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1.
J Clin Biochem Nutr ; 73(3): 255-261, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37970549

RESUMEN

Lysophosphatidic acid is composed of lysophosphatidic acid (LPA) molecules with varied chemical forms. The present cross-sectional study was conducted to investigate the associations of various LPA molecules with liver fibrosis. Forty-six patients affected by various types of liver disease who underwent an ultrasound-guided liver biopsy were recruited for this study. Liver fibrosis was evaluated using histological grading, as well as shear wave velocity (Vs) and serum level of type IV collagen 7S (T4c7s). Serum levels of LPA molecules were determined using liquid-chromatography tandem mass-spectrometry (LC-MSMS). Total LPA showed a significant positive association with fibrosis severity evaluated based on histological grading, Vs, and T4c7s used as parameters, following adjustment for other confounding factors, including disease type, age, gender, body mass index, and high-sensitivity C-reactive protein. This association was replicated when 16:0-LPA was substituted for total LPA. In contrast, when 20:4-LPA was substituted for total LPA, no significant association with liver fibrosis was observed. In conclusion, the degree of association varied among the different LPA molecule chemical forms, suggesting different pathophysiological roles of individual LPA molecules, although total LPA concentration was shown to be associated with liver fibrosis.

2.
J Biol Chem ; 295(10): 2932-2947, 2020 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-31980461

RESUMEN

The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.


Asunto(s)
Diacilglicerol Quinasa/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Cromatografía Líquida de Alta Presión , Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/genética , Humanos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Alineación de Secuencia , Motivo alfa Estéril , Espectrometría de Masas en Tándem , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética
3.
Biochem Biophys Res Commun ; 513(4): 1035-1040, 2019 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-31010675

RESUMEN

Diacylglycerol kinase (DGK) δ, which is a key enzyme in the pathogenesis of type 2 diabetes (T2D), preferentially generates saturated fatty acid (SFA)- and/or monounsaturated fatty acid (MUFA)-containing phosphatidic acids (PAs) such as 16:0/16:0-PA and 16:0/18:1-PA, but not polyunsaturated fatty acid (PUFA)-containing PAs, in glucose-stimulated myoblast cells. Here, we searched for the target proteins of 16:0/16:0-PA in the mouse skeletal muscle and identified an energy metabolizing enzyme, creatine kinase muscle type (CKM), which is correlated with T2D. CKM bound to 16:0/16:0-PA with the highest affinity (dissociation constant: 2.0 µM) among all the PA-binding proteins reported thus far. Intriguingly, CKM preferentially interacted with SFA- and/or MUFA-containing PAs, but not with PUFA-containing PAs. Notably, CKM exclusively interacted with PA, whereas the protein did not bind to other lipids such as diacylglycerol, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol (3,4,5)-trisphosphate and cardiolipin. Taken together, these results demonstrate that CKM is a very unique PA-binding protein that possesses exceedingly high affinity for PA, exceptional preference for SFA/MUFA-PA and extremely high specificity to PA and suggest that SFA/MUFA-PAs produced by DGKδ are novel regulators of CKM function.


Asunto(s)
Creatina Quinasa/metabolismo , Diacilglicerol Quinasa/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Ácidos Grasos/metabolismo , Músculo Esquelético/enzimología , Ácidos Fosfatidicos/metabolismo , Animales , Diabetes Mellitus Tipo 2/enzimología , Ácidos Grasos/química , Ácidos Grasos Monoinsaturados/química , Ratones , Unión Proteica
4.
Biochem Biophys Res Commun ; 504(1): 54-60, 2018 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-30170729

RESUMEN

The ß-isoform of diacylglycerol kinase (DGK) localizes predominantly to neurons and induces neurite outgrowth and spine formation. However, the detailed molecular mechanisms underlying the functions of DGKß remain elusive. During the course of studies on other DGK isozymes, we unexpectedly found that the overexpression of wild-type DGKß in COS-7 cells markedly induced filopodium formation. Because filopodium formation is closely related to neurite outgrowth and spine formation, we constructed various DGKß mutants and compared their abilities to induce filopodium formation in order to elucidate the structure-function relationships of DGKß. We found that the C-terminal, C1 and catalytic domains and catalytic activity were indispensable for filopodium formation, but the recoverin homology domain and EF-hand motifs were not. Moreover, the extent of plasma membrane localization and F-actin colocalization were positively correlated with filopodium formation. Intriguingly, DGKß selectively interacted and colocalized at the plasma membrane with a Rac1-GTPase-activating protein, ß2-chimaerin, which is an inducer of filopodia; it also interacted, to lesser extent, with α2-chimaerin, but not with α1- or ß1-chimaerin. Moreover, DGKß enhanced the plasma membrane localization of ß2-chimaerin. These results suggest that DGKß plays an important role in neurite outgrowth and spine formation in neurons via its ability to induce filopodium formation.


Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Lipoproteína Lipasa/metabolismo , Proteínas de Neoplasias/metabolismo , Seudópodos/fisiología , Animales , Células COS , Dominio Catalítico , Chlorocebus aethiops , Lipoproteína Lipasa/química , Lipoproteína Lipasa/genética , Mutación , Dominios Proteicos , Seudópodos/ultraestructura
5.
J Biol Chem ; 291(15): 8150-61, 2016 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-26887948

RESUMEN

Type II diacylglycerol kinase (DGK) isozymes (δ, η, and κ) have a pleckstrin homology domain (PH) at their N termini. Here, we investigated the lipid binding properties of the PHs of type II DGK isozymes using protein-lipid overlay and liposome binding assays. The PH of DGKη showed the most pronounced binding activity to phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) among the various glycero- and sphingolipids including PI 3,4,5-trisphosphate, PI 3,4-bisphosphate, PI 3-phosphate, PI 4-phosphate, and PI 5-phosphate. Moreover, the PI(4,5)P2binding activity of the DGKη-PH was significantly stronger than that of other type II DGK isozymes. Notably, compared with the PH of phospholipase C (PLC) δ1, which is generally utilized as a cellular PI(4,5)P2- probe, the DGKη-PH is equal to or superior than the PLCδ1-PH in terms of affinity and selectivity for PI(4,5)P2 Furthermore, in COS-7 cells, GFP-fused wild-type DGKη1 and its PH partly translocated from the cytoplasm to the plasma membrane where the PLCδ1-PH was co-localized in response to hyperosmotic stress in an inositol 5-phosphatase-sensitive manner, whereas a PH deletion mutant did not. Moreover, K74A and R85A mutants of DGKη-PH, which lack the conserved basic amino acids thought to ligate PI(4,5)P2, were indeed unable to bind to PI(4,5)P2and co-localize with the PLCδ1-PH even in osmotically shocked cells. Overexpression of wild-type DGKη1 enhanced EGF-dependent phosphorylation of ERK, whereas either K74A or R85A mutant did not. Taken together, these results indicate that the DGKη-PH preferentially interacts with PI(4,5)P2and has crucial roles in regulating the subcellular localization and physiological function of DGKη. Moreover, the DGKη-PH could serve as an excellent cellular sensor for PI(4,5)P2.


Asunto(s)
Diacilglicerol Quinasa/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Diacilglicerol Quinasa/análisis , Humanos , Sistema de Señalización de MAP Quinasas , Datos de Secuencia Molecular , Presión Osmótica , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia
6.
Biochim Biophys Acta ; 1864(9): 1170-1176, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27346717

RESUMEN

Diacylglycerol kinase (DGK) consists of ten isozymes and is involved in a wide variety of patho-physiological events. However, the enzymological properties of DGKs have not been fully understood. In this study, we performed a comprehensive analysis on the 1-monoacylglycerol kinase (MGK) and 2-MGK activities of ten DGK isozymes. We revealed that type I (α, ß and γ), type II (δ, η and κ) and type III (ε) DGKs have 7.9-19.2% 2-MGK activity compared to their DGK activities, whereas their 1-MGK activities were <3.0%. Both the 1-MGK and 2-MGK activities of the type IV DGKs (ζ and ι) were <1% relative to their DGK activities. Intriguingly, type V DGKθ has approximately 6% 1-MGK activity and <2% 2-MGK activity compared to its DGK activity. Purified DGKθ exhibited the same results, indicating that its 1-MGK activity is intrinsic. Therefore, DGK isozymes are categorized into three types with respect to their 1-MGK and 2-MGK activities: those having (1) 2-MGK activity relatively stronger than their 1-MGK activity (types I-III), (2) only negligible 1-MGK and 2-MGK activities (type IV), and (3) 1-MGK activity stronger than its 2-MGK activity (type V). The 1-MGK activity of DGKθ and the 2-MGK activity of DGKα were stronger than those of the acylglycerol kinase reported as 1-MGK and 2-MGK to date. The presence or absence of 1-MGK and 2-MGK activities may be essential to the patho-physiological functions of each DGK isozyme.


Asunto(s)
Diacilglicerol Quinasa/metabolismo , Diglicéridos/metabolismo , Monoglicéridos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Células COS , Chlorocebus aethiops , Diacilglicerol Quinasa/clasificación , Diacilglicerol Quinasa/genética , Expresión Génica , Humanos , Isoenzimas/clasificación , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratones , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Recombinantes de Fusión/genética , Especificidad por Sustrato , Porcinos
7.
J Epidemiol ; 27(12): 568-573, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28623056

RESUMEN

BACKGROUND: Noncommunicable disease (NCD) has become the leading cause of mortality and disease burden worldwide. METHODS: A cross-sectional survey was carried out to investigate the prevalence of NCDs and risk factor control on dietary behaviors and dietary intake in China, Japan, and Korea. RESULTS: There were significant differences among the three countries on the prevalence of hypertension (24.5% in China, 17.6% in Korea, and 15.2% in Japan), diabetes (8.9% in China, 5.7% in Korea, and 4.8% in Japan), hyperlipidemia (13.1% in China, 9.2% in Korea, and 6.9% in Japan), and angina pectoris (3.6% in China, 1.7% in Korea, and 1.5% in Japan). The prevalence rate of hypertension, diabetes, hyperlipidemia, and angina pectoris was highest in China and lowest in Japan. However, 82.2%, 48.4%, and 64.4% of Chinese, Koreans, and Japanese presented good dietary behavior, respectively. Multivariable logistic regression analysis found that sex, age, and marital status were predictors of good dietary behavior. In addition, in comparison with subjects without hypertension, diabetes, or hyperlipidemia, subjects with hypertension, diabetes, or hyperlipidemia significantly improved their dietary behaviors and controlled their intake of salt, sugar, and oil. CONCLUSIONS: The prevalence of NCDs and trends in major modifiable risk factor control in China, Korea, and Japan remain troubling. Public efforts to introduce healthy lifestyle changes and systematic NCDs prevention programs are necessary to reduce the epidemic of NCDs in these three Asian countries.


Asunto(s)
Angina de Pecho/epidemiología , Diabetes Mellitus/epidemiología , Dieta/psicología , Hiperlipidemias/epidemiología , Hipertensión/epidemiología , Adulto , Distribución por Edad , China/epidemiología , Estudios Transversales , Dieta/estadística & datos numéricos , Femenino , Humanos , Japón/epidemiología , Masculino , Estado Civil/estadística & datos numéricos , Persona de Mediana Edad , Prevalencia , República de Corea/epidemiología , Factores de Riesgo , Distribución por Sexo , Adulto Joven
8.
J Lipid Res ; 57(3): 368-79, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26768655

RESUMEN

Diacylglycerol kinase (DGK) consists of 10 isozymes. The α-isozyme enhances the proliferation of cancer cells. However, DGKα facilitates the nonresponsive state of immunity known as T-cell anergy; therefore, DGKα enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGKα activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl)amino]2-thioxo-1,3-thiazolidin-4-one) (CU-3), which selectively inhibited DGKα with an IC50 value of 0.6 µM. CU-3 targeted the catalytic region, but not the regulatory region, of DGKα. CU-3 competitively reduced the affinity of DGKα for ATP, but not diacylglycerol or phosphatidylserine. Moreover, this compound induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells while simultaneously enhancing the interleukin-2 production of Jurkat T cells. Taken together, these results indicate that CU-3 is a selective and potent inhibitor for DGKα and can be an ideal anticancer drug candidate that attenuates cancer cell proliferation and simultaneously enhances immune responses including anticancer immunity.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Diacilglicerol Quinasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Rodanina/análogos & derivados , Sulfonamidas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Tiazoles/farmacología , Animales , Células COS , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Concentración 50 Inhibidora , Interleucina-2/biosíntesis , Isoenzimas/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Rodanina/farmacología , Especificidad por Sustrato , Linfocitos T/metabolismo
9.
J Neurochem ; 138(3): 448-56, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27167678

RESUMEN

The η isozyme of diacylglycerol kinase (DGK) is highly expressed in the hippocampus and Purkinje cells in the central nervous system. Recently, several genome-wide association studies have implicated DGKη in the etiology of bipolar disorder (BPD). However, it is still unknown whether DGKη is indeed related to BPD. In this study, we generated DGKη-knockout (KO) mice and performed behavioral tests such as the open field test, the elevated plus maze test and tail suspension test using the KO mice to investigate the effects of DGKη deficits on psychomotor behavior. Intriguingly, DGKη-KO mice displayed an overall behavioral profile that is similar to human mania, including hyperactivity, less anxiety and less depression-like behavior. In addition, these phenotypes were significantly attenuated by the administration of a BPD (mania) remedy, namely, lithium. Moreover, DGKη-KO mice showed impairment in glycogen synthase kinase (GSK) 3ß signaling, which is closely related to BPD. These findings clearly support the linkage between BPD and DGKη that is implicated by genome-wide association studies. Moreover, this study provides DGKη-KO mice as a previously unrecognized model that reflects several features of human BPD with manic episodes and revealed an important role for DGKη in regulating behavior and mood through, at least in part, GSK3ß signaling. Several genome-wide association studies have implicated diacylglycerol kinase (DGK) η gene in the etiology of bipolar disorder (BPD). In this study, we revealed that DGKη-knockout (KO) mice displayed an overall behavioral profile that is similar to mania of BPD and is lithium (BPD (mania) remedy)-sensitive. DGKη may regulate behavior and mood through, at least in part, glycogen synthase kinase (GSK) 3ß signaling.


Asunto(s)
Conducta/efectos de los fármacos , Trastorno Bipolar/metabolismo , Diacilglicerol Quinasa/metabolismo , Hipercinesia/metabolismo , Litio/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Ansiedad/genética , Ansiedad/metabolismo , Depresión/genética , Depresión/metabolismo , Diacilglicerol Quinasa/deficiencia , Estudio de Asociación del Genoma Completo/métodos , Masculino , Ratones , Ratones Noqueados , Transducción de Señal/fisiología
10.
J Biol Chem ; 289(38): 26607-26617, 2014 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-25112873

RESUMEN

Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels.


Asunto(s)
Diacilglicerol Quinasa/metabolismo , Diglicéridos/metabolismo , Glucosa/fisiología , Ácido Palmítico/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Línea Celular , Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/genética , Expresión Génica , Metabolismo de los Lípidos , Ratones , Mioblastos/enzimología , Fosforilación
11.
J Biol Chem ; 289(26): 18097-109, 2014 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-24821721

RESUMEN

Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells.


Asunto(s)
Lipopolisacáridos/metabolismo , Sistema de Señalización de MAP Quinasas , Monocitos/metabolismo , Neutrófilos/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Factor de Transcripción Sp1/genética , Receptor Toll-Like 4/metabolismo , Animales , Secuencia de Bases , Regulación hacia Abajo , Humanos , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Receptor Toll-Like 4/genética
12.
BMC Dev Biol ; 15: 6, 2015 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-25613821

RESUMEN

BACKGROUND: We have revealed that the type II diacylglycerol kinases (DGKs) δ, η and κ were expressed in the testis and ovary. However, these enzymes' functions in the reproductive organs remain unknown. RESULTS: In this study, we first identified the expression sites of type II DGKs in the mouse reproductive organs in detail. Reverse transcription-polymerase chain reaction and Western blotting confirmed that DGKδ2 (splicing variant 2) but not DGKδ1 (splicing variant 1) and DGKκ were expressed in the testis, ovary and uterus. DGKη1 (splicing variant 1) but not DGKη2 (splicing variant 2) was strongly detected in the ovary and uterus. Interestingly, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the testis. Moreover, we investigated the distribution of type II DGKs in the testis, ovary and uterus through in situ hybridization. DGKδ2 was distributed in the primary spermatocytes of the testis and ovarian follicles. DGKη1 was distributed in the oviductal epithelium of the ovary and the luminal epithelium of the uterus. Intriguingly, DGKη3 was strongly expressed in the secondary spermatocytes and round spermatids of the testis. DGKκ was distributed in the primary and secondary spermatocyte of the testis. CONCLUSION: These results indicate that the expression patterns of the type II DGK isoforms δ2, η1, η3 and κ differ from each other, suggesting that these DGK isoforms play specific roles in distinct compartments and developmental stages of the reproductive organs, especially in the processes of spermatogenesis and oocyte maturation.


Asunto(s)
Diacilglicerol Quinasa/metabolismo , Isoenzimas/metabolismo , Ovario/enzimología , Útero/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diacilglicerol Quinasa/genética , Femenino , Isoenzimas/genética , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética
13.
Biochem Biophys Res Commun ; 444(4): 617-21, 2014 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-24486543

RESUMEN

There are ten isozymes of diacylglycerol kinase (DGK), and they regulate diverse patho-physiological functions. Here, we investigated the lipid-binding properties of DGK isozymes using protein-lipid overlay and liposome-binding assays. DGKγ showed a strong binding activity compared with other DGK isozymes for phosphatidic acid (PA) among the various glycerophospholipids tested. However, DGKγ failed to interact with DG and lyso-PA. Moreover, the isozyme was capable of binding to ceramide-1-phosphate but not to ceramide or sphingosine-1-phosphate. The isozyme bound more strongly to PA containing unsaturated fatty acid than to PA having only saturated fatty acid. An analysis using a series of deletion mutants of DGKγ revealed that the N-terminal region, which contains a recoverin homology domain and EF-hand motifs, is responsible for the PA binding activity of DGKγ. Taken together, these results indicate that DGKγ is an anionic phospholipid binding protein that preferably interacts with a small highly charged head group that is very close to the glycerol or sphingosine backbone.


Asunto(s)
Diacilglicerol Quinasa/metabolismo , Fosfolípidos/metabolismo , Animales , Aniones/metabolismo , Sitios de Unión , Células COS , Ceramidas/metabolismo , Chlorocebus aethiops , Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Lisofosfolípidos/metabolismo , Ácidos Fosfatidicos/metabolismo , Unión Proteica , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Transfección
14.
Adv Biol Regul ; : 101054, 2024 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-39368888

RESUMEN

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprise ten isozymes (α-κ) that regulate a wide variety of physiological and pathological events. Recently, we revealed that DGK isozymes use saturated fatty acid (SFA)/monosaturated fatty acid (MUFA)-containing and docosahexaenoic acid (22:6)-containing DG species, but not phosphatidylinositol (PI) turnover-derived 18:0/20:4-DG. For example, DGKδ, which is involved in the pathogenesis of type 2 diabetes, preferentially uses SFA/MUFA-containing DG species, such as 16:0/16:0- and 16:0/18:1-DG species, in high glucose-stimulated skeletal muscle cells. Moreover, DGKδ, which destabilizes the serotonin transporter (SERT) and regulates the serotonergic system in the brain, primarily generates 18:0/22:6-PA. Furthermore, 16:0/16:0-PA is produced by DGKζ in Neuro-2a cells during neuronal differentiation. We searched for SFA/MUFA-PA- and 18:0/22:6-PA-selective binding proteins (candidate downstream targets of DGKδ) and found that SFA/MUFA-PA binds to and activates the creatine kinase muscle type, an energy-metabolizing enzyme, and that 18:0/22:6-PA interacts with and activates Praja-1, an E3 ubiquitin ligase acting on SERT, and synaptojanin-1, a key player in the synaptic vesicle cycle. Next, we searched for SFA/MUFA-DG-generating enzymes upstream of DGKδ. We found that sphingomyelin synthase (SMS)1, SMS2, and SMS-related protein (SMSr) commonly act as phosphatidylcholine (PC)-phospholipase C (PLC) and phosphatidylethanolamine (PE)-PLC, generating SFA/MUFA-DG species, in addition to SMS and ceramide phosphoethanolamine synthase. Moreover, the orphan phosphatase PHOSPHO1 showed PC- and PE-PLC activities that produced SFA/MUFA-DG. Although PC- and PE-PLC activities were first described 70-35 years ago, their proteins and genes were not identified for a long time. We found that DGKδ interacts with SMSr and PHOSPHO1, and that DGKζ binds to SMS1 and SMSr. Taken together, these results strongly suggest that there are previously unrecognized signal transduction pathways that include DGK isozymes and generate and utilize SFA/MUFA-DG/PA or 18:0/22:6-DG/PA but not PI-turnover-derived 18:0/20:4-DG/PA.

15.
Biochim Biophys Acta ; 1823(12): 2210-6, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22974639

RESUMEN

The type II diacylglycerol kinases (DGKs) contain several functional domains such as a pleckstrin homology (PH) domain, two C1 domains and a sterile α-motif (SAM) domain. It was previously revealed that DGKδ contributes to hyperglycemia-induced peripheral insulin resistance and thereby exacerbate the severity of type 2 diabetes. Moreover, a high extracellular concentration of glucose activated DGKδ in skeletal muscle cells, which was followed by a reduction in the intracellular diacylglycerol levels and the inactivation of protein kinase Cα, the enzyme that phosphorylates and inactivates the insulin receptor. However, the intracellular behavior of DGKδ upon high glucose stimulation remains unclear. In this study, we found that DGKδ1, but not a splice variant DGKδ2 or the other type II DGKη1/2, translocated from the cytoplasm to the plasma membrane in human embryonic kidney HEK293 and mouse myoblast C2C12 cells within 5 min in response to high glucose levels. The translocation was inhibited by phosphatidylinositol 3-kinase inhibitors, LY294002 and GDC-0941, suggesting that the event is regulated via the phosphatidylinositol 3-kinase pathway. Moreover, we revealed that the PH and C1 domains are responsible for the plasma membrane translocation and that the SAM domain negatively regulates the translocation. These results indicate that DGKδ1 is the sole type II DGK isoform that responds rapidly and dynamically to high glucose levels.


Asunto(s)
Membrana Celular/efectos de los fármacos , Diacilglicerol Quinasa/metabolismo , Glucosa/farmacología , Transporte de Proteínas/efectos de los fármacos , Edulcorantes/farmacología , Animales , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas , Riñón/citología , Riñón/efectos de los fármacos , Riñón/metabolismo , Ratones , Estructura Terciaria de Proteína
16.
Mol Cell Biochem ; 373(1-2): 85-93, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23054194

RESUMEN

Type I diacylglycerol kinase (DGK) isozymes (α, ß, and γ) contain recoverin homology domains and calcium-binding EF-hand motifs at their N-termini. The γ-isoform of DGK is abundantly expressed in retinal and Purkinje cells; however, its function in neuronal cells remains unknown. Here, we report that the mRNA and protein levels of DGKγ, but not DGKα or ß, were markedly increased in N1E-115 neuroblastoma cells upon cellular differentiation by serum starvation. Interestingly, overexpression of wild-type DGKγ, which was partially located at the plasma membrane, considerably induced the formation of slender, filopodia-like cytoplasmic projections from N1E-115 cell bodies. Deletion of the recoverin homology domain and the EF-hand motifs, which potentiated the plasma membrane localization of the isozyme, significantly enhanced the formation of the filopodia-like protrusions. Intriguingly, the catalytic activity of the isozyme is not essential for the protrusion formation. The N-terminal half of the catalytic domain and a short stretch of amino acid residues at the C-terminus are responsible for plasma membrane localization and filopodia-like process formation. Taken together, we have described a potentially novel morphological function of the C-terminal DGKγ catalytic region that is independent of its enzymatic activity.


Asunto(s)
Diacilglicerol Quinasa/metabolismo , Seudópodos/enzimología , Animales , Dominio Catalítico , Diferenciación Celular , Línea Celular Tumoral , Membrana Celular/enzimología , Medio de Cultivo Libre de Suero , Diacilglicerol Quinasa/genética , Diacilglicerol Quinasa/fisiología , Expresión Génica , Isoenzimas/metabolismo , Ratones , Neuroblastoma , Transporte de Proteínas , Eliminación de Secuencia
17.
Pharmacology ; 92(1-2): 99-107, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23949095

RESUMEN

Ten mammalian diacylglycerol kinase (DGK) isozymes (α-κ) have been identified. Recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of pathophysiological functions. Thus, it is important to be able to easily check DGK activity in each pathophysiological event. Moreover, the conventional DGK assay is quite laborious because it requires the use of a radioisotope and thin-layer chromatography including multiple extraction steps. In order to minimize the laborious procedures, we established a non-radioactive, single well, two-step DGK assay system. We demonstrated that, compared to the conventional method, the new assay system has comparable sensitivity and much higher efficiency, and is effective in detecting potential agents with high reliability (Z'-factor = 0.69 ± 0.12; n = 3). Using the newly developed assay, we comprehensively evaluated the DGK isozyme selectivities of commercially available DGK inhibitors, R59022 and R59949, in vitro. We found that among 10 isozymes, R59022 strongly inhibited type I DGKα and moderately attenuated type III DGKε and type V DGKθ, and that R59949 strongly inhibited type I DGK α and γ, and moderately attenuated type II DGK δ and κ.


Asunto(s)
Diacilglicerol Quinasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Piperidinas/farmacología , Pirimidinonas/farmacología , Quinazolinonas/farmacología , Tiazoles/farmacología , Animales , Células COS , Chlorocebus aethiops , Isoenzimas/antagonistas & inhibidores
18.
Yakugaku Zasshi ; 143(10): 847-855, 2023.
Artículo en Japonés | MEDLINE | ID: mdl-37779015

RESUMEN

In 2006, Japan's pharmaceutical science education was revised to a 6-year enrollment course, placing greater emphasis on cultivating practical clinical ability. Quality Assurance (QA) measures have been implemented including offering education based on a model core curriculum and third-party assessments. In August 2021, Ministry of Education, Culture, Sports, Science and Technology (MEXT) launched an investigative commission to review the above. For QA, the commission summarized a comprehensive report in August 2022 for items including: modality of selecting entrants; revising enrollment limits; securing education management; information disclosure; corresponding to pharmaceutical education assessments. For revising the model core curriculum, the commission is reviewing correspondence to: demographic changes due to decreasing birthrates, an ageing population, changes in the structure of diseases; rising and emerging infectious diseases; utilizing Big Data and artificial intelligence (AI). As Japan's ageing population is forecast to peak in 2040s, pharmacists must be fostered to provide safe and quality medicine towards a drastically changing future. Medical care is provided through the collaboration of various professions. In such changing demographics, team medicine is crucial to provide quality medical care. Moreover, towards all medical professions sharing the same vision, revisions to the model core curricula for medical and dental education are also being reviewed. The commission is now reviewing detailed curricula to foster pharmacists with competencies to: comprehensively assess patients and ordinary citizens; utilize information science and technology; professionalism. Towards securing quality pharmaceutical education, pharmaceutical departments at universities must also organize and implement an educational curriculum based on the Model Core Curriculum for Pharmaceutical Education. This paper will introduce the investigative commission's review.


Asunto(s)
Inteligencia Artificial , Farmacéuticos , Humanos , Curriculum , Escolaridad , Preparaciones Farmacéuticas
19.
Brain Res ; 1800: 148197, 2023 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-36481236

RESUMEN

Phospholipid levels are reported to be decreased in Alzheimer's disease (AD). For a better understanding, we investigated the time-dependent changes of phospholipids species in a mouse model of AD. The levels of phospholipids in the hippocampus and prefrontal cortex of wild-type and APP-Tg (J20) mice were measured by LC-ESI-MS/MS. Compared to wild-type, total phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lysophosphatidylcholine (LPC) were Increased at 3 months but decreased at 6 months in the cortex of J20 mice. Total lysophosphatidylethanolamine (LPE) was decreased both at 3 and 6 months. PC was decreased and LPC was increased at 6 months, resulting in an increased LPC/PC ratio in the hippocampus of J20 mice. At species levels, PCA analysis could discriminate wild-type and J20 based on PC and LPC distribution at 6 months. At 6 months, several highly abundant PC including PC (16:0/16:0), PC (16:0/18:0), PC (16:0/18:1), and PC (18:0/18:1) were decreased in the cortex and hippocampus of J20. Conversely, LPC species including LPC 16:0, LPC 18:1, and LPC 20:4 were increased especially in the hippocampal area. Increased activation of phospholipid-metabolizing enzyme cPLA2 was seen in the hippocampus and cortex of J20 mice at 9 months. On the other hand, ROS levels started to increase as early as 3 months. Compared to 3 months, ROS levels were higher at 6 months in J20 mice. Thus, we demonstrated here a time- and area-dependent alteration of phospholipid composition during the early stage of AD, which could be important in understanding the pathological process.


Asunto(s)
Enfermedad de Alzheimer , Fosfolípidos , Ratones , Animales , Enfermedad de Alzheimer/patología , Especies Reactivas de Oxígeno , Espectrometría de Masas en Tándem , Encéfalo/patología
20.
Biochem Biophys Res Commun ; 423(3): 571-6, 2012 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-22695121

RESUMEN

The type I diacylglycerol kinase (DGK) isozymes (α, ß and γ) contain a shared recoverin homology (RVH) domain, a tandem repeat of Ca2+-binding EF-hand motifs, two cysteine-rich C1 domains, and the catalytic domain. We previously reported that a DGKα mutant lacking the RVH domain and EF-hands was constitutively active, implying that the N-terminal region (NTR) of DGKα, consisting of the RVH domain and EF-hand motifs, intramolecularly interacts with and masks the activity of the C-terminal region (CTR), containing the C1 and catalytic domains. In this study, we demonstrate that a glutathione S-transferase (GST)-fused DGKα-NTR construct physically binds to a green fluorescent protein (GFP)-fused DGKα-CTR construct. Moreover, co-precipitation of GFP-DGKα-CTR with GST-DGKα-NTR was clearly attenuated by the addition of 1 µM Ca2+. This result indicates that Ca2+ induces dissociation of the physical interaction between DGKα-NTR and DGKα-CTR. In addition to previously reported calcium-dependent changes in the hydrophobicity and net surface charge, Ca2+ also appeared to induce a decrease in the α-helical content of DGKα-NTR. These results suggest that Ca2+-induced conformational changes in the NTR release the intramolecular association between the NTR and the CTR of DGKα.


Asunto(s)
Calcio/química , Dominio Catalítico , Diacilglicerol Quinasa/química , Secuencias de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Diacilglicerol Quinasa/genética , Interacciones Hidrofóbicas e Hidrofílicas , Mutación , Porcinos
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