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991.
992.
Identification of a novel serine phosphorylation site in human glutamine:fructose-6-phosphate amidotransferase isoform 1 总被引:1,自引:0,他引:1
Li Y Roux C Lazereg S LeCaer JP Laprévote O Badet B Badet-Denisot MA 《Biochemistry》2007,46(45):13163-13169
Glutamine:fructose-6-phosphate amidotransferase (Gfat) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway. The increasing amount of evidence that links excess hexosamine biosynthesis with pathogenic complications of type II diabetes highlights the need to understand the regulation of Gfat. Previous studies showed that eukaryotic Gfat is subjected to feedback inhibition by UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) and to phosphorylation by cAMP-activated protein kinase A (PKA). In this study, overexpression of human Gfat isoform 1 (hGfat1) in insect cells revealed that hGfat1 is phosphorylated in vivo. Using matrix-assisted laser desorption/ionization and electrospray tandem mass spectrometry, we have identified Ser243 as a novel phosphorylation site. Biochemical properties of the wild type and the Ser243Glu mutant of hGfat1 overexpressed in Escherichia coli were compared. Our results provide evidence that phosphorylation at Ser243 stimulates glucosamine 6-phosphate-synthesizing activity, lowers amidohydrolyzing activity in the absence of fructose 6-phosphate (F6P) (glutaminase activity), and lowers Km(F6P) 2-fold, but has no effect on UDP-GlcNAc inhibition. On the basis of the sequence consensus, AMP-activated protein kinase and calcium/calmodulin-dependent kinase II were identified to phosphorylate specifically Ser243 in vitro. Phosphorylation by these two kinases results in an increase of enzymatic activity by 1.4-fold. These findings suggest for the first time that hGfat1 may be regulated by kinases other than PKA. 相似文献
993.
994.
Ken Yamada Samuel Hildebrand Sarah M Davis Rachael Miller Faith Conroy Ellen Sapp Jillian Caiazzi Julia F Alterman Loic Roux Dimas Echeverria Matthew R Hassler Edith L Pfister Marian DiFiglia Neil Aronin Anastasia Khvorova 《Nucleic acids research》2021,49(21):12069
Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (iE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for iE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the iE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of iE-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington''s disease as an example, iE-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the iE-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs. 相似文献
995.
The interaction of the 5-14, 1-14, and 1-24 fragments of ACTH with sonicated phospholipid bilayers containing egg yolk phosphatidylcholine (EPC) either pure or mixed with 10 mole % phosphatidic acid (EPA), was investigated by proton nuclear magnetic resonance (1H-nmr). The effects observed with zwitterionic EPC vesicles were small, indicating a low binding of the ACTH derivatives. The N-terminal aromatic resonances of the ACTH peptides were markedly broadened in the presence of negatively charged vesicles (EPC/EPA 9:1 M/M), while those of the C-terminal end were barely affected, showing that ACTH interacts with its N-terminal fragment. The choline resonance of the EPC molecules of the outer monolayer was shifted and broadened upon ACTH binding to the lipid vesicles, while that of the inner layer was not affected, suggesting that the peptide molecules interact only with the external leaflet of the lipid bilayer. The C2H and C4H resonances of the histidine-6 side chain were both shifted downfield upon peptide binding to the negatively charged lipid interface. In the case of the 1–24 derivative, these resonances were also split into two signals reflecting two different species of membrane-bound ACTH 1–24. Analysis of the line width and chemical shift variations of the ACTH and lipid resonances observed upon peptide binding shows that the membrane-binding potency of the shorter 5–14+1 fragment, which presents a +1 net charge, is roughly similar to that of the highly cationic 1–24+6 (net charge +6) derivative, implying that the 15–24+5 segment is not essential for membrane binding. The nmr measurements at a fixed lipid-to-peptide ratio in the presence of increasing amounts of spin-labeled lipids demonstrate that the N-terminal fragment of ACTH does not penetrate the hydrophobic core of the bilayer, and should lie parallel to the membrane surface. © 1997 John Wiley & Sons, Inc. Biopoly 42: 731–744, 1997 相似文献
996.
Osteoclast activation is a critical cellular process for pathological bone resorption, such as erosions in rheumatoid arthritis (RA) or generalized bone loss. Among many factors triggering excessive osteoclast activity, cytokines such as IL-1 or tumour necrosis factor (TNF)-α play a central role. New members of the TNF receptor ligand family (namely receptor activator of nuclear factor-κB [RANK] and RANK ligand [RANKL]) have been discovered whose cross-interaction is mandatory for the differentiation of osteoclasts from hemopoietic precursors, in both physiological and pathological situations. Osteoprotegerin, a decoy receptor which blocks this interaction, decreases osteoclast activity and could have a fascinating therapeutic potential in conditions associated with upregulated bone resorption. 相似文献
997.
998.
Anglique Desplat Virginie Penalba Emeline Gros Thibaud Parpaite Bertrand Coste Patrick Delmas 《The Journal of general physiology》2021,153(12)
Cholangiocytes actively contribute to the final composition of secreted bile. These cells are exposed to abnormal mechanical stimuli during obstructive cholestasis, which has a deep impact on their function. However, the effects of mechanical insults on cholangiocyte function are not understood. Combining gene silencing and pharmacological assays with live calcium imaging, we probed molecular candidates essential for coupling mechanical force to ATP secretion in mouse cholangiocytes. We show that Piezo1 and Pannexin1 are necessary for eliciting the downstream effects of mechanical stress. By mediating a rise in intracellular Ca2+, Piezo1 acts as a mechanosensor responsible for translating cell swelling into activation of Panx1, which triggers ATP release and subsequent signal amplification through P2X4R. Co-immunoprecipitation and pull-down assays indicated physical interaction between Piezo1 and Panx1, which leads to stable plasma membrane complexes. Piezo1–Panx1–P2X4R ATP release pathway could be reconstituted in HEK Piezo1 KO cells. Thus, our data suggest that Piezo1 and Panx1 can form a functional signaling complex that controls force-induced ATP secretion in cholangiocytes. These findings may foster the development of novel therapeutic strategies for biliary diseases. 相似文献
999.
Dexamethasone Regulation of P-Glycoprotein Activity in an Immortalized Rat Brain Endothelial Cell Line, GPNT 总被引:5,自引:0,他引:5
A Régina I A Romero J Greenwood P Adamson J M Bourre P O Couraud F Roux 《Journal of neurochemistry》1999,73(5):1954-1963
The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain capillary endothelial cells, thus forming a functional barrier to lipid-soluble drugs, notably, antitumor agents. It is of interest to develop an in vitro BBB model that stably expresses P-gp to investigate the mechanisms of regulation in expression and activity. The rat brain endothelial cell line, GPNT, was derived from a previously characterized rat brain endothelial cell line. A strong expression of P-gp was found in GPNT monocultures, whereas the multidrug resistance-associated pump Mrp1 was not expressed. The transendothelial permeability coefficient of the P-gp substrate vincristine across GPNT monolayers was close to the permeability coefficient of bovine brain endothelial cells cocultured with astrocytes, a previously documented in vitro BBB model. Furthermore, the P-gp blocker cyclosporin A induced a large increase in apical to basal permeability of vincristine. Thus, P-gp is highly functional in GPNT cells. A 1-h treatment of GPNT cells with dexamethasone resulted in decreased uptake of vincristine without any increase in P-gp expression. This effect could be mimicked by protein kinase C (PKC) activation and prevented by PKC inhibition, strongly suggesting that activation of P-gp function may involve a PKC-dependent pathway. These results document the GPNT cell line as a valuable in vitro model for studying drug transport and P-gp function at the BBB and suggest that activation of P-gp activity at the BBB might be considered in chemotherapeutic treatment of cancer patients. 相似文献
1000.
Different functions for the thyroid hormone receptors TRalpha and TRbeta in the control of thyroid hormone production and post-natal development. 总被引:17,自引:1,他引:16 下载免费PDF全文
K Gauthier O Chassande M Plateroti J P Roux C Legrand B Pain B Rousset R Weiss J Trouillas J Samarut 《The EMBO journal》1999,18(3):623-631
The biological activities of thyroid hormones are thought to be mediated by receptors generated by the TRalpha and TRbeta loci. The existence of several receptor isoforms suggests that different functions are mediated by specific isoforms and raises the possibility of functional redundancies. We have inactivated both TRalpha and TRbeta genes by homologous recombination in the mouse and compared the phenotypes of wild-type, and single and double mutant mice. We show by this method that the TRbeta receptors are the most potent regulators of the production of thyroid stimulating hormone (TSH). However, in the absence of TRbeta, the products of the TRalpha gene can fulfill this function as, in the absence of any receptors, TSH and thyroid hormone concentrations reach very high levels. We also show that TRbeta, in contrast to TRalpha, is dispensable for the normal development of bone and intestine. In bone, the disruption of both TRalpha and TRbeta genes does not modify the maturation delay observed in TRalpha -/- mice. In the ileum, the absence of any receptor results in a much more severe impairment than that observed in TRalpha -/- animals. We conclude that each of the two families of proteins mediate specific functions of triiodothyronin (T3), and that redundancy is only partial and concerns a limited number of functions. 相似文献