Effect of human acetylcholinesterase subunit assembly on its circulatory residence

2001 ◽  
Vol 354 (3) ◽  
pp. 613-625 ◽  
Author(s):  
Theodor CHITLARU ◽  
Chanoch KRONMAN ◽  
Baruch VELAN ◽  
Avigdor SHAFFERMAN
Keyword(s):  
Laser Desorption Ionization ◽  
Wild Type ◽  
Ionization Time ◽  
Hek 293 ◽  
Enzyme Preparations ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Differential Behaviour ◽  
Oligomeric Forms

Sialylated recombinant human acetylcholinesterase (rHuAChE), produced by stably transfected cells, is composed of a mixed population of monomers, dimers and tetramers and manifests a time-dependent circulatory enrichment of the higher-order oligomeric forms. To investigate this phenomenon further, homogeneous preparations of rHuAChE differing in their oligomerization statuses were generated: (1) monomers, represented by the oligomerization-impaired C580A-rHuAChE mutant, (2) wild-type (WT) dimers and (3) tetramers of WT-rHuAChE generated in vitro by complexation with a synthetic ColQ-derived proline-rich attachment domain (‘PRAD’) peptide. Three different series of each of these three oligoform preparations were produced: (1) partly sialylated, derived from HEK-293 cells; (2) fully sialylated, derived from engineered HEK-293 cells expressing high levels of sialyltransferase; and (3) desialylated, after treatment with sialidase to remove sialic acid termini quantitatively. The oligosaccharides associated with each of the various preparations were extensively analysed by matrix-assisted laser desorption ionization–time-of-flight MS. With the enzyme preparations comprising the fully sialylated series, a clear linear relationship between oligomerization and circulatory mean residence time (MRT) was observed. Thus monomers, dimers and tetramers exhibited MRTs of 110, 195 and 740min respectively. As the level of sialylation decreased, this differential behaviour became less pronounced; eventually, after desialylation all oligoforms had the same MRT (5min). These observations suggest that multiple removal systems contribute to the elimination of AChE from the circulation. Here we also demonstrate that by the combined modulation of sialylation and tetramerization it is possible to generate a rHuAChE displaying a circulatory residence exceeding that of all other known forms of native or recombinant human AChE.

Identification and characterization of four novel phosphorylation sites (Ser31, Ser325, Thr336 and Thr366) on LKB1/STK11, the protein kinase mutated in Peutz–Jeghers cancer syndrome

2002 ◽  
Vol 362 (2) ◽  
pp. 481-490 ◽  
Author(s):  
Gopal P. SAPKOTA ◽  
Jérôme BOUDEAU ◽  
Maria DEAK ◽  
Agnieszka KIELOCH ◽  
Nick MORRICE ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Wild Type ◽  
Cancer Syndrome ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Increased Risk ◽  
In Cells

Peutz—Jeghers syndrome is an inherited cancer syndrome, which results in a greatly increased risk of developing tumours in those affected. The causative gene encodes a nuclear-localized protein kinase, termed LKB1, which is predicted to function as a tumour suppressor. The mechanism by which LKB1 is regulated in cells is not known, and nor have any of its physiological substrates been identified. Recent studies have demonstrated that LKB1 is phosphorylated in cells. As a first step towards identifying the roles that phosphorylation of LKB1 play, we have mapped the residues that are phosphorylated in human embryonic kidney (HEK)-293 cells, as well as the major in vitro autophosphorylation sites. We demonstrate that LKB1 expressed in HEK-293 cells, in addition to being phosphorylated at Ser431, a previously characterized phosphorylation site, is also phosphorylated at Ser31, Ser325 and Thr366. Incubation of wild-type LKB1, but not a catalytically inactive mutant, with manganese-ATP in vitro resulted in the phosphorylation of LKB1 at Thr336 as well as at Thr366. We were unable to detect autophosphorylation at Thr189, a site previously claimed to be an LKB1 autophosphorylation site. A catalytically inactive mutant of LKB1 was phosphorylated at Ser31 and Ser325 in HEK-293 cells to the same extent as the wild-type enzyme, indicating that LKB1 does not phosphorylate itself at these residues. We show that phosphorylation of LKB1 does not directly affect its nuclear localization or its catalytic activity in vitro, but that its phosphorylation at Thr336, and perhaps to a lesser extent at Thr366, inhibits LKB1 from suppressing cell growth.

Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells

2007 ◽  
Vol 292 (3) ◽  
pp. F1028-F1034 ◽  
Author(s):  
W. Bruce Sneddon ◽  
Yanmei Yang ◽  
Jianming Ba ◽  
Lisa M. Harinstein ◽  
Peter A. Friedman
Keyword(s):  
Conditioned Media ◽  
Kidney Cells ◽  
Wild Type ◽  
Egfr Transactivation ◽  
Hek 293 ◽  
Erk Activation ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Extracellular Signal

The PTH receptor (PTH1R) activates multiple signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). The role of epidermal growth factor receptor (EGFR) transactivation in ERK1/2 activation by PTH in distal kidney cells, a primary site of PTH action, was characterized. ERK1/2 phosphorylation was stimulated by PTH and blocked by the EGFR inhibitor, AG1478. Upon PTH stimulation, metalloprotease cleavage of membrane-bound heparin-binding fragment (HB-EGF) induced EGFR transactivation of ERK. Conditioned media from PTH-treated distal kidney cells activated ERK in HEK-293 cells. AG1478 added to HEK-293 cells ablated transactivation by conditioned media. HB-EGF directly activated ERK1/2 in HEK-293 cells. Pretreatment of distal kidney cells with the metalloprotease inhibitor GM-6001 abolished transactivation of ERK1/2 by PTH. The role of the PTH1R COOH terminus in PTX-sensitive ERK1/2 activation was characterized in HEK-293 cells transfected with wild-type PTH1R, with a PTH1R mutated at its COOH terminus, or with PTH1R truncated at position 480. PTH stimulated ERK by wild-type, mutated and truncated PTH1Rs 21-, 27- and 57-fold, respectively. Thus, the PTH1R COOH terminus exerts an inhibitory effect on ERK activation. EBP50, a scaffolding protein that binds to the PDZ recognition domain of the PTH1R, impaired PTH but not isoproterenol or calcitonin-induced ERK activation. Pertussis toxin inhibited PTH-stimulated ERK1/2 by mutated and truncated PTH1Rs and abolished ERK1/2 activation by wild-type PTH1R. We conclude that ERK phosphorylation in distal kidney cells by PTH requires PTH1R activation of Gi, which leads to stimulation of metalloprotease-mediated cleavage of HB-EGF and transactivation of the EGFR and is regulated by EBP50.

The effects of zolpidem treatment and withdrawal on the in vitro expression of recombinant α1β2γ2s GABAA receptors expressed in HEK 293 cells

2010 ◽  
Vol 382 (3) ◽  
pp. 201-212 ◽  
Author(s):  
Josipa Vlainić ◽  
Maja Jazvinšćak Jembrek ◽  
Dubravka Švob Štrac ◽  
Danka Peričić
Keyword(s):  
Gabaa Receptors ◽  
Hek 293 ◽  
In Vitro Expression ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Critical roles for p22phox in the structural maturation and subcellular targeting of Nox3

2007 ◽  
Vol 403 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Yoko Nakano ◽  
Botond Banfi ◽  
Algirdas J. Jesaitis ◽  
Mary C. Dinauer ◽  
Lee-Ann H. Allen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Targeting ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Regulatory Subunits ◽  
Hek 293 Cells ◽  
293 Cells ◽  
A Subunit ◽  
And Function

Otoconia are small biominerals in the inner ear that are indispensable for the normal perception of gravity and motion. Normal otoconia biogenesis requires Nox3, a Nox (NADPH oxidase) highly expressed in the vestibular system. In HEK-293 cells (human embryonic kidney cells) transfected with the Nox regulatory subunits NoxO1 (Nox organizer 1) and NoxA1 (Nox activator 1), functional murine Nox3 was expressed in the plasma membrane and exhibited a haem spectrum identical with that of Nox2, the electron transferase of the phagocyte Nox. In vitro Nox3 cDNA expressed an ∼50 kDa primary translation product that underwent N-linked glycosylation in the presence of canine microsomes. RNAi (RNA interference)-mediated reduction of endogenous p22phox, a subunit essential for stabilization of Nox2 in phagocytes, decreased Nox3 activity in reconstituted HEK-293 cells. p22phox co-precipitated not only with Nox3 and NoxO1 from transfectants expressing all three proteins, but also with NoxO1 in the absence of Nox3, indicating that p22phox physically associated with both Nox3 and with NoxO1. The plasma membrane localization of Nox3 but not of NoxO1 required p22phox. Moreover, the glycosylation and maturation of Nox3 required p22phox expression, suggesting that p22phox was required for the proper biosynthesis and function of Nox3. Taken together, these studies demonstrate critical roles for p22phox at several distinct points in the maturation and assembly of a functionally competent Nox3 in the plasma membrane.

scholarly journals β-Arrestin mediates oxytocin receptor signaling, which regulates uterine contractility and cellular migration

2011 ◽  
Vol 300 (3) ◽  
pp. E468-E477 ◽  
Author(s):  
Chad A. Grotegut ◽  
Liping Feng ◽  
Lan Mao ◽  
R. Phillips Heine ◽  
Amy P. Murtha ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Mapk Signaling ◽  
Oxytocin Receptor ◽  
Cellular Migration ◽  
Wild Type ◽  
Uterine Contractility ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Uterine Muscle ◽  
293 Cells

Desensitization of the oxytocin receptor (OXTR) in the setting of prolonged oxytocin exposure may lead to dysfunctional labor, which increases the risk for cesarean delivery, and uterine atony, which may result in postpartum hemorrhage. The molecular mechanism for OXTR desensitization is through the agonist-mediated recruitment of the multifunctional protein β-arrestin. In addition to its desensitizing function, β-arrestins have recently been shown to simultaneously activate downstream signaling. We tested whether oxytocin stimulation promotes β-arrestin-mediated OXTR desensitization in vivo and activates β-arrestin-mediated mitogen-activated protein kinase (MAPK) growth signaling. Uterine muscle strips isolated from wild-type mice exhibited diminished uterine contractility following repeated exposure to oxytocin, whereas uterine muscle strips from β-arrestin-1 and β-arrestin-2 knockout mice showed no desensitization. Utilizing siRNA knockdown of β-arrestin-1 and β-arrestin-2 in HEK-293 cells expressing the OXTR, we demonstrated oxytocin-mediated MAPK signaling that was dependent on β-arrestin-1 and β-arrestin-2. Wild-type and β-arrestin-1 and β-arrestin-2 knockout mice receiving intravenous oxytocin also demonstrated oxytocin-mediated MAPK signaling that was dependent on β-arrestin-1 and β-arrestin-2. Finally, to test the significance of β-arrestin-mediated signaling from the OXTR, HEK-293 cells expressing the OXTR showed β-arrestin-dependent proliferation in a cell migration assay following oxytocin treatment. In conclusion, β-arrestin is a multifunctional scaffold protein that mediates both desensitization of the OXTR, leading to decreases in uterine contractility, and MAPK growth signaling following stimulation by oxytocin. The development of unique OXTR ligands that prevent receptor desensitization may be a novel approach in the treatment of adverse clinical events secondary to prolonged oxytocin therapy.

Antibodies against the melanocortin-4 receptor act as inverse agonists in vitro and in vivo

2007 ◽  
Vol 292 (6) ◽  
pp. R2151-R2158 ◽  
Author(s):  
Jean-Christophe Peter ◽  
Janet R. Nicholson ◽  
Déborah Heydet ◽  
Anne-Catherine Lecourt ◽  
Johan Hoebeke ◽  
...  
Keyword(s):  
Food Intake ◽  
Third Ventricle ◽  
Separate Experiment ◽  
Inverse Agonists ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Melanocortin 4 Receptor ◽  
293 Cells ◽  
Immunofluorescence Labeling

Functionally active antibodies (Abs) against central G-protein-coupled receptors have not yet been reported. We selected the hypothalamic melanocortin-4 receptor (MC4-R) as a target because of its crucial role in the regulation of energy homeostasis. A 15 amino acid sequence of the N-terminal (NT) domain was used as an antigen. This peptide showed functional activity in surface plasmon resonance experiments and in studies on HEK-293 cells overexpressing the human MC4-R (hMC4-R). Rats immunized against the NT peptide produced specific antibodies, which were purified and characterized in vitro. In HEK-293 cells, rat anti-NT Abs showed specific immunofluorescence labeling of hMC4-R. They reduced the production of cAMP under basal conditions and after stimulation with a synthetic MC4-R agonist. Rats immunized against the NT peptide developed a phenotype consistent with MC4-R blockade, that is, increased food intake and body weight, increased liver and fat pad weight, and elevated plasma triglycerides. In a separate experiment in rats, an increase in food intake could be produced after injection of purified Abs into the third ventricle. Similar results were obtained in rats injected with anti-NT Abs raised in rabbits. Our data show for the first time that active immunization of rats against the NT sequence of the MC4-R results in specific Abs, which appear to stimulate food intake by acting as inverse agonists in the hypothalamus.

scholarly journals Characterization of the reversible phosphorylation and activation of ERK8

2006 ◽  
Vol 394 (1) ◽  
pp. 365-373 ◽  
Author(s):  
Iva V. Klevernic ◽  
Margaret J. Stafford ◽  
Nicholas Morrice ◽  
Mark Peggie ◽  
Simon Morton ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Growth Factor ◽  
Protein Kinase ◽  
Insect Cells ◽  
Osmotic Shock ◽  
Tyrosine Phosphatase ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

ERK8 (extracellular-signal-regulated protein kinase 8) expressed in Escherichia coli or insect cells was catalytically active and phosphorylated at both residues of the Thr-Glu-Tyr motif. Dephosphorylation of the threonine residue by PP2A (protein serine/threonine phosphatase 2A) decreased ERK8 activity by over 95% in vitro, whereas complete dephosphorylation of the tyrosine residue by PTP1B (protein tyrosine phosphatase 1B) decreased activity by only 15–20%. Wild-type ERK8 expressed in HEK-293 cells was over 100-fold less active than the enzyme expressed in bacteria or insect cells, but activity could be increased by exposure to hydrogen peroxide, by incubation with the protein serine/threonine phosphatase inhibitor okadaic acid, or more weakly by osmotic shock. In unstimulated cells, ERK8 was monophosphorylated at Tyr-177, and exposure to hydrogen peroxide induced the appearance of ERK8 that was dually phosphorylated at both Thr-175 and Tyr-177. IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), PMA or anisomycin had little effect on activity. In HEK-293 cells, phosphorylation of the Thr-Glu-Tyr motif of ERK8 was prevented by Ro 318220, a potent inhibitor of ERK8 in vitro. The catalytically inactive mutants ERK8[D154A] and ERK8[K42A] were not phosphorylated in HEK-293 cells or E. coli, whether or not the cells had been incubated with protein phosphatase inhibitors or exposed to hydrogen peroxide. Our results suggest that the activity of ERK8 in transfected HEK-293 cells depends on the relative rates of ERK8 autophosphorylation and dephosphorylation by one or more members of the PPP family of protein serine/threonine phosphatases. The major residue in myelin basic protein phosphorylated by ERK8 (Ser-126) was distinct from that phosphorylated by ERK2 (Thr-97), demonstrating that, although ERK8 is a proline-directed protein kinase, its specificity is distinct from ERK1/ERK2.

scholarly journals Nephroprotective activity of Annona Squamosa leaves against paracetamol-induced nephrotoxicity in rats: in vitro and in vivo experiments

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
S. Neelima ◽  
P. Dwarakanadha Reddy ◽  
Chandra Sekhar Kothapalli Bannoth
Keyword(s):  
Kidney Tissue ◽  
Ethanolic Extract ◽  
Tissue Homogenate ◽  
Annona Squamosa ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
In Vivo Experiments ◽  
293 Cells

Abstract Background Paracetamol (PCM), being extensively adapted analgesic and anti-inflammatory drug all over the world, beyond therapeutic dosages, the oxidative stress-involved nephrotoxicity has been evidenced. However, herbal plants are the windfall for the humankind providing solution for most of the wellness breakdowns. Annona squamosa (AS) is one of such plants with enormous therapeutic and nutraceutical potencies. The main aspiration of the current investigation is to evaluate the nephroprotective ability of ethanolic extract of Annona squamosa (EEAS) leaves against paracetamol-induced nephrotoxicity using in vitro human embryonic kidney (HEK)-293 cells and in vivo experiments in Wistar rats through biochemical parameters, oxidative parameters, and histopathological findings. Results When HEK-293 cells were incubated with PCM, an increased cell death associated with alterations in the morphology of normal cells was observed. At variable concentrations, HEK-293 cells co-treated with PCM and EEAS extracts gave a significant improvement in cell growth on comparing with PCM treatment showing cytoprotective feature of EEAS with an IC50 28.75 μg/mL. In vivo nephroprotective property was assessed from the amount of blood urea nitrogen (BUN) along with creatinine and uric acid which were reduced (P < 0.001) within serum and compact levels of glutathione, catalase, and superoxide dismutase which were termed as GSH, CAT, and SOD, respectively, were increased (P < 0.001) in kidney tissue homogenate in the treated groups than the PCM alone group. Results were additionally supported by histopathological observations. Conclusion The results exhibited that EEAS has impending benefits against PCM-induced nephrotoxicity through in vitro and in vivo experiments.

scholarly journals In vitro 3D Spheroid Culture Developed on the Parafilm Surface Using HEK-293 Cells

2020 ◽  
Vol 3 (1) ◽  
pp. 220-227
Author(s):  
Erdal Eroğlu
Keyword(s):  
Three Dimensional ◽  
Culture Method ◽  
In Vivo Studies ◽  
Preclinical Research ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Hanging Drop Culture

Preclinical research to predict the effects of drugs and chemicals on humans is commonly carried out either by cell culture studies in vitro condition or on animals in vivo condition. While drug studies tested on cells cultured as a monolayer in plastic flasks are incompatible with realistic results, falsifying findings can also be achieved from in vivo studies performed on different species. In recent years, research on drug tests using spheroid cultures formed by growing cells in three-dimensional (3D) in vitro has attracted great interest. 3D spheroid structures are formed by growing the cells in a drop suspended on superhydrophobic surfaces. In this study, HEK-293 cells were investigated on parafilm surfaces displaying superhydrophobic properties by growing in 2 &amp;micro;l volume using hanging drop culture method in terms of spheroid formation. Light microscopy images from spheroid structures were taken on different incubation days and the area of spheroids was measured using the ImageJ program. Our study holds important findings for a chip platform that can be developed for use in vitro drug tests.

334 ADENOVIRAL VECTOR-MEDIATED EXPRESSION OF RECOMBINANT HUMAN GLUCOCEREBROSIDASE IN THE MAMMARY GLAND OF RATS

2013 ◽  
Vol 25 (1) ◽  
pp. 314
Author(s):  
K. C. S. Tavares ◽  
C. Feltrin ◽  
I. S. Carneiro ◽  
A. S. Morais ◽  
C. D. Medeiros ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Mammary Glands ◽  
Santa Cruz ◽  
Cmv Promoter ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Control Milk

Glucocerebrosidase is a lysosomal enzyme that plays a key role in sphingolipid cleavage, an intermediate in glycolipid metabolism. A recessive mutation in the glucocerebrosidase gene leads to the accumulation of glucosylceramide in macrophages (sphingolipidosis), a lysosomal storage disease known in humans as the Gaucher disease. The enzyme replacement treatment with recombinant human glucocerebrosidase (hGCase) dramatically reduces and reverses symptoms, with the need of lifelong treatment for patients to attain a normal life. Currently, hGCase is very costly, being produced through in vitro expression in Chinese hamster ovary cells or in vivo, in plants. The aim of this study was to develop a model for the production of hGCase in the mammary gland of rats transiently transduced with recombinant adenovirus. A replication-defective adenovirus carrying hGCase was generated using the AdEasy™ adenoviral vector system (Stratagene, La Jolla, CA, USA). The hGCase cDNA (NM_001005741) was in vitro-synthesized and ligated in the XhoI site of the pAdTrack-CMV vector (pAdT-hGCase). The resulting plasmid was recombined with the pAdEasy™ vector in BJ5183 electro-competent cells. The purified pAdE-pAdT-hGCase vector was linearized and transfected into HEK-293 cells for the production of a primary viral stock. Further amplifications and the titration assay were done in HEK-293 cells, monitoring the transduction by the qualitative evaluation of green fluorescent protein (GFP) expression. Following transfection, the HEK-293 cells increasingly expressed the GFP reporter, regulated by a CMV promoter, in tandem with the hGCase cDNA, under another CMV promoter. On Day 18 of gestation, a female rat (Rattus norvegicus) was anesthetized and the 2 left caudal mammary glands were infused with 109 GTU mL–1 of the pAdE-pAdT-hGCase in PBS solution supplemented with 36 mM EGTA. The 2 right caudal mammary glands were infused only with PBS-EGTA (control milk). Milk samples collected from Days 2 through 9 post-partum were mixed with separation buffer (10 mM Tris-HCl, pH 8.0; 10 mM CaCl2) and centrifuged, with the supernatant assayed for hGCase by Western blot using a monoclonal anti-human glucocerebrosidase antibody (sc-166407, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Relative quantification of the hGCase expression was done using the FluorChem FC2 system (Alpha Innotech, San Leandro, CA, USA), with hGCase band intensity being normalized against GAPDH expression. The in vivo expression assay confirmed the production of hGCase in the secreted portion of the rat milk, with a specific band between 50 to 60 kDa observed on the Western blot, and no detection of the protein in the control milk. The hGCase peak production occurred in Days 5 and 6 of lactation, with levels being 35 times greater than on Day 9. An ELISA quantification assay and an enzymatic activity assay for the recombinant hGCase are currently in development. In conclusion, the use of the rat for hGCase transient expression in the milk was proven a valid model for testing the potential use of a mammary gland expression system for the production of a functional human glucocerebrosidase protein.

Sign in / Sign up

Export Citation Format

Share Document