RESUMEN
Neovastat® is a standardized extract of marine cartilage, an avascular tissue, which contains many biologically active molecules and has multiple antiangiogenic properties. In addition to VEGFR2 and MMPs inhibition, shark cartilage extract (SCE) has recently been shown to induce tissue plasminogen activator gene (PLAT) expression in bovine endothelial cells in a TNF like manner, by inducing the typical mediators NF-κB and JNK. There is now compelling evidences that the NF-κB and JNK pathways are activated by cytokines induced generation of reactive oxygen species (ROS). We used macroarray genes expression analysis on human umbilical vein endothelial cells, to investigate if that mechanism could mediate the effect of SCE. Transcriptomic results showed that SCE induced expression of several cytokines. Their impact must be important, given that treatment of endothelial cells with the cytokine TNF-α was able to reproduce most of the effects of cartilage extract on genes expression. In addition, most of the genes, known to be inducible by NF-κB or JNK following cytokines stimulation, were less induced by SCE when endothelial cells were pretreated with the antioxidant N-Acetylcysteine (NAC), suggesting a role of ROS in endothelial cell activation by SCE. Finally, the possible effects of PLAT, PLG, SELE, IL8 and PRDX2 (those validated by q-PCR) on angiogenesis, will also be discussed.
Asunto(s)
Citocinas/metabolismo , Selectina E/biosíntesis , Plasminógeno/biosíntesis , Extractos de Tejidos/farmacología , Activador de Tejido Plasminógeno/biosíntesis , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Células Cultivadas , Citocinas/biosíntesis , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Especies Reactivas de Oxígeno/metabolismo , Extractos de Tejidos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Diaminopelargonic acid aminotransferase (DAPA-AT) and dethiobiotin synthetase (DTBS) catalyze the antepenultimate and the penultimate steps, respectively, of biotin synthesis. Whereas DAPA-AT and DTBS are encoded by distinct genes in bacteria, in biotin-synthesizing eukaryotes (plants and most fungi), both activities are carried out by a single enzyme encoded by a bifunctional gene originating from the fusion of prokaryotic monofunctional ancestor genes. In few angiosperms, including Arabidopsis thaliana, this chimeric gene (named BIO3-BIO1) also produces a bicistronic transcript potentially encoding separate monofunctional proteins that can be produced following an alternative splicing mechanism. The functional significance of the occurrence of a bifunctional enzyme in biotin synthesis pathway in eukaryotes and the relative implication of each of the potential enzyme forms (bifunctional versus monofunctional) in the plant biotin pathway are unknown. In this study, we demonstrate that the BIO3-BIO1 fusion protein is the sole protein form produced by the BIO3-BIO1 locus in Arabidopsis. The enzyme catalyzes both DAPA-AT and DTBS reactions in vitro and is targeted to mitochondria in vivo. Our biochemical and kinetic characterizations of the pure recombinant enzyme show that in the course of the reaction, the DAPA intermediate is directly transferred from the DAPA-AT active site to the DTBS active site. Analysis of several structures of the enzyme crystallized in complex with and without its ligands reveals key structural elements involved for acquisition of bifunctionality and brings, together with mutagenesis experiments, additional evidences for substrate channeling.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Biotina/biosíntesis , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo , Transaminasas/química , Transaminasas/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Biocatálisis , Vías Biosintéticas , Biotina/análogos & derivados , Biotina/química , Biotina/metabolismo , Dominio Catalítico , Holoenzimas/química , Holoenzimas/metabolismo , Cinética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Fracciones Subcelulares/enzimología , Especificidad por SustratoRESUMEN
In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO(2) fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO(2) through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts.
Asunto(s)
Arabidopsis/enzimología , Cloroplastos/enzimología , Fructosa-Bifosfato Aldolasa/metabolismo , Pisum sativum/enzimología , Procesamiento Proteico-Postraduccional/fisiología , Arabidopsis/genética , Cloroplastos/genética , Fructosa-Bifosfato Aldolasa/genética , Metilación , Pisum sativum/genética , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismoRESUMEN
OBJECTIVE: The roles of phosphatidylinositol 3 (PI3K) and mitogen-activated protein kinases (MAPK) have been widely studied in terms of the differentiation process induced by several drugs (phorbol ester, vitamin D-3, retinoic acid, etc.), but their exact functions in leukemic cells' phenotype and their potential therapeutic role remain incompletely clarified. MATERIALS AND METHODS: In order to investigate this query, leukemia cells were cultured in presence of kinase inhibitors (KIs). Proliferation, apoptosis, and differentiation were analyzed at the cellular and molecular levels, using flow cytometry and reverse transcriptase quantitative polymerase chain reaction. RESULTS: SB203580, a P38 MAPK inhibitor, had no effect on cell proliferation, whereas LY294002, a PI3K inhibitor, and PD098059, a selective inhibitor of mitogen-activated extracellular regulated kinase (MEK) phosphorylation, arrested cells in G(0)/G(1). However, LY294002 and PD098059 acted using different mechanisms: LY294002 decreased the expression of phosphorylated S6RP, whereas PD098059 increased P21/waf1 antigen expression. SP600125, an inhibitor of N-terminal c-jun kinases, arrested cells in G(2) and induced an endoreplicative process. SP600125 increased p21 at both the mRNA and protein levels. G(2) blockage is dependent on the PI3K pathway and the endoreplicative process is dependent on the PI3K and extracellular regulated kinase (ERK) pathways and mRNA synthesis. On the other hand, PD098059 potentiated the apoptotic process induced by either SP600125 or LY294002. Modulation of the expression of CD11, CD15, CD18, and CD54 was cell-dependent. CONCLUSION: Our results suggest that KIs modulate proliferation of leukemia cells and that the MEK/ERK inhibitor, PD098059, in combination with either SP600125 or LY294002, could have clinical value.
Asunto(s)
Leucemia/enzimología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Antracenos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Cromonas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Flavonoides/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Humanos , Imidazoles/farmacología , Leucemia/tratamiento farmacológico , Leucemia/patología , Monocitos/efectos de los fármacos , Monocitos/enzimología , Morfolinas/farmacología , Piridinas/farmacología , Células U937/efectos de los fármacos , Células U937/enzimologíaRESUMEN
Postprandial hypertriglyceridemia and low plasma HDL levels, which are principal features of the metabolic syndrome, are displayed by transgenic mice expressing human apolipoprotein A-II (hapoA-II). In these mice, hypertriglyceridemia results from the inhibition of lipoprotein lipase and hepatic lipase activities by hapoA-II carried on VLDL. This study aimed to determine whether the association of hapoA-II with triglyceride-rich lipoproteins (TRLs) is sufficient to impair their catabolism. To measure plasma TRL residence time, intestinal TRL production was induced by a radioactive oral lipid bolus. Radioactive and total triglyceride (TG) were rapidly cleared in control mice but accumulated in plasma of transgenic mice, in relation to hapoA-II concentration. Similar plasma TG accumulations were measured in transgenic mice with or without endogenous apoA-II expression. HapoA-II (synthesized in liver) was detected in chylomicrons (produced by intestine). The association of hapoA-II with TRL in plasma was further confirmed by the absence of hapoA-II in chylomicrons and VLDL of transgenic mice injected with Triton WR 1339, which prevents apolipoprotein exchanges. We show that the association of hapoA-II with TRL occurs in the circulation and induces postprandial hypertriglyceridemia.
Asunto(s)
Apolipoproteína A-II/sangre , Lipoproteínas/sangre , Triglicéridos/sangre , Animales , Apolipoproteína A-II/deficiencia , Apolipoproteína A-II/genética , Quilomicrones/metabolismo , Femenino , Humanos , Hiperglucemia/sangre , Hiperglucemia/etiología , Mucosa Intestinal/metabolismo , Lipoproteínas VLDL/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Recombinantes/sangre , Proteínas Recombinantes/genéticaRESUMEN
UM384 cells, derived from the human myeloid leukemia U937 cell line, fail to differentiate in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Using cDNA microarray and real-time quantitative PCR (RT-QPCR) approaches, we observed a difference in the response to TPA treatment: all the genes from U937 cells were continuously modulated from 2 to 24h. In UM384 cells, 60% of the genes were transiently modulated at 2h, then returned to control levels at 24h. Moreover, HuR, an AU-rich element-binding protein (ARE-BP), was differentially located in the two cell lines. Therefore, a defect of mRNA stabilization could be responsible for the resistance of UM384 cells to TPA-induced differentiation, suggesting a possible role for the post-transcriptional regulation in the leukemogenesis.