Photo-triggered fluorescent labelling of recombinant proteins in live cells

2015 ◽  
Vol 51 (47) ◽  
pp. 9670-9673 ◽  
Author(s):  
Deokho Jung ◽  
Kohei Sato ◽  
Kyoungmi Min ◽  
Akira Shigenaga ◽  
Juyeon Jung ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Live Cells ◽  
Fluorescent Labelling ◽  
Target Proteins ◽  
Spatiotemporal Control

A method to photo-chemically trigger fluorescent labelling of proteins in live cells is developed for background-free fluorescent labelling of target proteins with the necessary spatiotemporal control.

A cascading reaction sequence involving ligand-directed azaelectrocyclization and autooxidation-induced fluorescence recovery enables visualization of target proteins on the surfaces of live cells

2014 ◽  
Vol 12 (9) ◽  
pp. 1412-1418 ◽  
Author(s):  
Katsunori Tanaka ◽  
Masataka Kitadani ◽  
Ayumi Tsutsui ◽  
Ambara R. Pradipta ◽  
Rie Imamaki ◽  
...  
Keyword(s):  
Target Protein ◽  
Fluorescence Recovery ◽  
Live Cells ◽  
Reaction Sequence ◽  
Target Proteins

A general probe designed to induce a cascading sequence of reactions on a target protein was efficiently synthesized.

Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells

2016 ◽  
Vol 34 (1) ◽  
pp. 161-174 ◽  
Author(s):  
Ruth Röder ◽  
Jonas Helma ◽  
Tobias Preiß ◽  
Joachim O. Rädler ◽  
Heinrich Leonhardt ◽  
...  
Keyword(s):  
Intracellular Delivery ◽  
Live Cells ◽  
Target Proteins

Protein degradation through covalent inhibitor-based PROTACs

2020 ◽  
Vol 56 (10) ◽  
pp. 1521-1524 ◽  
Author(s):  
Gang Xue ◽  
Jiahui Chen ◽  
Lihong Liu ◽  
Danli Zhou ◽  
Yingying Zuo ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Live Cells ◽  
Target Proteins ◽  
Covalent Inhibitor ◽  
Application Scope

Covalent inhibitor-based PROTACs were successfully developed for the degradation of target proteins in live cells to further extend the application scope of PROTACs.

scholarly journals Regulatory Elements in Vectors for Efficient Generation of Cell Lines Producing Target Proteins

Acta Naturae ◽  
2015 ◽  
Vol 7 (3) ◽  
pp. 15-26 ◽  
Author(s):  
O. G. Maksimenko ◽  
N. B. Gasanov ◽  
P. G. Georgiev
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Cell Cultures ◽  
Recombinant Proteins ◽  
Regulatory Elements ◽  
Mechanisms Of Action ◽  
Expression Level ◽  
Target Proteins ◽  
Efficient Generation ◽  
Mammalian Cell Cultures

To date, there has been an increasing number of drugs produced in mammalian cell cultures. In order to enhance the expression level and stability of target recombinant proteins in cell cultures, various regulatory elements with poorly studied mechanisms of action are used. In this review, we summarize and discuss the potential mechanisms of action of such regulatory elements.

scholarly journals Development of an Acrylamide-Based Inhibitor of Protein S-Acylation

2021 ◽  
Author(s):  
Saara-Anne Azizi ◽  
Tong Lan ◽  
Clémence Delalande ◽  
Rahul Kathayat ◽  
Bryan Dickinson
Keyword(s):  
Cell Signaling ◽  
Human Disease ◽  
Cellular Signaling ◽  
Protein S ◽  
Live Cells ◽  
Synthesis And Characterization ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Cellular Events

<div><div><div><p>Protein S-acylation is a dynamic lipid post-translational modification that can modulate the localization and activity of target proteins. In humans, the installation of the lipid onto target proteins is catalyzed by a family of 23 Asp-His-His-Cys domain-containing protein acyltransferases (DHHC-PATs). DHHCs are increasingly recognized as critical players in cellular signaling events and in human disease. However, progress elucidating the functions and mechanisms of DHHC “writers” has been hampered by a lack of chemical tools to perturb their activity in live cells. Herein, we report the synthesis and characterization of PATi, a pan- DHHC inhibitor more potent than 2-bromopalmitate (2BP), the most commonly used DHHC inhibitor in the field. Possessing an acrylamide warhead, PATi pairs its gain in potency with decreases in both toxicity and inhibition of the S-acylation eraser enzymes – two of the major weaknesses of 2BP. Our studies show that PATi engages with DHHC family proteins in cells, inhibits protein S-acylation, and disrupts DHHC-regulated cellular events. PATi represents an improved chemical tool for untangling the complexities of DHHC-mediated cell signaling by protein S-acylation.</p></div></div></div>

scholarly journals Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

2020 ◽  
Author(s):  
Bo Meng ◽  
Pedro P. Vallejo Ramirez ◽  
Katharina M. Scherer ◽  
Ezra Bruggeman ◽  
Julia C. Kenyon ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Biological Process ◽  
Full Length ◽  
Live Cells ◽  
Fluorescent Labelling ◽  
Endosomal Membrane ◽  
Cellular Processes ◽  
N Terminus ◽  
Main Component

The scission of lipid membranes is a common biological process, often mediated by ESCRT complexes in concert with VPS4 assembling around the separation point. The functions of the ESCRT-I and ESCRT-III complexes are well established in certain of these cellular processes; however, the role of ESCRT-II remains contentious. Here, we devised a SNAP-tag fluorescent labelling strategy to understand the domain requirements of EAP45, the main component of ESCRT-II, in HIV egress, late endosome recruitment, and cytokinesis. We used TIRF microscopy to measure the spatial co-occurrence of the HIV structural polyprotein Gag with full length EAP45 in both fixed and live cells. Gag colocalises with the full length EAP45 comparably to ALIX, but this is lost on deletion of the EAP45 N terminus. Our findings reveal the H0 domain of the EAP45 protein is essential for linking to ESCRT-I during HIV budding and in anchoring at the late endosomal membrane, however in cytokinesis it is the Glue domain that is critical.

scholarly journals A new bioluminescence-based tool for modulating target proteins in live cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tetsuya Ishimoto ◽  
Hisashi Mori
Keyword(s):  
Reactive Oxygen Species ◽  
Structural Change ◽  
Fusion Protein ◽  
Cultured Cells ◽  
Firefly Luciferase ◽  
Live Cells ◽  
Oxygen Species ◽  
Characteristic Structure ◽  
Target Proteins ◽  
Low Levels

AbstractWe have developed a new genetically encoded tool designed to generate reactive oxygen species (ROS) at target proteins in cultured cells; it is designed using firefly luciferase and photosensitiser protein KillerRed. Targeting this fusion protein, KillerFirefly, to F-actin in live cells and treatment with luciferin induced a characteristic structure, previously reported as a cofilin-actin rod, which is seen in patients with Alzheimer’s disease. This structural change is considered to be elicited by the consistent generation of very low-level ROS by KillerFirefly in the vicinity of F-actin. Moreover, our results suggest the presence of an actin-regulating system, controlled by very low levels of endogenously generated ROS.

scholarly journals Catalytic Activation of Bioorthogonal Chemistry with Light (CABL) Enables Rapid, Spatiotemporally-controlled Labeling and No-Wash, Subcellular 3D-Patterning in Live Cells using Long Wavelength Light

2021 ◽  
Author(s):  
Andrew Jemas ◽  
Yixin Xie ◽  
Jessica Pigga ◽  
Jeffrey Caplan ◽  
Christopher am Ende ◽  
...  
Keyword(s):  
Diels Alder ◽  
Cell Labeling ◽  
Live Cells ◽  
Bioorthogonal Chemistry ◽  
Long Wavelength ◽  
Catalytic Activation ◽  
Spatially Resolved ◽  
Photon Excitation ◽  
Live Cell Labeling ◽  
Spatiotemporal Control

Described is the spatiotemporally controlled labeling and patterning of biomolecules in live cells through the catalytic activation of bioorthogonal chemistry with light, referred to as “CABL”. Here, an unreactive dihydrotetrazine (DHTz) is photocatalytically oxidized in the intracellular environment by ambient O2 to produce a tetrazine that immediately reacts with a trans-cyclooctene (TCO) dieno-phile. 6-(2-Pyridyl)-dihydrotetrazine-3-carboxamides were developed as stable, cell permeable DHTz reagents that upon oxidation pro-duce the most reactive tetrazines ever used in live cells with Diels-Alder kinetics exceeding k2 106 M-1s-1. CABL photocatalysts are based on fluorescein or silarhodamine dyes with activation at 470 or 660 nm. Strategies for limiting extracellular production of singlet oxygen are described that increase the cytocompatibility of photocatalysis. The HaloTag self-labeling platform was used to introduce DHTz tags to proteins localized in the nucleus, mitochondria, actin or cytoplasm, and high-yielding subcellular activation and labeling with a TCO-fluorophore was demonstrated. CABL is light-dose dependent, and 2-photon excitation promotes CABL at the sub-organelle level to selectively pattern live cells under no-wash conditions. CABL was also applied to spatially resolved live-cell labeling of an endogenous pro-tein target by using TIRF microscopy to selectively activate intracellular monoacylglycerol lipase tagged with DHTz-labeled small mole-cule covalent inhibitor. Beyond spatiotemporally controlled labeling, CABL also improves the efficiency of ‘ordinary’ tetrazine ligations by rescuing the reactivity of commonly used 3-aryl-6-methyltetrazine reporters that become partially reduced to DHTzs inside cells. The spatiotemporal control and fast rates of photoactivation and labeling of CABL should enable a range of biomolecular labeling applications in living systems.

scholarly journals Ligand-directed covalent labelling of a GPCR with a fluorescent tag in live cells

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Leigh A. Stoddart ◽  
Nicholas D. Kindon ◽  
Omolade Otun ◽  
Clare R. Harwood ◽  
Foteini Patera ◽  
...  
Keyword(s):  
Rational Design ◽  
Live Cells ◽  
Fluorescent Labelling ◽  
Non Invasive ◽  
Cellular Environment ◽  
Selective Approach ◽  
Fluorescent Ligands ◽  
Fluorescent Moiety ◽  
Fluorescent Tag ◽  
G Protein Coupled

AbstractTo study the localisation of G protein-coupled receptors (GPCR) in their native cellular environment requires their visualisation through fluorescent labelling. To overcome the requirement for genetic modification of the receptor or the limitations of dissociable fluorescent ligands, here we describe rational design of a compound that covalently and selectively labels a GPCR in living cells with a fluorescent moiety. We designed a fluorescent antagonist, in which the linker incorporated between pharmacophore (ZM241385) and fluorophore (sulfo-cyanine5) is able to facilitate covalent linking of the fluorophore to the adenosine A2A receptor. We pharmacologically and biochemically demonstrate irreversible fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.

Imaging of Conformational Changes of Proteins with a New Environment-Sensitive Fluorescent Probe Designed for Site-Specific Labeling of Recombinant Proteins in Live Cells

2001 ◽  
Vol 73 (13) ◽  
pp. 2920-2928 ◽  
Author(s):  
Jun Nakanishi ◽  
Takahiro Nakajima ◽  
Moritoshi Sato ◽  
Takeaki Ozawa ◽  
Kohji Tohda ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Recombinant Proteins ◽  
Conformational Changes ◽  
Live Cells ◽  
Site Specific
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