RESUMEN
Macrophage classical M1 activation via TLR4 triggers a variety of responses to achieve the elimination of foreign pathogens. During this process, there is also an increase in lipid droplets which contain large quantities of triacylglycerol (TAG) and phospholipid (PL). The functional consequences of this increment in lipid mass are poorly understood. Here, we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). Using bone marrow-derived macrophages (BMDMs) treated with Kdo2-lipid A, we showed that glycerolipid synthesis is induced during macrophage activation. GPAT4 protein level and GPAT3/GPAT4 enzymatic activity increase during this process, and these two isoforms were required for the accumulation of cell TAG and PL. The phagocytic capacity of Gpat3-/- and Gpat4-/- BMDM was impaired. Additionally, inhibiting fatty acid ß-oxidation reduced phagocytosis only partially, suggesting that lipid accumulation is not necessary for the energy requirements for phagocytosis. Finally, Gpat4-/- BMDM expressed and released more pro-inflammatory cytokines and chemokines after macrophage activation, suggesting a role for GPAT4 in suppressing inflammatory responses. Together, these results provide evidence that glycerolipid synthesis directed by GPAT4 is important for the attenuation of the inflammatory response in activated macrophages.
Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Lipogénesis , Macrófagos/enzimología , Fosfolípidos/biosíntesis , Triglicéridos/biosíntesis , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Animales , Glicerol-3-Fosfato O-Aciltransferasa/genética , Inflamación/enzimología , Inflamación/genética , Inflamación/patología , Activación de Macrófagos/genética , Macrófagos/patología , Ratones , Ratones Noqueados , Fosfolípidos/genética , Triglicéridos/genéticaRESUMEN
Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first and rate-limiting step in the de novo glycerolipid synthesis. The GPAT2 isoform differs from the other isoforms because its expression is restricted to male germ cells and cancer cells. It has been recently reported that GPAT2 expression in mouse testis fluctuates during sexual maturation and that it is regulated by epigenetic mechanisms in combination with vitamin A derivatives. Despite progress made in this field, information about GPAT2 role in the developing male germ cells remains unclear. The aim of the present study was to confirm the hypothesis that GPAT2 is required for the normal physiology of testes and male germ cell maturation. The gene was silenced in vivo by inoculating lentiviral particles carrying the sequence of a short-hairpin RNA targeting Gpat2 mRNA into mouse testis. Histological and gene expression analysis showed impaired spermatogenesis and arrest at the pachytene stage. Defects in reproductive fitness were also observed, and the analysis of apoptosis-related gene expression demonstrated the activation of apoptosis in Gpat2-silenced germ cells. These findings indicate that GPAT2 protein is necessary for the normal development of male gonocytes, and that its absence triggers apoptotic mechanisms, thereby decreasing the number of dividing germ cells.
Asunto(s)
Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Túbulos Seminíferos/metabolismo , Espermatogénesis , Espermatozoides/enzimología , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Glicerol-3-Fosfato O-Aciltransferasa/antagonistas & inhibidores , Glicerol-3-Fosfato O-Aciltransferasa/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Masculino , Ratones Endogámicos BALB C , Microscopía Fluorescente , Fase Paquiteno , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Túbulos Seminíferos/citología , Túbulos Seminíferos/crecimiento & desarrollo , Espermatozoides/citología , Espermatozoides/metabolismoRESUMEN
Our understanding of the synthesis and remodeling of mitochondrial phospholipids remains incomplete. Two isoforms of glycerol-3-phosphate acyltransferase (GPAT1 and 2) and two isoforms of acylglycerol-3-phosphate acyltransferase (AGPAT4 and 5) are located on the outer mitochondrial membrane, suggesting that both lysophosphatidic acid and phosphatidic acid are synthesized in situ for de novo glycerolipid biosynthesis. However, it is believed that the phosphatidic acid substrate for cardiolipin and phosphatidylethanolamine biosynthesis is produced at the endoplasmic reticulum whereas the phosphatidic acid synthesized in the mitochondria must be transferred to the endoplasmic reticulum before it undergoes additional steps to form the mature phospholipids that are trafficked back to the mitochondria. It is unclear whether mitochondrial phospholipids are remodeled by mitochondrial acyltransferases or whether lysophospholipids must return to the endoplasmic reticulum or to the mitochondrial associated membrane for reesterification. In this review we will focus on the few glycerolipid acyltransferases that are known to be mitochondrial. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.
Asunto(s)
Aciltransferasas/metabolismo , Glicerofosfolípidos/metabolismo , Mitocondrias/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Glicerol-3-Fosfato O-Aciltransferasa , Humanos , Metabolismo de los Lípidos/fisiología , Transporte de Proteínas/fisiologíaRESUMEN
Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as encoding a glycerol-3-phosphate acyltransferase by sequence homology to Gpat1, GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs (piwi-interacting RNAs), a group of small RNAs that protect the germ cell genome from retrotransposable elements. In order to understand the relationship between GPAT2 and its role in the testis, we focused on Gpat2 expression during the first wave of mouse spermatogenesis. Gpat2 expression was analysed by qPCR (quantitative real-time PCR), in situ hybridization, immunohistochemistry and Western blotting. Gpat2 mRNA content and protein expression were maximal at 15 dpp (days post-partum) and were restricted to pachytene spermatocytes. To achieve this transient expression, both epigenetic mechanisms and trans-acting factors are involved. In vitro assays showed that Gpat2 expression correlates with DNA demethylation and histone acetylation and that it is up-regulated by retinoic acid. Epigenetic regulation by DNA methylation was confirmed in vivo in germ cells by bisulfite sequencing of the Gpat2 promoter. Consistent with the initiation of meiosis at 11 dpp, methylation decreased dramatically. Thus, Gpat2 is expressed at a specific stage of spermatogenesis, consistent with piRNA synthesis and meiosis I prophase, and its on-off expression pattern responds predominantly to epigenetic modifications.
Asunto(s)
Metilación de ADN/fisiología , Epigénesis Genética/fisiología , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Profase Meiótica I/fisiología , Fase Paquiteno/fisiología , Regiones Promotoras Genéticas/fisiología , Espermatocitos/metabolismo , Espermatogénesis/fisiología , Animales , Glicerol-3-Fosfato O-Aciltransferasa/genética , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Espermatocitos/citologíaRESUMEN
Reverse cholesterol transport is a process of high antiatherogenic relevance in which apolipoprotein AI (apoA-I) plays an important role. The interaction of apoA-I with peripheral cells produces through mechanisms that are still poorly understood the mobilization of intracellular cholesterol depots toward plasma membrane. In macrophages, these mechanisms seem to be related to the modulation of the activity of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme responsible for the intracellular cholesterol ester biosynthesis that is stored in lipid droplets. The activation of ACAT and the accumulation of lipid droplets play a key role in the transformation of macrophages into foam cells, leading to the formation of atheroma or atherosclerotic plaque. ApoA-I Helsinki (or ∆K107) is a natural apoA-I variant with a lysine deletion in the central protein region, carriers of which have increased atherosclerosis risk. We herein show that treatment of cultured RAW macrophages or CHOK1 cells with ∆K107, but not with wild-type apoA-I or a variant containing a similar deletion at the C-terminal region (∆K226), lead to a marked increase (more than 10 times) in the intracellular ACAT1 protein level as detected by western blot analysis. However, we could only detect a slight increase in cholesteryl ester produced by ∆K107 mainly when Chol loading was supplied by low-density lipoprotein (LDL). Although a similar choline-phospholipid efflux is evoked by these apoA-I variants, the change in phosphatidylcholine/sphyngomyelin distribution produced by wild-type apoA-I is not observed with either ∆K107 or ∆K226.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/metabolismo , Apolipoproteína A-I/fisiología , Acetil-CoA C-Acetiltransferasa/genética , Animales , Línea Celular , Membrana Celular/metabolismo , Supervivencia Celular , Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , LDL-Colesterol/fisiología , Expresión Génica , Humanos , Ratones , Fosfolípidos/metabolismo , Triglicéridos/metabolismoRESUMEN
ß-Carbolines (ßCs) are a group of alkaloids present in many plants and animals. It has been suggested that these alkaloids participate in a variety of significant photosensitized processes. Despite their well-established natural occurrence, the main biological role of these alkaloids and the mechanisms involved are, to date, poorly understood. In the present work, we examined the capability of three important ßCs (norharmane, harmane and harmine) and two of its derivatives (N-methyl-norharmane and N-methyl-harmane) to induce DNA damage upon UV-A excitation, correlating the type and extent of the damage with the photophysical characteristics and DNA binding properties of the compounds. The results indicate that DNA damage is mostly mediated by a direct type-I photoreaction of the protonated ßCs after non-intercalative electrostatic binding. Reactive oxygen species such as singlet oxygen and superoxide are not involved to a major extent, as indicated by the only small influence of D(2)O and of superoxide dismutase on damage generation. An analysis with repair enzymes revealed that oxidative purine modifications such as 8-oxo-7,8-dihydroguanine, sites of base loss and single-strand breaks (SSB) are generated by all ßCs, while only photoexcited harmine gives rise to the formation of cyclobutane pyrimidine dimers as well.
Asunto(s)
Carbolinas/química , ADN/química , Fármacos Fotosensibilizantes/química , Cinética , Estructura Molecular , Procesos Fotoquímicos , Especies Reactivas de Oxígeno , Timidina/químicaRESUMEN
Gliomas are the most frequent solid tumors in children. Among these, high-grade gliomas are less common in children than in adults, though they are similar in their aggressive clinical behavior. In adults, glioblastoma is the most lethal tumor of the central nervous system. Insulin-like growth factor 1 receptor (IGF1R) plays an important role in cancer biology, and its nuclear localization has been described as an adverse prognostic factor in different tumors. Previously, we have demonstrated that, in pediatric gliomas, IGF1R nuclear localization is significantly associated with high-grade tumors, worst clinical outcome, and increased risk of death. Herein we explore the role of IGF1R intracellular localization by comparing two glioblastoma cell lines that differ only in their IGF1R capacity to translocate to the nucleus. In vitro, IGF1R nuclear localization enhances glioblastoma cell motility and metabolism without affecting their proliferation. In vivo, IGF1R has the capacity to translocate to the nucleus and allows not only a higher proliferation rate and the earlier development of tumors but also renders the cells sensitive to OSI906 therapy. With this work, we provide evidence supporting the implications of the presence of IGF1R in the nucleus of glioma cells and a potential therapeutic opportunity for patients harboring gliomas with IGF1R nuclear localization.
Asunto(s)
Glioblastoma , Glioma , Adulto , Carcinogénesis/metabolismo , Núcleo Celular/metabolismo , Niño , Glioblastoma/metabolismo , Glioma/metabolismo , Humanos , Receptores de Somatomedina/metabolismoRESUMEN
Metal ions and metal complexes are important components of nucleic acid biochemistry, participating both in regulation of gene expression and as therapeutic agents. Three new transition metal complexes of copper(II), zinc(II) and oxidovanadium(IV) with a ligand derived from o-vanillin and thiophene were previously synthesized and their antitumor properties were studied in our laboratory. To elucidate some molecular mechanisms tending to explain the cytotoxic effects observed over tumor cells, we investigated the interaction of these complexes with DNA by gel electrophoresis, UV-Vis spectroscopy, docking studies and molecular dynamics simulations. Our spectroscopy and computational results have shown that all of them were able to bind to DNA, Cu(II) complex is located in the minor groove while Zn(II) and oxidovanadium(IV) complexes act as major groove binding molecules. Interestingly, only the Cu(II) complex caused double-strand DNA nicks, consistent with its higher cytotoxic activities previously observed in tumor cell lines. We propose that the DNA-complex interaction destabilize the molecule either disrupting the phosphodiester bonds or impairing DNA replication, giving those complexes strong antitumor potential.
Asunto(s)
Cobre/química , ADN/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Vanadatos/química , Zinc/química , Bases de SchiffRESUMEN
BACKGROUND AND AIMS: The transition of macrophage to foam cells is a major hallmark of early stage atherosclerotic lesions. This process is characterized by the accumulation of large cytoplasmic lipid droplets containing large quantities of cholesterol esters (CE), triacylglycerol (TAG) and phospholipid (PL). Although cholesterol and CE metabolism during foam cell formation has been broadly studied, little is known about the role of the glycerolipids (TAG and PL) in this context. Here we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). METHODS: We used RAW 264.7 cells and bone marrow derived macrophages (BMDM) treated with oxidized LDL (oxLDL). RESULTS: We showed that TAG synthesis is induced during the macrophage to foam cell transition. The expression and activity of GPAT3 and GPAT4 also increased during this process, and these two isoforms were required for the accumulation of cell TAG and PL. Compared to cells from wildtype mice after macrophage to foam cell transition, Gpat4-/- BMDM released more pro-inflammatory cytokines and chemokines, suggesting that the activity of GPAT4 could be associated with a decrease in the inflammatory response, probably by sequestering signaling precursors into lipid droplets. CONCLUSIONS: Our results provide evidence that TAG synthesis directed by GPAT3 and GPAT4 is required for lipid droplet formation and the modulation of the inflammatory response during the macrophage-foam cell transition.
Asunto(s)
Células Espumosas , Gotas Lipídicas , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Animales , Glicerol , Glicerol-3-Fosfato O-Aciltransferasa/genética , Lipoproteínas LDL , Macrófagos , Ratones , Fosfatos , TriglicéridosRESUMEN
UV-A radiation (320-400 nm) induces damages to the DNA molecule and its components through photosensitized reactions. Beta-carbolines (betaCs), heterocyclic compounds widespread in biological systems, participate in several biological processes and are able to act as photosensitizers. The photosensitization of plasmidic DNA by norharmane in aqueous solution under UV-A radiation was studied. The effect of pH was evaluated and the participation of reactive oxygen species (ROS), such as hydroxyl radical (HO*), superoxide anion (O(2)(*-)) and singlet oxygen ((1)O(2)) was investigated. A strong dependence of the photosensitized DNA relaxation on the pH was observed. The extent of the reaction was shown to be higher in the experiments performed at pH 4.7 than those performed at pH 10.2. As was expected, an intermediate extent of the reaction was observed at physiological pH (pH 7.4). Kinetic studies using ROS scavengers revealed that the chemical reactions between ROS and DNA are not the main pathways responsible for the damage of DNA. Consequently, the predominant mechanism yielding the DNA strand break takes place most probably via a type I mechanism (electron transfer) from the single excited state (S(1)) of the protonated form of norharmane ((1)[nHoH(+)]*). Additional information about the nature of the norharmane electronic excited states involved in the photocleavage reaction was obtained by using the N-methyl derivative of norharmane (N-methyl-norharmane).
Asunto(s)
Carbolinas/farmacología , ADN , Harmina/análogos & derivados , Luz , Plásmidos , ADN/efectos de los fármacos , Aductos de ADN/farmacología , Daño del ADN/efectos de los fármacos , Harmina/farmacología , Concentración de Iones de Hidrógeno , Fotoquímica , Plásmidos/efectos de los fármacos , Soluciones/química , Espectrofotometría , Agua/químicaRESUMEN
Glycerol 3-phosphate acyltransferase-1 (GPAT1), catalyzes the committed step in phospholipid and triacylglycerol synthesis. Because both GPAT1 and carnitine-palmitoyltransferase 1 are located on the outer mitochondrial membrane (OMM) it has been suggested that their reciprocal regulation controls acyl-CoA metabolism at the OMM. To determine whether GPAT1, like carnitine-palmitoyltransferase 1, is enriched in both mitochondrial contact sites and OMM, and to correlate protein location and enzymatic function, we used Percoll and sucrose gradient fractionation of rat liver to obtain submitochondrial fractions. Most GPAT1 protein was present in a vesicular membrane fraction associated with mitochondria (MAV) but GPAT specific activity in this fraction was low. In contrast, highest GPAT1 specific activity was present in purified mitochondria. Contact sites from crude mitochondria, which contained markers for both endoplasmic reticulum (ER) and mitochondria, also showed high expression of GPAT1 protein but low specific activity, whereas contact sites isolated from purified mitochondria lacked ER markers and expressed highly active GPAT1. To determine how GPAT1 is targeted to mitochondria, recombinant protein was synthesized in vitro and its incorporation into crude and purified mitochondria was assayed. GPAT1 was rapidly incorporated into mitochondria, but not into microsomes. Incorporation was ATP-driven, and lack of GPAT1 removal by alkali and a chaotropic agent showed that GPAT1 had become an integral membrane protein after incorporation. These results demonstrate that two pools of GPAT1 are present in rat liver mitochondria: an active one, located in OMM and a less active one, located in membranes (ER-contact sites and mitochondrial associated vesicles) associated with both mitochondria and ER.
Asunto(s)
Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Mitocondrias Hepáticas/enzimología , Membranas Mitocondriales/enzimología , Partículas Submitocóndricas/enzimología , Adenosina Trifosfato/farmacología , Animales , Biomarcadores/metabolismo , Fraccionamiento Celular , Mezclas Complejas , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/ultraestructura , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Partículas Submitocóndricas/efectos de los fármacos , Partículas Submitocóndricas/ultraestructuraRESUMEN
In mammalian cells, de novo glycerolipid synthesis begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferases (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions, and overexpressed in several types of cancers and cancer-derived human cell lines where its expression contributes to the tumor phenotype. Using gene silencing and atomic force microscopy, we studied the correlation between GPAT2 expression and cell surface topography, roughness and membrane permeability in MDA-MB-231 cells. In addition, we analyzed the glycerolipid composition by gas-liquid chromatography. GPAT2 expression altered the arachidonic acid content in glycerolipids, and the lack of GPAT2 seems to be partially compensated by the overexpression of another arachidonic-acid-metabolizing enzyme, AGPAT11. GPAT2 expressing cells exhibited a rougher topography and less membrane damage than GPAT2 silenced cells. Pore-like structures were present only in GPAT2 subexpressing cells, correlating with higher membrane damage evidenced by lactate dehydrogenase release. These GPAT2-induced changes are consistent with its proposed function as a tumor-promoting gene, and might be used as a phenotypic differentiation marker. AFM provides the basis for the identification and quantification of those changes, and demonstrates the utility of this technique in the study of cancer cell biology.
Asunto(s)
Neoplasias de la Mama/patología , Permeabilidad de la Membrana Celular , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Microscopía de Fuerza Atómica/métodos , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Femenino , Silenciador del Gen , Glicerol-3-Fosfato O-Aciltransferasa/genética , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Two lipovitellin (LV) forms containing the same apoproteins but differing in their lipid composition were isolated from Macrobrachium borelii eggs at early (LVe) and late (LVI) embryogenic stages and characterized. These two forms of LV, as well as liposomes prepared with lipids extracted from them, were used as simpler models to study the effect of the pesticide fenitrothion (FS) on their structures and functions. Rotational diffusion and fluorescence lifetime of two fluorescent probes [1,6-diphenyl-1,3,5-hexatriene (DPH) and 3-(p-(6-phenyl)-1,3,5-hexatrienal)phenylpropionic acid (DPH-PA)] were used to obtain information on structural changes induced by FS in the inner and outer regions of the LV, respectively. Comparison of the rotational behavior of these probes in native LV and liposomes (LP) from extracted LV lipids suggests that apoprotein-lipid interactions result in an ordered neutral lipid core. FS increased the lipid phase polarity of both LV and LP forms. The rotation of these probes in LP was not affected, suggesting a dependence of FS action on lipid-protein interactions. DPH-PA steady-state anisotropy showed that, unlike the LVe form, the LVI form was sensitive to extremely low FS concentrations. The ability of both LV to transfer palmitic acid to albumin was increased, but in a dissimilar manner, by the presence of FS. Such differences in the sensitivity of the LV at different steps of embryogenesis to FS influence the toxic action of this insecticide.
Asunto(s)
Proteínas Dietéticas del Huevo , Proteínas del Huevo , Fenitrotión/farmacología , Insecticidas/farmacología , Óvulo , Palaemonidae/embriología , Animales , Proteínas del Huevo/química , Proteínas del Huevo/metabolismo , Proteínas Dietéticas del Huevo/metabolismo , Femenino , Polarización de Fluorescencia , Metabolismo de los Lípidos , Lípidos/química , Óvulo/química , Óvulo/efectos de los fármacos , Ácido Palmítico/metabolismoRESUMEN
The de novo synthesis of glycerolipids in mammalian cells begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferase (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions. Because it is aberrantly expressed in multiple myeloma, it has been proposed as a novel cancer testis gene. Using a bioinformatics approach, we found that GPAT2 is highly expressed in melanoma, lung, prostate and breast cancer, and we validated GPAT2 expression at the protein level in breast cancer by immunohistochemistry. In this case GPAT2 expression correlated with a higher histological grade. 5-Aza-2' deoxycytidine treatment of human cells lines induced GPAT2 expression suggesting epigenetic regulation of gene expression. In order to evaluate the contribution of GPAT2 to the tumor phenotype, we silenced its expression in MDA-MB-231 cells. GPAT2 knockdown diminished cell proliferation, anchorage independent growth, migration and tumorigenicity, and increased staurosporine-induced apoptosis. In contrast, GPAT2 over-expression increased cell proliferation rate and resistance to staurosporine-induced apoptosis. To understand the functional role of GPAT2, we performed a co-expression analysis in mouse and human testis and found a significant association with semantic terms involved in cell cycle, DNA integrity maintenance, piRNA biogenesis and epigenetic regulation. Overall, these results indicate the GPAT2 would be directly associated with the control of cell proliferation. In conclusion, we confirm GPAT2 as a cancer testis gene and that its expression contributes to the tumor phenotype of MDA-MB-231 cells.
Asunto(s)
Neoplasias de la Mama/patología , Carcinogénesis/genética , Glicerol-3-Fosfato O-Aciltransferasa/genética , Testículo/metabolismo , Animales , Azacitidina/análogos & derivados , Azacitidina/farmacología , Neoplasias de la Mama/genética , Carcinogénesis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Transformación Celular Neoplásica , Simulación por Computador , Decitabina , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Glicerol-3-Fosfato O-Aciltransferasa/deficiencia , Humanos , Masculino , Ratones , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
BACKGROUND: De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid. METHODS AND RESULTS: Incubation of GPAT2-transfected CHO-K1 cells with [1-(14)C]arachidonate for 3 h increased incorporation of [(14)C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2's role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein. CONCLUSIONS: These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Células Germinativas/metabolismo , Glicerol-3-Fosfato O-Aciltransferasa/biosíntesis , Espermatozoides/metabolismo , Acilcoenzima A/metabolismo , Animales , Células CHO , Catálisis , Cricetinae , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Glicerol-3-Fosfato O-Aciltransferasa/genética , Humanos , Masculino , Ratones , Isoformas de Proteínas , Ratas , Especificidad por Sustrato , Testículo/metabolismo , Factores de Tiempo , Distribución TisularRESUMEN
GPAT1, one of four known glycerol-3-phosphate acyltransferase isoforms, is located on the mitochondrial outer membrane, allowing reciprocal regulation with carnitine palmitoyltransferase-1. GPAT1 is upregulated transcriptionally by insulin and SREBP-1c and downregulated acutely by AMP-activated protein kinase, consistent with a role in triacylglycerol synthesis. Knockout and overexpression studies suggest that GPAT1 is critical for the development of hepatic steatosis and that steatosis initiated by overexpression of GPAT1 causes hepatic, and perhaps also peripheral, insulin resistance. Future questions include the function of GPAT1 in relation to the other GPAT isoforms and whether the lipid intermediates synthesized by GPAT and downstream enzymes in the pathway of glycerolipid biosynthesis participate in intracellular signaling pathways.
Asunto(s)
Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Insulina/fisiología , Mitocondrias Hepáticas/enzimología , Triglicéridos/biosíntesis , Acilcoenzima A/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Femenino , Resistencia a la Insulina/fisiología , Isoenzimas/metabolismo , Lisofosfolípidos/metabolismo , Ratones , Membranas Mitocondriales/metabolismo , Ratas , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/fisiologíaRESUMEN
Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including N-ethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open-reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 microM; 0.25 microCi) [1-(14)C]oleate for 6h increased incorporation of [(14)C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an approximately 89-kDa band in liver and testis from GPAT1 null mice and both 89- and 80-kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis.
Asunto(s)
Glicerol-3-Fosfato O-Aciltransferasa/química , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Mitocondrias/enzimología , Testículo/enzimología , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular/métodos , Activación Enzimática , Glicerol-3-Fosfato O-Aciltransferasa/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Distribución TisularRESUMEN
Glycerol phosphate acyltransferase (GPAT) catalyzes the formation of 1-acyl-sn-glycerol-3-phosphate from glycerol-3-phosphate and long chain fatty acyl-CoA substrates. We previously determined the topography of the mitochondrial GPAT1 isoform (mtGPAT1, 828 amino acids). mtGPAT1 has two transmembrane domains (TMDs) (aa 472-493 and aa 576-592) with both the N- and C-termini facing the cytosol and a loop (aa 494-575) facing the intermembrane space. Alignment of amino acid sequences from mtGPAT1 and other acyltransferases and site directed mutagenesis studies have demonstrated that the active site of the enzyme resides in the N-terminal domain of the protein. In this study, we sequentially truncated the C-terminal domain and characterized the properties of the resulting mutants expressed in CHO cells. Although the mutants were overexpressed, none of them conferred GPAT activity. The loss of activity was not due to the miss-targeting of the proteins since immunofluorescence experiments demonstrated their mitochondrial localization. Instead, chemical crosslinking and protein cleavage studies demonstrated that the N- and C-termini of the protein interact. These results suggest that the C-terminal domain is necessary for mtGPAT1 activity, and probably contributes to catalysis or substrate binding.
Asunto(s)
Glicerol-3-Fosfato O-Aciltransferasa/fisiología , Mitocondrias/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Cricetinae , Cricetulus , Glicerol-3-Fosfato O-Aciltransferasa/genética , Cinética , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
Recent studies suggest that the long-chain acyl-CoA synthetases (ACS) may play a role in channeling fatty acids either toward complex lipid synthesis and storage or toward oxidation. Each of the five members of the ACS family that has been cloned has a distinct tissue distribution and subcellular location, and is regulated independently during cellular differentiation and by diverse hormones and nuclear transcription factors including adrenocorticotropic hormone (ACTH), peroxisomal proliferator-activated receptor-alpha (PPARalpha) and sterol regulatory element binding protein. Taken as a whole, these features suggest that in liver, ACS1 and ACS5 may provide acyl-CoA destined primarily for triacylglycerol synthesis or for mitochondrial oxidation, respectively. ACS4 may provide acyl-CoA for both synthesis and peroxisomal oxidation, depending on whether the enzyme is associated with the mitochondrial-associated membrane or with peroxisomes. It should be emphasized that although the data for acyl-CoA channeling are strong, they are indirect. Rigorous testing of these predictions will be required.