scholarly journals Bimolecular Complementation of Paramyxovirus Fusion and Hemagglutinin-Neuraminidase Proteins Enhances Fusion: Implications for the Mechanism of Fusion Triggering

2009 ◽  
Vol 83 (21) ◽  
pp. 10857-10868 ◽  
Author(s):  
Sarah A. Connolly ◽  
George P. Leser ◽  
Theodore S. Jardetzky ◽  
Robert A. Lamb
Keyword(s):  
Receptor Binding ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cell Types ◽  
Bimolecular Fluorescence Complementation ◽  
Dependent Manner ◽  
Viral Fusion ◽  
Concentration Dependent Manner ◽  
Cytoplasmic Tails ◽  
Multiple Cell

ABSTRACT For paramyxoviruses, entry requires a receptor-binding protein (hemagglutinin-neuraminidase [HN], H, or G) and a fusion protein (F). Like other class I viral fusion proteins, F is expressed as a prefusion metastable protein that undergoes a refolding event to induce fusion. HN binding to its receptor triggers F refolding by an unknown mechanism. HN may serve as a clamp that stabilizes F in its prefusion state until HN binds the target cell (the “clamp model”). Alternatively, HN itself may undergo a conformational change after receptor binding that destabilizes F and causes F to trigger (the “provocateur model”). To examine F-HN interactions by bimolecular fluorescence complementation (BiFC), the cytoplasmic tails of parainfluenza virus 5 (PIV5) F and HN were fused to complementary fragments of yellow fluorescent protein (YFP). Coexpression of the BiFC constructs resulted in fluorescence; however, coexpression with unrelated BiFC constructs also produced fluorescence. The affinity of the two halves of YFP presumably superseded the F-HN interaction. Unexpectedly, coexpression of the BiFC F and HN constructs greatly enhanced fusion in multiple cell types. We hypothesize that the increase in fusion occurs because the BiFC tags bring F and HN together more frequently than occurs in a wild-type (wt) scenario. This implies that normally much of wt F is not associated with wt HN, in conflict with the clamp model for activation. Correspondingly, we show that wt PIV5 fusion occurs in an HN concentration-dependent manner. Also inconsistent with the clamp model are the findings that BiFC F does not adopt a postfusion conformation when expressed in the absence of HN and that HN coexpression does not provide resistance to the heat-induced triggering of F. In support of a provocateur model of F activation, we demonstrate by analysis of the morphology of soluble F trimers that the hyperfusogenic mutation S443P has a destabilizing effect on F.

scholarly journals MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro

2013 ◽  
Vol 304 (8) ◽  
pp. L511-L518 ◽  
Author(s):  
Shijing Fang ◽  
Anne L. Crews ◽  
Wei Chen ◽  
Joungjoa Park ◽  
Qi Yin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Airway Epithelial Cells ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Mucin Secretion ◽  
Concentration Dependent Manner

Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.

scholarly journals DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain

2015 ◽  
Vol 37 (1) ◽  
pp. 193-200 ◽  
Author(s):  
Takeshi Kanno ◽  
Ayako Tsuchiya ◽  
Tadashi Shimizu ◽  
Miyuki Mabuchi ◽  
Akito Tanaka ◽  
...  
Keyword(s):  
Signal Intensity ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Reversed Phase ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Pc 12 Cells ◽  
Substrate Peptide ◽  
Carboxyl Terminal

Background/Aims: The linoleic acid derivative DCP-LA selectively and directly activates PKCε. The present study aimed at understanding the mechanism of DCP-LA-induced PKCε activation. Methods: Point mutation in the C2-like domain on PKCε was carried out, and each kinase activity was monitored in PC-12 cells using a föerster resonance energy transfer (FRET) probe with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) at the N- and C-terminal ends of PKCε, respectively, or in the cell-free systems using a reversed phase high-performance liquid chromatography (HPLC). Intracellular PKCε mobilization was monitored in PC-12 cells using mRuby-conjugated PKCε. DCP-LA binding to PKCε was assayed using a fluorescein conjugated to DCP-LA at the carboxyl-terminal end (Fluo-DCP). Uptake of DCP-LA into cells was measured in PC-12 ells. Results: In the FRET analysis, DCP-LA decreased the ratio of YFP signal intensity/CFP signal intensity in PC-12 cells and in the cell-free kinase assay, DCP-LA increased area of phosphorylated PKC substrate peptide, indicating DCP-LA-induced PKCε activation. These effects were significantly suppressed by replacing Arg50 and Ile89 by Ala or Asn in the C2-like domain of PKCε. In the fluorescent cytochemistry, DCP-LA did not affect intracellular PKCε distribution. In the cell-free binding assay, Fluo-DCP, that had no effect on the potential for PKCε activation, bound to PKCε, and the binding was inhibited only by mutating Ile89. Extracellularly applied DCP-LA was taken up into cells in a concentration-dependent manner. Although no activation was obtained in the cell-free kinase assay, the broad PKC activator PMA activated PKCε in PC-12 cells in association with translocation towards the cell surface, which was inhibited by mutating I89A. Conclusion: Unlike PMA DCP-LA activates cytosolic PKCε by binding to the phosphatidylserine binding/associating sites Arg50 and Ile89, possibly at the carboxyl-terminal end and the cyclopropane rings, respectively.

Coupling of GABAB receptor GABAB2 subunit to G proteins: evidence from Xenopus oocyte and baby hamster kidney cell expression system

2006 ◽  
Vol 290 (1) ◽  
pp. C200-C207 ◽  
Author(s):  
Yasuhito Uezono ◽  
Masato Kanaide ◽  
Muneshige Kaibara ◽  
Rachel Barzilai ◽  
Nathan Dascal ◽  
...  
Keyword(s):  
G Proteins ◽  
Xenopus Oocytes ◽  
Fluorescent Protein ◽  
Gabab Receptor ◽  
Expression System ◽  
Yellow Fluorescent Protein ◽  
Dependent Manner ◽  
Baby Hamster Kidney ◽  
Concentration Dependent Manner ◽  
Bhk Cells

Coupling of functional GABAB receptors (GABABR) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing GABAB1a receptor (GB1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABAB2 receptor (GB2R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB1aR-Cerulean and GB2R-Venus form a heterodimer. The GABABR agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K+ currents in a concentration-dependent manner in oocytes expressing GB1aR and GB2R, or GB1aR-Cerulean and GB2R-Venus, together with G protein-activated inward-rectifying K+ channels (GIRKs), but not in oocytes expressing GB1aR alone or GB2R alone together with GIRKs. Oocytes coexpressing GB1aR + Gαi2-fused GB2R (GB2R-Gαi2) caused faster K+ currents in response to baclofen. Furthermore, oocytes coexpressing GB1aR + GB2R fused to Gαqi5 (a chimeric Gαq protein that activates PLC pathways) caused PLC-mediated Ca2+-activated Cl− currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB1aR-Gαi2 or GB1aR-Gαqi5 together with GB2R. BHK cells and Xenopus oocytes coexpressing GB1aR-Cerulean + a triplet tandem of GB2R-Venus-Gαqi5 caused FRET and Ca2+-activated Cl− currents, respectively, with a similar potency in BHK cells coexpressing GB1aR-Cerulean + GB2R-Venus and in oocytes coexpressing GB1aR + GB2R-Gαqi5. Our results indicate that functional GABABR forms a heterodimer composed of GB1R and GB2R and that the signal transducing G proteins are directly coupled to GB2R but not to GB1R.

scholarly journals Glucose-dependent blood flow dynamics in murine pancreatic islets in vivo

2010 ◽  
Vol 298 (4) ◽  
pp. E807-E814 ◽  
Author(s):  
Lara R. Nyman ◽  
Eric Ford ◽  
Alvin C. Powers ◽  
David W. Piston
Keyword(s):  
Blood Flow ◽  
Blood Glucose ◽  
Pancreatic Islets ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cell Types ◽  
Β Cells ◽  
Islet Blood Flow ◽  
Significant Difference

Pancreatic islets are highly vascularized and arranged so that regions containing β-cells are distinct from those containing other cell types. Although islet blood flow has been studied extensively, little is known about the dynamics of islet blood flow during hypoglycemia or hyperglycemia. To investigate changes in islet blood flow as a function of blood glucose level, we clamped blood glucose sequentially at hyperglycemic (∼300 mg/dl or 16.8 mM) and hypoglycemic (∼50 mg/dl or 2.8 mM) levels while simultaneously imaging intraislet blood flow in mouse models that express green fluorescent protein in the β-cells or yellow fluorescent protein in the α-cells. Using line scanning confocal microscopy, in vivo blood flow was assayed after intravenous injection of fluorescent dextran or sulforhodamine-labeled red blood cells. Regardless of the sequence of hypoglycemia and hyperglycemia, islet blood flow is faster during hyperglycemia, and apparent blood volume is greater during hyperglycemia than during hypoglycemia. However, there is no change in the order of perfusion of different islet endocrine cell types in hypoglycemia compared with hyperglycemia, with the islet core of β-cells usually perfused first. In contrast to the results in islets, there was no significant difference in flow rate in the exocrine pancreas during hyperglycemia compared with hypoglycemia. These results indicate that glucose differentially regulates blood flow in the pancreatic islet vasculature independently of blood flow in the rest of the pancreas.

scholarly journals Generation of a Quantitative Luciferase Reporter for Sox9 SUMOylation

2020 ◽  
Vol 21 (4) ◽  
pp. 1274
Author(s):  
Hideka Saotome ◽  
Atsumi Ito ◽  
Atsushi Kubo ◽  
Masafumi Inui
Keyword(s):  
Cell Types ◽  
Live Cells ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Post Translational Modifications ◽  
Extracellular Signal ◽  
Multiple Cell ◽  
Sox9 Activity ◽  
Reporter Signal

Sox9 is a master transcription factor for chondrogenesis, which is essential for chondrocyte proliferation, differentiation, and maintenance. Sox9 activity is regulated by multiple layers, including post-translational modifications, such as SUMOylation. A detection method for visualizing the SUMOylation in live cells is required to fully understand the role of Sox9 SUMOylation. In this study, we generated a quantitative reporter for Sox9 SUMOylation that is based on the NanoBiT system. The simultaneous expression of Sox9 and SUMO1 constructs that are conjugated with NanoBiT fragments in HEK293T cells induced luciferase activity in SUMOylation target residue of Sox9-dependent manner. Furthermore, the reporter signal could be detected from both cell lysates and live cells. The signal level of our reporter responded to the co-expression of SUMOylation or deSUMOylation enzymes by several fold, showing dynamic potency of the reporter. The reporter was active in multiple cell types, including ATDC5 cells, which have chondrogenic potential. Finally, using this reporter, we revealed a extracellular signal conditions that can increase the amount of SUMOylated Sox9. In summary, we generated a novel reporter that was capable of quantitatively visualizing the Sox9-SUMOylation level in live cells. This reporter will be useful for understanding the dynamism of Sox9 regulation during chondrogenesis.

scholarly journals A fate-mapping approach reveals the composite origin of the connecting tubule and alerts on “single-cell”-specific KO model of the distal nephron

2016 ◽  
Vol 311 (5) ◽  
pp. F901-F906 ◽  
Author(s):  
Francesco Trepiccione ◽  
Christelle Soukaseum ◽  
Anna Iervolino ◽  
Federica Petrillo ◽  
Miriam Zacchia ◽  
...  
Keyword(s):  
Single Cell ◽  
Kidney Development ◽  
Fluorescent Protein ◽  
Collecting Duct ◽  
Yellow Fluorescent Protein ◽  
Cell Types ◽  
Intercalated Cells ◽  
Distal Nephron ◽  
Principal Cells ◽  
Different Cell Types

The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl−cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.

A light-regulated bZIP module, photozipper, induces the binding of fused proteins to the target DNA sequence in a blue light-dependent manner

2015 ◽  
Vol 14 (11) ◽  
pp. 1998-2006 ◽  
Author(s):  
Osamu Hisatomi ◽  
Keigo Furuya
Keyword(s):  
Dna Sequence ◽  
Blue Light ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Dependent Manner ◽  
Target Dna

Yellow fluorescent protein or mCherry protein fused with the Photozipper underwent blue light-induced dimerization, which enhanced their affinities for the target DNA.

scholarly journals The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2336-2344 ◽  
Author(s):  
Masako Shimada ◽  
Matthew J. Mahon ◽  
Peter A. Greer ◽  
Gino V. Segre
Keyword(s):  
Parathyroid Hormone ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Subunit ◽  
Hek293 Cells ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Intact Cells ◽  
Calcium Dependent

Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

scholarly journals Inhibition of Orbivirus Replication by Aurintricarboxylic Acid

2020 ◽  
Vol 21 (19) ◽  
pp. 7294
Author(s):  
Celia Alonso ◽  
Sergio Utrilla-Trigo ◽  
Eva Calvo-Pinilla ◽  
Luis Jiménez-Cabello ◽  
Javier Ortego ◽  
...  
Keyword(s):  
Cell Types ◽  
Dependent Manner ◽  
Biting Midges ◽  
Lethal Challenge ◽  
Concentration Dependent Manner ◽  
Antiviral Compound ◽  
Aurintricarboxylic Acid ◽  
And Control

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging to the Orbivirus genus, which are transmitted between hosts primarily by biting midges of the genus Culicoides. With recent BTV and AHSV outbreaks causing epidemics and important economy losses, there is a pressing need for efficacious drugs to treat and control the spread of these infections. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antiviral activity. Here, we evaluated ATA as a potential antiviral compound against Orbivirus infections in both mammalian and insect cells. Notably, ATA was able to prevent the replication of BTV and AHSV in both cell types in a time- and concentration-dependent manner. In addition, we evaluated the effect of ATA in vivo using a mouse model of infection. ATA did not protect mice against a lethal challenge with BTV or AHSV, most probably due to the in vivo effect of ATA on immune system regulation. Overall, these results demonstrate that ATA has inhibitory activity against Orbivirus replication in vitro, but further in vivo analysis will be required before considering it as a potential therapy for future clinical evaluation.

In vivo associations of Escherichia coli NarJ with a peptide of the first 50 residues of nitrate reductase catalytic subunit NarG

2009 ◽  
Vol 55 (2) ◽  
pp. 179-188 ◽  
Author(s):  
Haiming Li ◽  
Raymond J. Turner
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Catalytic Subunit ◽  
Bimolecular Fluorescence Complementation ◽  
E Coli ◽  
N Terminus ◽  
Tat System

The catalytic subunit of many Escherichia coli redox enzymes bares a twin-arginine translocation (Tat)-dependent signal peptide in its precursor, which directs the redox enzyme complex to this Sec-independent pathway. NarG of the E. coli nitrate reductase NarGHI complex possesses a vestige twin-arginine motif at its N terminus. During the cofactor insertion, and assembly and folding of the NarG–NarH complex, a chaperone protein, NarJ, is thought to interact with the N terminus and an unknown second site of NarG. Our previous in vitro study provided evidence that NarJ’s role shows some Tat system dependence. In this work, we investigated the associations of NarJ with a peptide of the first 50 residues of NarG (NarG50) in living cells. Two approaches were used: the Förster resonance energy transfer (FRET) based on yellow fluorescent protein – cyan fluorescent protein (YFP–CFP) and the bimolecular fluorescence complementation (BiFC). Compared with the wild-type (WT) E. coli cotransformants expressing both NarJ–YFP and NarG50–CFP, tat gene mutants gave an apparent FRET efficiency (Eapp) that was on the order of 25%–40% lower. These experiments implied a Tat system dependency of the in vivo associations between NarJ and the NarG50 peptide. In the BiFC assay, a 4-fold lower specific fluorescence intensity was observed for the E. coli WT cotransformants expressing both NarJ–Yc and NarG50–Yn than for its tat mutants, again suggesting a Tat dependence of the interactions. Fluorescence microscopy showed a “dot”/unipolar distribution of the reassembled YFP–NarJ:NarG50 both in WT and tat mutants, demonstrating a distinct localization of the interaction. Thus, although the degree of the interaction shows Tat dependence, the cell localization is less so. Taken together, these data further support that NarJ’s activity on NarG may be assisted by the Tat system.

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