RESUMEN
Targeting phospholipid biosynthesis, specifically phosphatidylcholine (PC), which is enhanced in tumor cells, has been proven a suitable antitumor strategy. In fact, the overexpression of the choline kinase α1 (ChoKα1) isoform has been found in malignant cells and tumors, thus becoming an excellent antitumor target. ChoKα1 inhibitors are being synthesized at the present that show a large inhibitory activity. Two of them have been chosen in this study as representatives of different structural families: a biscationic biphenyl derivative of thieno[3,2-d]pyrimidinium substituted with a cyclic amine (here referred to as Fa22) and a biscationic biphenyl thioethano derivative of 7-chloro-quinolinium substituted with a pyrrolidinic moiety (here referred to as PL48). However, the potential use of these types of compounds in systemic treatments is hampered because of their low specificity. In fact, to enter the cell and reach their target, these inhibitors use choline transporters and inhibit choline uptake, being that one of the causes of their toxicity. One way to solve this problem could be allowing their entrance into the cells by alternative ways. With this goal, MamC-mediated magnetic nanoparticles (BMNPs), already proven effective drug nanocarriers, have been used to immobilize Fa22 and PL48. The idea is to let BMNPs enter the cell (they enter the cell by endocytosis) carrying these molecules, and, therefore, offering another way in for these compounds. In the present study, we demonstrate that the coupling of Fa22 and PL48 to BMNPs allows these molecules to enter the tumoral cell without completely inhibiting choline uptake, so, therefore, the use of Fa22 and PL48 in these nanoformulations reduces the toxicity compared to that of the soluble drugs. Moreover, the nanoassemblies Fa22-BMNPs and PL48-BMNPs allow the combination of chemotherapy and local hyperthermia therapies for a enhanced cytotoxic effect on the tumoral HepG2 cell line. The consistency of the results, independently of the drug structure, may indicate that this behavior could be extended to other ChoKα1 inhibitors, opening up a possibility for their potential use in clinics.
Asunto(s)
Colina Quinasa , Humanos , Colina Quinasa/antagonistas & inhibidores , Colina Quinasa/metabolismo , Estructura Molecular , Nanopartículas de Magnetita/química , Relación Estructura-Actividad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Materiales Biomiméticos/síntesis química , Proliferación Celular/efectos de los fármacosRESUMEN
Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in CHKB, encoding the first step in the synthesis of PC, is the cause of a rostrocaudal muscular dystrophy in both humans and mice. Loss of sarcolemma integrity is a hallmark of muscular dystrophies; however, how this occurs in the absence of choline kinase function is not known. We determine that in Chkb -/- mice there is a failure of the α7ß1 integrin complex that is specific to affected muscle. We observed that in Chkb -/- hindlimb muscles there is a decrease in sarcolemma association/abundance of the PI(4,5)P2 binding integrin complex proteins vinculin, and α-actinin, and a decrease in actin association with the sarcolemma. In cells, pharmacological inhibition of choline kinase activity results in internalization of a fluorescent PI(4,5)P2 reporter from discrete plasma membrane clusters at the cell surface membrane to cytosol, this corresponds with a decreased vinculin localization at plasma membrane focal adhesions that was rescued by overexpression of CHKB.
Asunto(s)
Colina Quinasa , Integrinas , Ratones Noqueados , Distrofias Musculares , Sarcolema , Vinculina , Animales , Ratones , Vinculina/metabolismo , Vinculina/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/genética , Integrinas/metabolismo , Colina Quinasa/metabolismo , Colina Quinasa/genética , Sarcolema/metabolismo , Humanos , Adhesiones Focales/metabolismo , Membrana Celular/metabolismo , Actinina/metabolismo , Actinina/genética , Músculo Esquelético/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Actinas/metabolismo , Modelos Animales de EnfermedadAsunto(s)
Cardiomiopatía Dilatada , Insuficiencia Cardíaca , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , Regulación hacia Abajo , Corazón , Insuficiencia Cardíaca/genética , Aldehído Deshidrogenasa Mitocondrial/genética , Colina Quinasa/genética , Colina Quinasa/metabolismoRESUMEN
PURPOSE: Entamoeba histolytica is one of the death-causing parasites in the world. Study on its lipid composition revealed that it is predominated by phosphatidylcholine and phosphatidylethanolamine. Further study revealed that its phosphorylated metabolites might be produced by the Kennedy pathway. Here, we would like to report on the characterizations of enzymes from this pathway that would provide information for the design of novel inhibitors against these enzymes in future. METHODOLOGY: E. histolytica HM-1:IMSS genomic DNA was isolated and two putative choline/ethanolamine kinase genes (EhCK1 and EhCK2) were cloned and expressed from Escherichia coli BL21 strain. Enzymatic characterizations were further carried out on the purified enzymes. RESULTS: EhCK1 and EhCK2 were identified from E. histolytica genome. The deduced amino acid sequences were more identical to its homologues in human (35-48%) than other organisms. The proteins were clustered as ethanolamine kinase in the constructed phylogeny tree. Sequence analysis showed that they possessed all the conserved motifs in choline kinase family: ATP-binding loop, Brenner's phosphotransferase motif, and choline kinase motif. Here, the open reading frames were cloned, expressed, and purified to apparent homogeneity. EhCK1 showed activity with choline but not ethanolamine. The biochemical characterization showed that it had a Vmax of 1.9 ± 0.1 µmol/min/mg. Its Km for choline and ATP was 203 ± 26 µM and 3.1 ± 0.4 mM, respectively. In contrast, EhCK2 enzymatic activity was only detected when Mn2+ was used as the co-factor instead of Mg2+ like other choline/ethanolamine kinases. Highly sensitive and specific antibody against EhCK1 was developed and used to confirm the endogenous EhCK1 expression using immunoblotting. CONCLUSIONS: With the understanding of EhC/EK importance in phospholipid metabolism and their unique characteristic, EhC/EK could be a potential target for future anti-amoebiasis study.
Asunto(s)
Colina Quinasa , Entamoeba histolytica , Filogenia , Entamoeba histolytica/genética , Entamoeba histolytica/enzimología , Colina Quinasa/genética , Colina Quinasa/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Clonación Molecular , Secuencia de Aminoácidos , Escherichia coli/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Etanolaminas/metabolismo , Colina/metabolismoRESUMEN
The synovial intimal lining is mainly governed by fibroblast-like synoviocytes (FLS), which portray a transformed tumor-like phenotype in rheumatoid arthritis (RA). Among the diverse cytokines that engender FLS, interleukin-21 (IL-21) was reported to stimulate hyperproliferation and perpetuate inflammation. Recently, choline kinase (ChoKα) has been reported to be an essential enzyme aiding RA-FLS hyperproliferation by altering phosphatidylcholine biosynthesis. The current study aimed to elucidate the therapeutic efficacy of myricetin, a flavonoid, in abating the IL-21-induced tumor-like phenotype of adjuvant-induced arthritis (AIA)-FLS via the ChoKα signaling cascade. Our results showed that myricetin suppressed IL-21 receptor expression and activation of the ChoKα signaling cascade (N-Ras, Ral-GDS, and PI3K) in IL-21-induced AIA-FLS. Consequently, myricetin treatment decreased ChoKα and PLD2 enzymatic activity and inhibited the proliferative, migratory, and invasive properties of AIA-FLSs. Our results demonstrated that myricetin could be a promising anti-arthritic compound by abating IL-21-induced hyperproliferation, migration, and invasive behavior of AIA-FLS by downregulating the ChoKα signaling cascade.
Asunto(s)
Artritis Experimental , Artritis Reumatoide , Neoplasias , Sinoviocitos , Animales , Sinoviocitos/metabolismo , Membrana Sinovial/metabolismo , Colina Quinasa/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Flavonoides/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/metabolismo , Fibroblastos/metabolismo , Células CultivadasRESUMEN
Choline kinase (CK) is reportedly overexpressed in various malignancies. Among its isoforms, CKα overexpression is presumably related to oncogenic change. Choline positron emission tomography (PET) is reportedly useful for detecting and evaluating therapy outcomes in malignancies. In this study, we investigated the correlation between CKα expression and 11C-choline accumulation in breast cancer cells. We also compared the CKα expression level with other pathological findings for investigating tumour activity. Fifty-six patients with breast cancer (mean age: 51 years) who underwent their first medical examination between May 2007 and December 2008 were enrolled. All the patients underwent 11C-choline PET/computed tomography imaging prior to surgery. The maximum standardised uptake value was recorded for evaluating 11C-choline accumulation. The intensity of CKα expression was classified using immunostaining. A significant correlation was observed between CKα expression and 11C-choline accumulation (P < 0.0001). A comparison of breast cancer mortality demonstrated that strong CKα expression was associated with a shorter survival time (P < 0.0001). 11C-choline accumulation was also negatively correlated with survival time (P < 0.0001). Tumours with strong CKα expression are reportedly highly active in breast cancer. A correlation was observed between CKα expression and 11C-choline accumulation, suggesting their role as prognostic indicators of breast cancer.
Asunto(s)
Neoplasias de la Mama , Colina Quinasa , Femenino , Humanos , Persona de Mediana Edad , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Radioisótopos de Carbono , Colina , Colina Quinasa/genética , Colina Quinasa/metabolismo , Tomografía Computarizada por Tomografía de Emisión de Positrones , Tomografía de Emisión de Positrones , Radiofármacos , Estudios Retrospectivos , Tomografía Computarizada por Rayos XRESUMEN
The de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica is largely dependent on the CDP-choline and CDP-ethanolamine pathways. Although the first enzymes of these pathways, EhCK1 and EhCK2, have been previously characterized, their enzymatic activity was found to be low and undetectable, respectively. This study aimed to identify the unusual characteristics of these enzymes in this deadly parasite. The discovery that EhCKs prefer Mn2+ over the typical Mg2+ as a metal ion cofactor is intriguing for CK/EK family of enzymes. In the presence of Mn2+, the activity of EhCK1 increased by approximately 108-fold compared to that in Mg2+. Specifically, in Mg2+, EhCK1 exhibited a Vmax and K0.5 of 3.5 ± 0.1 U/mg and 13.9 ± 0.2 mM, respectively. However, in Mn2+, it displayed a Vmax of 149.1 ± 2.5 U/mg and a K0.5 of 9.5 ± 0.1 mM. Moreover, when Mg2+ was present at a constant concentration of 12 mM, the K0.5 value for Mn2+ was ~ 2.4-fold lower than that in Mn2+ alone, without affecting its Vmax. Although the enzyme efficiency of EhCK1 was significantly improved by about 25-fold in Mn2+, it is worth noting that its Km for choline and ATP were higher than in equimolar of Mg2+ in a previous study. In contrast, EhCK2 showed specific activity towards ethanolamine in Mn2+, exhibiting Michaelis-Menten kinetic with ethanolamine (Km = 312 ± 27 µM) and cooperativity with ATP (K0.5 = 2.1 ± 0.2 mM). Additionally, we investigated the effect of metal ions on the substrate recognition of human choline and ethanolamine kinase isoforms. Human choline kinase α2 was found to absolutely require Mg2+, while choline kinase ß differentially recognized choline and ethanolamine in Mg2+ and Mn2+, respectively. Finally, mutagenesis studies revealed that EhCK1 Tyr129 was critical for Mn2+ binding, while Lys233 was essential for substrate catalysis but not metal ion binding. Overall, these findings provide insight into the unique characteristics of the EhCKs and highlight the potential for new approaches to treating amoebiasis. Amoebiasis is a challenging disease for clinicians to diagnose and treat, as many patients are asymptomatic. However, by studying the enzymes involved in the CDP-choline and CDP-ethanolamine pathways, which are crucial for de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica, there is great potential to discover new therapeutic approaches to combat this disease.
Asunto(s)
Amebiasis , Entamoeba histolytica , Humanos , Colina/metabolismo , Colina Quinasa/metabolismo , Fosfatidiletanolaminas/metabolismo , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Etanolaminas/metabolismo , Etanolamina , Citidina Difosfato Colina/metabolismo , Fosfatidilcolinas , Isoformas de Proteínas , Adenosina Trifosfato , CinéticaRESUMEN
Twist (TWIST1) is a gene required for cell fate specification in embryos and its expression in mammary epithelium can initiate tumorigenesis through the epithelial-mesenchymal transition. To identify downstream target genes of Twist in breast cancer, we performed microarray analysis on the transgenic breast cancer cell line, MCF-7/Twist. One of the targets identified was choline kinase whose upregulation resulted in increased cellular phosphocholine and total choline containing compounds-a characteristic observed in highly aggressive metastatic cancers. To study the interactions between Twist, choline kinase, and their effect on the microenvironment, we used 1H magnetic resonance spectroscopy and found significantly higher phosphocholine and total choline, as well as increased phosphocholine/glycerophosphocholine ratio in MCF-7/Twist cells. We also observed significant increases in extracellular glucose, lactate, and [H +] ion concentrations in the MCF-7/Twist cells. Magnetic resonance imaging of MCF-7/Twist orthotopic breast tumors showed a significant increase in vascular volume and permeability surface area product compared to control tumors. In addition, by reverse transcription-quantitative polymerase chain reaction, we discovered that Twist upregulated choline kinase expression in estrogen receptor negative breast cancer cell lines through FOXA1 downregulation. Moreover, using The Cancer Genome Atlas database, we observed a significant inverse relationship between FOXA1 and choline kinase expression and propose that it could act as a modulator of the Twist/choline kinase axis. The data presented indicate that Twist is a driver of choline kinase expression in breast cancer cells via FOXA1 resulting in the generation of an aggressive breast cancer phenotype.
Asunto(s)
Colina Quinasa , Fosforilcolina , Línea Celular Tumoral , Colina/metabolismo , Colina Quinasa/genética , Colina Quinasa/metabolismo , Fenotipo , Fosforilcolina/metabolismo , Microambiente Tumoral , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
Entamoeba histolytica is the parasite responsible for amoebiasis, which can result in amoebic colitis or amoebic liver abscess. Metronidazole has been the conventional treatment for intestinal amoebiasis, but concerns regarding resistance have emerged due to the identification of resistance pathways in E. histolytica. This study investigates a novel anti-amoebic approach targeting the CDP-choline pathway. Inhibition studies were conducted using potential choline kinase (CK) inhibitors to inhibit the EhCK enzyme, and RNA interference was employed to knock down the EhCK gene. Km and Vmax of purified EhCK and hCKa2 proteins were determined by pyruvate kinase-lactate dehydrogenase (PK-LDH) coupled assay. The IC50 values for EhCK and hCKa2 were determined with several commercial CK inhibitors. Selected inhibitors were incubated with E. histolytica trophozoites for 48 hours to determine the EC50 for each inhibitor. Silencing of gene encoding EhCK was carried out using duplex siRNA and the gene expression level was measured by real-time qPCR. Based on the IC50 values, three of the inhibitors, namely CK37, flavopiridol and H-89 were more potent against EhCK than hCKa2. Trophozoites growth inhibition showed that only HDTAB, H-89 and control drug metronidazole could penetrate and induce cell death after 48-hour incubation. siRNA concentration of 10 µg/mL was used for the transfection of positive control GAPDH, EhCK, and non-targeting GFP siRNAs. RNAi experiment concluded with positive control GAPDH downregulated by 99% while the level of EhCK mRNA was downregulated by 47%. In this study, potential inhibitors of EhCK and siRNA have been identified, paving the way for further refinement and testing to enhance their potency against EhCK while sparing hCK. The utilization of these specific inhibitors and siRNA targeting EhCK represents a novel approach to impede the growth of E. histolytica by disrupting its phospholipid synthesis pathway.
Asunto(s)
Amebiasis , Entamoeba histolytica , Entamebiasis , Isoquinolinas , Sulfonamidas , Humanos , Entamebiasis/tratamiento farmacológico , Metronidazol/farmacología , Colina Quinasa/metabolismo , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Choline kinase (ChoK) has been well documented as a major enzyme involved in the anomalous cellular lipid metabolic profile of chronic inflammatory disorders. However, new research has now been unveiled that helps us to better understand how changes in lipid metabolism influence the transformational phenotype, drug resistance, and antiapoptotic characteristics of invasive cells, leading to rheumatoid arthritis (RA) disease progression. It is still unknown how ChoK modulates the lipid metabolic aberrations that may promote altered cell phenotype and functionality in RA. Herein, we review the current understanding of ChoK's role in altered metabolism in diverse cell types involved in RA progression, and for the first time, we take a step forward to complete the puzzle and summarise striking facts that link choline metabolism to its transformed phenotype, in order to postulate ChoK as a robust therapeutic target in RA. This review forms a foundation on which ChoK can be tackled as a potential biomarker, opening doors for RA diagnosis and prognosis. It frameworks several ChoK inhibitors that rewire the lipid metabolic profile in the inflammatory disease landscape and envisages its being translated to clinics.
Asunto(s)
Artritis Reumatoide , Colina Quinasa , Humanos , Colina Quinasa/genética , Colina Quinasa/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Colina/metabolismo , Apoptosis , Lípidos , Membrana Sinovial/metabolismoRESUMEN
The short-chain dehydrogenase/reductase (SDR) superfamily has essential roles in lipid metabolism and redox sensing. In recent years, accumulating evidence highlights the emerging association between SDR family enzymes and cancer. Dehydrogenase/reductase member 2(DHRS2) belongs to the NADH/NADPH-dependent SDR family, and extensively participates in the regulation of the proliferation, migration, and chemoresistance of cancer cells. However, the underlying mechanism has not been well defined. In the present study, we have demonstrated that DHRS2 inhibits the growth and metastasis of ovarian cancer (OC) cells in vitro and in vivo. Mechanistically, the combination of transcriptome and metabolome reveals an interruption of choline metabolism by DHRS2. DHRS2 post-transcriptionally downregulates choline kinase α (CHKα) to inhibit AKT signaling activation and reduce phosphorylcholine (PC)/glycerophosphorylcholine (GPC) ratio, impeding choline metabolism reprogramming in OC. These actions mainly account for the tumor-suppressive role of DHRS2 in OC. Overall, our findings establish the mechanistic connection among metabolic enzymes, metabolites, and the malignant phenotype of cancer cells. This could result in further development of novel pharmacological tools against OC by the induction of DHRS2 to disrupt the choline metabolic pathway.
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Colina Quinasa , Neoplasias Ováricas , Carbonil Reductasa (NADPH)/genética , Carbonil Reductasa (NADPH)/metabolismo , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Proliferación Celular , Colina/metabolismo , Colina Quinasa/genética , Colina Quinasa/metabolismo , Regulación hacia Abajo , Femenino , Glicerilfosforilcolina/metabolismo , Humanos , NAD/metabolismo , NADP/metabolismo , Neoplasias Ováricas/genética , Oxidorreductasas/genética , Fosforilcolina/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
BACKGROUND: Choline kinase beta (CHKB) catalyzes the first step in the de novo biosynthesis of phosphatidyl choline and phosphatidylethanolamine via the Kennedy pathway. Derangement of this pathway might also influence the homeostasis of mitochondrial membranes. Autosomal recessive CHKB mutations cause a rare form of congenital muscular dystrophy known as megaconial congenital muscular dystrophy (MCMD). CASE PRESENTATION: We describe a novel proband presenting MCMD due to unpublished CHKB mutations. The patient is a 6-year-old boy who came to our attention for cognitive impairment and slowly progressive muscular weakness. He was the first son of non-consanguineous healthy parents from Sri Lanka. Neurological examination showed proximal weakness at four limbs, weak osteotendinous reflexes, Gowers' maneuver, and waddling gate. Creatine kinase levels were mildly increased. EMG and brain MRI were normal. Left quadriceps skeletal muscle biopsy showed a myopathic pattern with nuclear centralizations and connective tissue increase. Histological and histochemical staining suggested subsarcolemmal localization and dimensional increase of mitochondria. Ultrastructural analysis confirmed the presence of enlarged ("megaconial") mitochondria. Direct sequencing of CHKB identified two novel defects: the c.1060G > C (p.Gly354Arg) substitution and the c.448-56_29del intronic deletion, segregating from father and mother, respectively. Subcloning of RT-PCR amplicons from patient's muscle RNA showed that c.448-56_29del results in the partial retention (14 nucleotides) of intron 3, altering physiological splicing and transcript stability. Biochemical studies showed reduced levels of the mitochondrial fission factor DRP1 and the severe impairment of mitochondrial respiratory chain activity in patient's muscle compared to controls. CONCLUSIONS: This report expands the molecular findings associated with MCMD and confirms the importance of considering CHKB variants in the differential diagnosis of patients presenting with muscular dystrophy and mental retardation. The clinical outcome of MCMD patients seems to be influenced by CHKB molecular defects. Histological and ultrastructural examination of muscle biopsy directed molecular studies and allowed the identification and characterization of an intronic mutation, usually escaping standard molecular testing.
Asunto(s)
Colina Quinasa , Distrofias Musculares , Niño , Colina Quinasa/genética , Colina Quinasa/metabolismo , Creatina Quinasa , Humanos , Masculino , Músculo Esquelético/metabolismo , Distrofias Musculares/congénito , Distrofias Musculares/diagnóstico , Distrofias Musculares/genética , Mutación , Nucleótidos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , ARN/metabolismoRESUMEN
CHKB encodes one of two mammalian choline kinase enzymes that catalyze the first step in the synthesis of the membrane phospholipid phosphatidylcholine. In humans and mice, inactivation of the CHKB gene (Chkb in mice) causes a recessive rostral-to-caudal muscular dystrophy. Using Chkb knockout mice, we reveal that at no stage of the disease is phosphatidylcholine level significantly altered. We observe that in affected muscle a temporal change in lipid metabolism occurs with an initial inability to utilize fatty acids for energy via mitochondrial ß-oxidation resulting in shunting of fatty acids into triacyglycerol as the disease progresses. There is a decrease in peroxisome proliferator-activated receptors and target gene expression specific to Chkb-/- affected muscle. Treatment of Chkb-/- myocytes with peroxisome proliferator-activated receptor agonists enables fatty acids to be used for ß-oxidation and prevents triacyglyerol accumulation, while simultaneously increasing expression of the compensatory choline kinase alpha (Chka) isoform, preventing muscle cell injury.
Asunto(s)
Enfermedades Musculares , Distrofias Musculares , Animales , Colina Quinasa/genética , Colina Quinasa/metabolismo , Ácidos Grasos , Metabolismo de los Lípidos/genética , Mamíferos/metabolismo , Ratones , Distrofias Musculares/genética , Distrofias Musculares/terapia , Fosfatidilcolinas/metabolismoRESUMEN
The CHKB gene encodes choline kinase ß, which catalyzes the first step in the biosynthetic pathway for the major phospholipid phosphatidylcholine. Homozygous loss-of-function variants in human CHKB are associated with a congenital muscular dystrophy. Dilated cardiomyopathy is present in some CHKB patients and can cause heart failure and death. Mechanisms underlying a cardiac phenotype due to decreased CHKB levels are not well characterized. We determined that there is cardiac hypertrophy in Chkb-/- mice along with a decrease in left ventricle size, internal diameter, and stroke volume compared with wildtype and Chkb+/- mice. Unlike wildtype mice, 60% of the Chkb+/- and all Chkb-/- mice tested displayed arrhythmic events when challenged with isoproterenol. Lipidomic analysis revealed that the major change in lipid level in Chkb+/- and Chkb-/- hearts was an increase in the arrhythmogenic lipid acylcarnitine. An increase in acylcarnitine level is also associated with a defect in the ability of mitochondria to use fatty acids for energy and we observed that mitochondria from Chkb-/- hearts had abnormal cristae and inefficient electron transport chain activity. Atrial natriuretic peptide (ANP) is a hormone produced by the heart that protects against the development of heart failure including ventricular conduction defects. We determined that there was a decrease in expression of ANP, its receptor NPRA, as well as ventricular conduction system markers in Chkb+/- and Chkb-/- mice.
Asunto(s)
Arritmias Cardíacas , Colina Quinasa , Insuficiencia Cardíaca , Animales , Arritmias Cardíacas/enzimología , Arritmias Cardíacas/genética , Factor Natriurético Atrial/genética , Colina Quinasa/deficiencia , Colina Quinasa/genética , Colina Quinasa/metabolismo , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/genética , Humanos , Ratones , Fosfatidilcolinas/metabolismoRESUMEN
Multiple noncoding natural antisense transcripts (ncNAT) are known to modulate key biological events such as cell growth or differentiation. However, the actual impact of ncNATs on cancer progression remains largely unknown. In this study, we identified a complete list of differentially expressed ncNATs in hepatocellular carcinoma. Among them, a previously undescribed ncNAT HNF4A-AS1L suppressed cancer cell growth by regulating its sense gene HNF4A, a well-known cancer driver, through a promoter-specific mechanism. HNF4A-AS1L selectively activated the HNF4A P1 promoter via HNF1A, which upregulated expression of tumor suppressor P1-driven isoforms, while having no effect on the oncogenic P2 promoter. RNA-seq data from 23 tissue and cancer types identified approximately 100 ncNATs whose expression correlated specifically with the activity of one promoter of their associated sense gene. Silencing of two of these ncNATs ENSG00000259357 and ENSG00000255031 (antisense to CERS2 and CHKA, respectively) altered the promoter usage of CERS2 and CHKA. Altogether, these results demonstrate that promoter-specific regulation is a mechanism used by ncNATs for context-specific control of alternative isoform expression of their counterpart sense genes. SIGNIFICANCE: This study characterizes a previously unexplored role of ncNATs in regulation of isoform expression of associated sense genes, highlighting a mechanism of alternative promoter usage in cancer.
Asunto(s)
Carcinoma Hepatocelular/patología , Colina Quinasa/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Neoplasias Hepáticas/patología , Proteínas de la Membrana/metabolismo , Regiones Promotoras Genéticas , ARN sin Sentido/genética , Esfingosina N-Aciltransferasa/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Colina Quinasa/antagonistas & inhibidores , Colina Quinasa/genética , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 4 del Hepatocito/antagonistas & inhibidores , Factor Nuclear 4 del Hepatocito/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Ratones SCID , Pronóstico , Esfingosina N-Aciltransferasa/antagonistas & inhibidores , Esfingosina N-Aciltransferasa/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.
Asunto(s)
Adenosina Trifosfato/antagonistas & inhibidores , Colina Quinasa/antagonistas & inhibidores , Ciclohexanos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Adenosina Trifosfato/metabolismo , Colina Quinasa/metabolismo , Ciclohexanos/síntesis química , Ciclohexanos/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
An evaluation of the impact of vitamin E deficiency on expression of the alpha-tocopherol transfer protein (α-TTP) and related CRAL_TRIO genes was undertaken using livers from adult zebrafish based on the hypothesis that increased lipid peroxidation would modulate gene expression. Zebrafish were fed either a vitamin E sufficient (E+) or deficient (E-) diet for 9 months, then fish were euthanized, and livers were harvested. Livers from the E+ relative to E- fish contained 40-times more α-tocopherol (P <0.0001) and one fourth the malondialdehyde (P = 0.0153). RNA was extracted from E+ and E- livers, then subject to evaluation of gene expression of ttpa and other genes of the CRAL_TRIO family, genes of antioxidant markers, and genes related to lipid metabolism. Ttpa expression was not altered by vitamin E status. However, one member of the CRAL_TRIO family, tyrosine-protein phosphatase non-receptor type 9 gene (ptpn9a), showed a 2.4-fold increase (P = 0.029) in E- relative to E+ livers. Further, we identified that the gene for choline kinase alpha (chka) showed a 3.0-fold increase (P = 0.010) in E- livers. These outcomes are consistent with our previous findings that show vitamin E deficiency increased lipid peroxidation causing increases in phospholipid turnover.
Asunto(s)
Proteínas Portadoras/genética , Expresión Génica , Hígado/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/genética , Deficiencia de Vitamina E/genética , Proteínas de Pez Cebra/genética , Animales , Antioxidantes , Proteínas Portadoras/metabolismo , Colina Quinasa/genética , Colina Quinasa/metabolismo , Metabolismo de los Lípidos/genética , Malondialdehído/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Deficiencia de Vitamina E/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismo , alfa-Tocoferol/metabolismoRESUMEN
Lipid droplets are important for cancer cell growth and survival. However, the mechanism underlying the initiation of lipid droplet lipolysis is not well understood. We demonstrate here that glucose deprivation induces the binding of choline kinase (CHK) α2 to lipid droplets, which is sequentially mediated by AMPK-dependent CHKα2 S279 phosphorylation and KAT5-dependent CHKα2 K247 acetylation. Importantly, CHKα2 with altered catalytic domain conformation functions as a protein kinase and phosphorylates PLIN2 at Y232 and PLIN3 at Y251. The phosphorylated PLIN2/3 dissociate from lipid droplets and are degraded by Hsc70-mediated autophagy, thereby promoting lipid droplet lipolysis, fatty acid oxidation, and brain tumor growth. In addition, levels of CHKα2 S279 phosphorylation, CHKα2 K247 acetylation, and PLIN2/3 phosphorylation are positively correlated with one another in human glioblastoma specimens and are associated with poor prognosis in glioblastoma patients. These findings underscore the role of CHKα2 as a protein kinase in lipolysis and glioblastoma development.
Asunto(s)
Colina Quinasa/metabolismo , Glioblastoma/enzimología , Gotas Lipídicas/enzimología , Lipólisis , Proteínas de Neoplasias/metabolismo , Proteínas Quinasas/metabolismo , Acetilación , Línea Celular Tumoral , Colina Quinasa/genética , Glioblastoma/genética , Humanos , Proteínas de Neoplasias/genética , Proteínas Quinasas/genéticaRESUMEN
Upregulated choline metabolism, characterized by an increase in phosphocholine (PCho), is a hallmark of oncogenesis and tumor progression. Choline kinase (ChoK), the enzyme responsible for PCho synthesis, has consequently become a promising drug target for cancer therapy and as such a significant number of ChoK inhibitors have been developed over the last few decades. More recently, due to the role of this enzyme in other pathologies, ChoK inhibitors have also been used in new therapeutic approaches against malaria and rheumatoid arthritis. Here, we review research results in the field of ChoKα inhibitors from their synthesis to the molecular basis of their binding mode. Strategies for the development of inhibitors and their selectivity on ChoKα over ChoKß, the plasticity of the choline-binding site, the discovery of new exploitable binding sites, and the allosteric properties of this enzyme are highlighted. The outcomes summarized in this review will be a useful guide to develop new multifunctional potent drugs for the treatment of various human diseases.