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 Endocytic Down-Regulation of ErbB2 Is Stimulated by Cleavage of Its C-Terminus

2007 ◽  
Vol 18 (9) ◽  
pp. 3656-3666 ◽  
Author(s):  
Mads Lerdrup ◽  
Silas Bruun ◽  
Michael V. Grandal ◽  
Kirstine Roepstorff ◽  
Malene M. Kristensen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Retention Mechanism ◽  
Full Length ◽  
Dependent Manner ◽  
Lysosomal Degradation ◽  
Down Regulation ◽  
C Terminus ◽  
Early Endosomes

High ErbB2 levels are associated with cancer, and impaired endocytosis of ErbB2 could contribute to its overexpression. Therefore, knowledge about the mechanisms underlying endocytic down-regulation of ErbB2 is warranted. The C-terminus of ErbB2 can be cleaved after various stimuli, and after inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the N- and C-terminus of ErbB2, respectively (YFP-ErbB2-CFP). After geldanamycin stimulation YFP-ErbB2-CFP became cleaved in nonapoptotic cells in a proteasome-dependent manner, and a markedly larger relative amount of cleaved YFP-ErbB2-CFP was observed in early endosomes than in the plasma membrane. Furthermore, cleavage took place at the plasma membrane, and cleaved ErbB2 was internalized and degraded far more efficiently than full-length ErbB2. Concordantly, a C-terminally truncated ErbB2 was also readily endocytosed and degraded in lysosomes compared with full-length ErbB2. Altogether, we suggest that geldanamycin leads to C-terminal cleavage of ErbB2, which releases the receptor from a retention mechanism and causes endocytosis and lysosomal degradation of ErbB2.

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 The Agrobacterium rhizogenes GALLS Gene Encodes Two Secreted Proteins Required for Genetic Transformation of Plants

2008 ◽  
Vol 191 (1) ◽  
pp. 355-364 ◽  
Author(s):  
Larry D. Hodges ◽  
Lan-Ying Lee ◽  
Henry McNett ◽  
Stanton B. Gelvin ◽  
Walt Ream
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Plant Cells ◽  
Tumor Formation ◽  
Plant Genome ◽  
Start Codon ◽  
Dependent Manner ◽  
Single Stranded Dna ◽  
Nuclear Localization Signals

ABSTRACT Agrobacterium tumefaciens and Agrobacterium rhizogenes are related pathogens that cause crown gall and hairy root diseases, which result from integration and expression of bacterial genes in the plant genome. Single-stranded DNA (T strands) and virulence proteins are translocated into plant cells by a type IV secretion system. VirD2 nicks a specific DNA sequence, attaches to the 5′ end, and pilots the DNA into plant cells. A. tumefaciens translocates single-stranded DNA-binding protein VirE2 into plant cells where it likely binds T strands and may aid in targeting them into the nucleus. Although some A. rhizogenes strains lack VirE2, they transfer T strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant for tumor formation. Unlike VirE2, full-length GALLS (GALLS-FL) contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. GALLS-FL and VirE2 contain nuclear localization signals (NLS) and secretion signals. Mutations in any of these domains abolish the ability of the GALLS gene to substitute for virE2. Here, we show that the GALLS gene encodes two proteins from one open reading frame: GALLS-FL and a protein comprised of the C-terminal domain, which initiates at an internal in-frame start codon. On some hosts, both GALLS proteins were required to substitute for VirE2. GALLS-FL tagged with yellow fluorescent protein localized to the nucleus of tobacco cells in an NLS-dependent manner. In plant cells, the GALLS proteins interacted with themselves, VirD2, and each other. VirD2 interacted with GALLS-FL and localized inside the nucleus, where its predicted helicase activity may pull T strands into the nucleus.

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 Spectroscopic and Structural Analysis of Cu2+-Induced Fluorescence Quenching of ZsYellow

Biosensors ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 29 ◽  
Author(s):  
In Jung Kim ◽  
Yongbin Xu ◽  
Ki Hyun Nam
Keyword(s):  
Structural Analysis ◽  
Fluorescence Quenching ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Fluorescence Emission ◽  
Spectroscopic Properties ◽  
Dependent Manner ◽  
Quenching Analysis ◽  
High Degree

Fluorescent proteins exhibit fluorescence quenching by specific transition metals, suggesting their potential as fluorescent protein-based metal biosensors. Each fluorescent protein exhibits unique spectroscopic properties and mechanisms for fluorescence quenching by metals. Therefore, the metal-induced fluorescence quenching analysis of various new fluorescent proteins would be important step towards the development of such fluorescent protein-based metal biosensors. Here, we first report the spectroscopic and structural analysis of the yellow fluorescent protein ZsYellow, following its metal-induced quenching. Spectroscopic analysis showed that ZsYellow exhibited a high degree of fluorescence quenching by Cu2+. During Cu2+-induced ZsYellow quenching, fluorescence emission was recovered by adding EDTA. The crystal structure of ZsYellow soaked in Cu2+ solution was determined at a 2.6 Å resolution. The electron density map did not indicate the presence of Cu2+ around the chromophore or the β-barrel surface, which resulted in fluorescence quenching without Cu2+ binding to specific site in ZsYellow. Based on these results, we propose the fluorescence quenching to occur in a distance-dependent manner between the metal and the fluorescent protein, when these components get to a closer vicinity at higher metal concentrations. Our results provide useful insights for future development of fluorescent protein-based metal biosensors.

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