíaRESUMEN
BACKGROUND: Activation of peroxisome proliferator-activated receptor (PPAR)α and PPARδ causes an elevation of tissue carnitine concentrations through induction of genes involved in carnitine uptake [novel organic cation transporter 2, (OCTN2)], and carnitine biosynthesis [γ-butyrobetaine dioxygenase (BBD), 4-N-trimethyl-aminobutyraldehyde dehydrogenase (TMABA-DH)]. Recent studies showed that administration of the plasma lipid-lowering drug niacin causes activation of PPARα and/or PPARδ in tissues of obese Zucker rats, which have a compromised carnitine status and an impaired fatty acid oxidation capacity. Thus, we hypothesized that niacin administration to obese Zucker rats is also able to improve the diminished carnitine status of obese Zucker rats through PPAR-mediated stimulation of genes involved in carnitine uptake and biosynthesis. METHODS: To test this hypothesis, we used plasma, muscle and liver samples from a recent experiment with obese Zucker rats, which were fed either a niacin-adequate diet (30 mg niacin/kg diet) or a diet with a pharmacological niacin dose (780 mg niacin/kg diet), and determined concentrations of carnitine in tissues and mRNA and protein levels of genes critical for carnitine homeostasis (OCTN2, BBD, TMABA-DH). Statistical data analysis of all data was done by one-way ANOVA, and Fisher's multiple range test. RESULTS: Rats of the obese niacin group had higher concentrations of total carnitine in plasma, skeletal muscle and liver, higher mRNA and protein levels of OCTN2, BBD, and TMABA-DH in the liver and higher mRNA and protein levels of OCTN2 in skeletal muscle than those of the obese control group (P < 0.05), whereas rats of the obese control group had lower concentrations of total carnitine in plasma and skeletal muscle than lean rats (P < 0.05). CONCLUSION: The results show for the first time that niacin administration stimulates the expression of genes involved in carnitine uptake and biosynthesis and improves the diminished carnitine status of obese Zucker rats. We assume that the induction of genes involved in carnitine uptake and biosynthesis by niacin administration is mediated by PPAR-activation.
Asunto(s)
Carnitina/metabolismo , Expresión Génica/genética , Niacina/farmacología , Aldehído Oxidorreductasas/genética , Animales , Secuencia de Bases , Carnitina/biosíntesis , Carnitina/sangre , Cartilla de ADN , Hígado/metabolismo , Músculo Esquelético/metabolismo , Niacina/administración & dosificación , Obesidad/genética , Obesidad/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Ratas , Ratas Zucker , Reacción en Cadena en Tiempo Real de la Polimerasa , Miembro 5 de la Familia 22 de Transportadores de Solutos , gamma-Butirobetaína Dioxigenasa/genéticaRESUMEN
γ-Butyrobetaine hydroxylase (BBOX) catalyzes the conversion of gamma butyrobetaine (GBB) to l-carnitine, which is involved in the generation of metabolic energy from long-chain fatty acids. BBOX inhibitor 3-(1,1,1-trimethylhydrazin-1-ium-2-yl)propanoate (mildronate), which is an approved, clinically used cardioprotective drug, is a relatively poor BBOX inhibitor and requires high daily doses. In this paper we describe the design, synthesis, and properties of 51 compounds, which include both GBB and mildronate analogues. We have discovered novel BBOX inhibitors with improved IC50 values; the best examples are in the nanomolar range and about 2 orders of magnitude better when compared to mildronate. For six inhibitors, crystal structures in complex with BBOX have been solved to explain their activities and pave the way for further inhibitor design.
Asunto(s)
Carnitina/antagonistas & inhibidores , Carnitina/biosíntesis , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , Calorimetría , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Indicadores y Reactivos , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Metilhidrazinas/química , Metilhidrazinas/farmacología , Modelos Moleculares , Conformación Molecular , Unión Proteica , Proteínas Recombinantes/química , Relación Estructura-Actividad , gamma-Butirobetaína Dioxigenasa/genéticaRESUMEN
BACKGROUND: Carnitine is essential for mitochondrial ß-oxidation of long-chain fatty acids. Deficiency of carnitine leads to severe gut atrophy, ulceration and inflammation in animal models of carnitine deficiency. Genetic studies in large populations have linked mutations in the carnitine transporters OCTN1 and OCTN2 with Crohn's disease (CD), while other studies at the same time have failed to show a similar association and report normal serum carnitine levels in CD patients. METHODS: In this report, we have studied the expression of carnitine-synthesizing enzymes in intestinal epithelial cells to determine the capability of these cells to synthesize carnitine de novo. We studied expression of five enzymes involved in carnitine biosynthesis, namely 6-N-trimethyllysine dioxygenase (TMLD), 4-trimethylaminobutyraldehyde dehydrogenase (TMABADH), serine hydroxymethyltransferase 1 and 2 (SHMT1 and 2) and γ-butyrobetaine hydroxylase (BBH) by real-time PCR in mice (C3H strain). We also measured activity of γ-BBH in the intestine using an ex vivo assay and localized its expression by in situ hybridization. RESULTS: Our investigations show that mouse intestinal epithelium expresses all five enzymes required for de novo carnitine biosynthesis; the expression is localized mainly in villous surface epithelial cells throughout the intestine. The final rate-limiting enzyme γ-BBH is highly active in the small intestine; its activity was 9.7 ± 3.5 pmol/mg/min, compared to 22.7 ± 7.3 pmol/mg/min in the liver. CONCLUSIONS: We conclude that mouse gut epithelium is able to synthesize carnitine de novo. This capacity to synthesize carnitine in the intestine may play an important role in gut health and can help explain lack of clinical carnitine deficiency signs in subjects with mutations with OCTN transporters.
Asunto(s)
Carnitina/biosíntesis , Enterocitos/enzimología , Mucosa Intestinal/enzimología , Intestino Delgado/enzimología , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Animales , Carnitina/análisis , Expresión Génica , Glicina Hidroximetiltransferasa/genética , Glicina Hidroximetiltransferasa/metabolismo , Hibridación in Situ , Mucosa Intestinal/química , Intestino Delgado/química , Ratones , Ratones Endogámicos C3H , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , gamma-Butirobetaína Dioxigenasa/genética , gamma-Butirobetaína Dioxigenasa/metabolismoRESUMEN
OBJECTIVE: Gamma-butyrobetaine hydroxylase is an enzyme that catalyzes the last step in the biosynthesis of L-carnitine. We cloned, expressed and characterized a gamma-butyrobetaine hydroxylase gene bbh from Pseudomonas sp. L-1, to facilitate the production of L-carnitine using microorganisms. METHODS: We cloned bbh gene by PCR, and then cloned the open reading frame of bbh into pET-15b vector and expressed by Isopropyl beta-D-1-thiogalactopyranoside (IPTG) induction. After His-Bind Resin purification, the characteristics of BBH were studied. The three-dimensional structure of BBH monomer was modeled by SWISS-MODEL Workspace and resting cells were used for L-carnitine transformation. RESULTS: We cloned a gamma-butyrobetaine hydroxylase gene bbh (GenBank: JQ250036) from Pseudomonas sp. L-1 and expressed the gene in Escherichia coli BL21(DE3). BBH fusion protein was a homodimer, and the molecular weight of subunit was about 46.5kDa. The optimal temperature and pH was 30 degrees C and pH 7.5. The enzyme was stable below 45 degrees C. The enzyme was most stable at pH 6.0. We used resting cells of recombinant E. coli for L-carnitine biotransformation, after incubated at 30 degrees C and pH 7.0 for 31 h, the concentration of L-carnitine reached 12.7 mmol/L. CONCLUSION: The bbh gene from Pseudomonas sp. L-1 strain is remarkably different from that of reported one. The gamma-butyrobetaine hydroxylase expressed by this gene could effectively transform gamma-butyrobetaine for L-carnitine production. Beside by reporting of a bbh gene from bacteria, this research also provided a new process for biotransformation of L-carnitine.
Asunto(s)
Pseudomonas/enzimología , gamma-Butirobetaína Dioxigenasa/química , gamma-Butirobetaína Dioxigenasa/genética , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Pseudomonas/genética , Pseudomonas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Temperatura , gamma-Butirobetaína Dioxigenasa/biosíntesis , gamma-Butirobetaína Dioxigenasa/aislamiento & purificaciónRESUMEN
BACKGROUND: In rodents and pigs, it has shown that carnitine synthesis and uptake of carnitine into cells are regulated by peroxisome proliferator-activated receptor α (PPARA), a transcription factor which is physiologically activated during fasting or energy deprivation. Dairy cows are typically in a negative energy balance during early lactation. We investigated the hypothesis that genes of carnitine synthesis and uptake in dairy cows are enhanced during early lactation. RESULTS: mRNA abundances of PPARA and some of its classical target genes and genes involved in carnitine biosynthesis [trimethyllysine dioxygenase (TMLHE), 4-N-trimethylaminobutyraldehyde dehydrogenase (ALDH9A1), γ-butyrobetaine dioxygenase (BBOX1)] and uptake of carnitine [novel organic cation transporter 2 (SLC22A5)] as well as carnitine concentrations in liver biopsy samples of 20 dairy cows in late pregnancy (3 wk prepartum) and early lactation (1 wk, 5 wk, 14 wk postpartum) were determined. From 3 wk prepartum to 1 wk postpartum, mRNA abundances of PPARΑ and several PPARΑ target genes involved in fatty acid uptake, fatty acid oxidation and ketogenesis in the liver were strongly increased. Simultaneously, mRNA abundances of enzymes of carnitine synthesis (TMLHE: 10-fold; ALDH9A1: 6-fold; BBOX1: 1.8-fold) and carnitine uptake (SLC22A5: 13-fold) and the concentration of carnitine in the liver were increased from 3 wk prepartum to 1 wk postpartum (P < 0.05). From 1 wk to 5 and 14 wk postpartum, mRNA abundances of these genes and hepatic carnitine concentrations were declining (P < 0.05). There were moreover positive correlations between plasma concentrations of non-esterified fatty acids (NEFA) and hepatic carnitine concentrations at 1 wk, 5 wk and 14 wk postpartum (P < 0.05). CONCLUSIONS: The results of this study show for the first time that the expression of hepatic genes of carnitine synthesis and cellular uptake of carnitine is enhanced in dairy cows during early lactation. These changes might provide an explanation for increased hepatic carnitine concentrations observed in 1 wk postpartum and might be regarded as a physiologic means to provide liver cells with sufficient carnitine required for transport of excessive amounts of NEFA during a negative energy balance.
Asunto(s)
Carnitina/metabolismo , Bovinos/genética , Regulación Enzimológica de la Expresión Génica , Lactancia/genética , Hígado/metabolismo , PPAR alfa/genética , Aldehído Oxidorreductasas/biosíntesis , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Animales , Carnitina/sangre , Bovinos/metabolismo , Ingestión de Alimentos/fisiología , Femenino , Lactancia/metabolismo , Hígado/enzimología , Leche/química , Proteínas de Transporte de Catión Orgánico/biosíntesis , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , PPAR alfa/biosíntesis , PPAR alfa/metabolismo , Embarazo , ARN Mensajero/química , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Análisis de Regresión , gamma-Butirobetaína Dioxigenasa/biosíntesis , gamma-Butirobetaína Dioxigenasa/genética , gamma-Butirobetaína Dioxigenasa/metabolismoRESUMEN
The concentration of carnitine in plasma is generally increased with exercise training, suggesting that either carnitine biosynthesis is stimulated or renal reabsorption of carnitine is enhanced, or both. Carnitine, an essential cofactor in the oxidation of fatty acids, is released into the plasma following hydroxylation by γ-butyrobetaine hydroxylase (BBH), the final enzyme in the biosynthetic pathway found primarily in the liver. The organic cation transporter (OCTN2), the carnitine transporter found in kidney, is important in the distribution of carnitine by facilitating its renal reabsorption from urine. In this study, we tested the hypothesis that exercise training increases gene and protein expression of BBH and OCTN2, resulting in enhanced plasma carnitine levels. Male Wistar rats were subjected to 2 daily exercise sessions of treadmill running, 5 days per week, for a 10-week period. The concentration of total carnitine in plasma was significantly increased in trained rats compared with sedentary rats. In trained rats, mRNA and protein expression of BBH were increased in liver, whereas only BBH mRNA expression was increased in kidney. Liver of trained rats demonstrated increased mRNA and protein expression of OCTN2 compared with sedentary rats. In kidney of trained rats, however, only an increase in mRNA expression of OCTN2 was observed. Our results suggest that the improved plasma carnitine status in the trained rat is associated with increased carnitine biosynthesis in liver and kidney. The observation that OCTN2 expression was increased in kidney suggests a potential role of the kidney in the reabsorption of carnitine from the urine.
Asunto(s)
Regulación de la Expresión Génica , Riñón/metabolismo , Hígado/metabolismo , Actividad Motora , Proteínas de Transporte de Catión Orgánico/metabolismo , gamma-Butirobetaína Dioxigenasa/metabolismo , Animales , Carnitina/sangre , Carnitina/metabolismo , Riñón/enzimología , Hígado/enzimología , Masculino , Especificidad de Órganos , Proteínas de Transporte de Catión Orgánico/genética , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar , Miembro 5 de la Familia 22 de Transportadores de Solutos , Regulación hacia Arriba , gamma-Butirobetaína Dioxigenasa/genéticaRESUMEN
Convincing evidence from studies with peroxisome proliferator-activated receptor (PPAR)α-deficient mice suggested that the carnitine biosynthetic enzyme γ-butyrobetaine dioxygenase (BBD) is regulated by PPARα. However, the identification of BBD as a direct PPARα target gene as well as its exact regulation remained to be demonstrated. In silico-analysis of the mouse BBD promoter revealed seven putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using mutated and non-mutated serial 5'-truncation BBD promoter reporter constructs revealed that one PPRE located at -75 to -87 relative to the transcription start site in the proximal BBD promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα/RXRα heterodimer to this PPRE confirming that it is functional. In conclusion, the present study clearly shows that mouse BBD is a direct PPARα target gene and that transcriptional up-regulation of mouse BBD by PPARα is likely mediated by binding of the PPARα/RXR heterodimer to one PPRE located in its proximal promoter region. The results confirm emerging evidence from recent studies that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in both, carnitine synthesis and carnitine uptake.
Asunto(s)
PPAR alfa/fisiología , Regiones Promotoras Genéticas , Elementos de Respuesta/fisiología , gamma-Butirobetaína Dioxigenasa/genética , Animales , Carnitina/metabolismo , Células Hep G2 , Humanos , Ratones , Multimerización de Proteína , Receptor alfa X Retinoide/fisiologíaRESUMEN
Gamma-butyrobetaine hydroxylase (GBBH) is a 2-ketoglutarate-dependent dioxygenase that catalyzes the biosynthesis of l-carnitine by hydroxylation of gamma-butyrobetaine (GBB). l-carnitine is required for the transport of long-chain fatty acids into mitochondria for generating metabolic energy. The only known synthetic inhibitor of GBBH is mildronate (3-(2,2,2-trimethylhydrazinium) propionate dihydrate), which is a non-hydroxylatable analog of GBB. To aid in the discovery of novel GBBH inhibitors by rational drug design, we have solved the three-dimensional structure of recombinant human GBBH at 2.0A resolution. The GBBH monomer consists of a catalytic double-stranded beta-helix (DBSH) domain, which is found in all 2KG oxygenases, and a smaller N-terminal domain. Extensive interactions between two monomers confirm earlier observations that GBBH is dimeric in its biological state. Although many 2KG oxygenases are multimeric, the dimerization interface of GBBH is very different from that of related enzymes. The N-terminal domain of GBBH has a similar fold to the DUF971 superfamily, which consists of several short bacterial proteins with unknown function. The N-terminal domain has a bound Zn ion, which is coordinated by three cysteines and one histidine. Although several other 2KG oxygenases with known structures have more than one domain, none of them resemble the N-terminal domain of GBBH. The N-terminal domain may facilitate dimer formation, but its precise biological role remains to be discovered. The active site of the catalytic domain of GBBH is similar to that of other 2KG oxygenases, and Fe(II)-binding residues form a conserved His-X-Asp-X(n)-His triad, which is found in all related enzymes.
Asunto(s)
gamma-Butirobetaína Dioxigenasa/química , Dominio Catalítico , Cristalografía , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Metilhidrazinas/farmacología , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Zinc/química , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , gamma-Butirobetaína Dioxigenasa/genéticaRESUMEN
In contrast to other species, less is known about carnitine homeostasis in the pig. This study was performed to yield information about the site of carnitine synthesis and carnitine concentrations in various tissues of pigs (Sus scrofa). We found that among several pig tissues, a considerable activity of gamma-butyrobetaine dioxygenase (BBD), the last enzyme of carnitine synthesis, exists, like in humans and several other species, only in liver and kidney. Activity of that enzyme in liver and kidney was lower at birth than in the subsequent weeks of life. Highest carnitine concentrations were found in skeletal muscle and heart. Carnitine concentrations in plasma, liver and kidney at birth were higher than in the subsequent weeks of life in spite of the low BBD activity at birth. In conclusion, this study shows that liver and kidney are the major sites of carnitine synthesis and that neonatal pigs do not have an insufficient carnitine status.
Asunto(s)
Carnitina/análisis , Porcinos/metabolismo , gamma-Butirobetaína Dioxigenasa/metabolismo , Animales , Betaína/análogos & derivados , Betaína/análisis , Carnitina/biosíntesis , Dieta , Femenino , Corazón , Riñón/enzimología , Riñón/metabolismo , Hígado/enzimología , Hígado/metabolismo , Masculino , Datos de Secuencia Molecular , Músculo Esquelético/química , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular , gamma-Butirobetaína Dioxigenasa/genéticaRESUMEN
Carnitine is an essential metabolite that enables intracellular transport of fatty acids and acetyl units. Here we show that the yeast Candida albicans can synthesize carnitine de novo, and we identify the 4 genes of the pathway. Null mutants of orf19.4316 (trimethyllysine dioxygenase), orf19.6306 (trimethylaminobutyraldehyde dehydrogenase), and orf19.7131 (butyrobetaine dioxygenase) lacked their respective enzymatic activities and were unable to utilize fatty acids, acetate, or ethanol as a sole carbon source, in accordance with the strict requirement for carnitine-mediated transport under these growth conditions. The second enzyme of carnitine biosynthesis, hydroxytrimethyllysine aldolase, is encoded by orf19.6305, a member of the threonine aldolase (TA) family in C. albicans. A strain lacking orf19.6305 showed strongly reduced growth on fatty acids and was unable to utilize either acetate or ethanol, but TA activity was unaffected. Growth of the null mutants on nonfermentable carbon sources is restored only by carnitine biosynthesis intermediates after the predicted enzymatic block in the pathway, which provides independent evidence for a specific defect in carnitine biosynthesis for each of the mutants. In conclusion, we have genetically characterized a complete carnitine biosynthesis pathway in C. albicans and show that a TA family member is mainly involved in the aldolytic cleavage of hydroxytrimethyllysine in vivo.
Asunto(s)
Candida albicans/metabolismo , Carnitina/biosíntesis , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Secuencia de Aminoácidos , Candida albicans/genética , Candida albicans/crecimiento & desarrollo , Carnitina/química , Genes Fúngicos , Cinética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Aminoácido , gamma-Butirobetaína Dioxigenasa/genética , gamma-Butirobetaína Dioxigenasa/metabolismoRESUMEN
The kidney synthesizes L-carnitine and reabsorbs it via the Na(+)/L-carnitine cotransporter OCTN2. This study investigates the ontogeny of OCTN2, gamma-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) and gamma-butyrobetaine hydroxylase (BBH) in rat kidneys. Foetuses, newborn, suckling, weaning and adult rats were used. The apical membranes of foetal and newborn rat kidneys express OCTN2 transport activity, which is up-regulated by age. Maturation significantly increased the V(max) of this transport system without changing the apparent K(t), which excludes a maturation-related expression of different transporter isoforms. Northern analysis showed a 3.7kb transcript for OCTN2 in all the ages tested. Northern and RT-PCR assays revealed that maturation increased renal expression of OCTN2 mRNA. Foetuses express TMABA-DH mRNA and this expression increased during postnatal life. BBH mRNA, however, was detected during the suckling period onwards and its abundance was not changed significantly by maturation. This study reports for the first time that, in rat kidneys: (i) an apical OCTN2 transporter is active in rat foetuses, (ii) ontogeny up-regulates OCTN2 activity by increasing the density and/or turnover of the transporters, (iii) the maturation-related changes in OCTN2 are in part mediated by transcriptional mechanism(s) and (iv) the expression of both, TMABA-DH and BBH mRNA is ontogenically regulated. Some of these results were published as an abstract (García-Delgado et al., 2003).