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.

Drug repurposing using similarity-based target prediction, docking studies and scaffold hopping of lefamulin

2020 ◽  
Vol 17 ◽  
Author(s):  
Shikha Sharma ◽  
Shweta Sharma ◽  
Vaishali Pathak ◽  
Parwinder Kaur ◽  
Rajesh Kumar Singh
Keyword(s):  
In Silico ◽  
Target Prediction ◽  
Drug Repurposing ◽  
Docking Studies ◽  
Target Protein ◽  
Future Perspective ◽  
Antibacterial Drug ◽  
Scaffold Hopping ◽  
Target Proteins ◽  
Renin Inhibitors

Aim: To investigate and validate the potential target proteins for drug repurposing of newly FDA approved antibacterial drug. Background: Drug repurposing is the process of assigning indications for drugs other than the one(s) that they were initially developed for. Discovery of entirely new indications from already approved drugs is highly lucrative as it minimizes the pipeline of the drug development process by reducing time and cost. In silico driven technologies made it possible to analyze molecules for different target proteins which are not yet explored. Objective: To analyze possible targets proteins for drug repurposing of lefamulin and their validation. Also, in silico prediction of novel scaffolds from lefamulin has been performed for assisting medicinal chemists in future drug design. Methods: A similarity-based prediction tool was employed for predicting target protein and further investigated using docking studies on PDB ID: 2V16. Besides, various in silico tools were employed for prediction of novel scaffolds from lefamulin using scaffold hopping technique followed by evaluation with various in silico parameters viz., ADME, synthetic accessibility and PAINS. Results: Based on the similarity and target prediction studies, renin is found as the most probable target protein for lefamulin. Further, validation studies using docking of lefamulin revealed the significant interactions of lefamulin with the binding pocket of the target protein. Also, three novel scaffolds were predicted using scaffold hopping technique and found to be in the limit to reduce the chances of drug failure in the physiological system during the last stage approval process. Conclusion: To encapsulate the future perspective, lefamulin may assist in the development of the renin inhibitors and, also three possible novel scaffolds with good pharmacokinetic profile can be developed into both as renin inhibitors and for bacterial infections.

scholarly journals Protein Sensing Device with Multi-Recognition Ability Composed of Self-Organized Glycopeptide Bundle

2020 ◽  
Vol 22 (1) ◽  
pp. 366
Author(s):  
Mao Arai ◽  
Tomohiro Miura ◽  
Yuriko Ito ◽  
Takatoshi Kinoshita ◽  
Masahiro Higuchi
Keyword(s):  
Binding Site ◽  
Lipid Bilayer ◽  
Structural Changes ◽  
Bilayer Membrane ◽  
Lipid Bilayer Membrane ◽  
Target Protein ◽  
Self Organization ◽  
Specific Response ◽  
Target Proteins ◽  
Recognition Ability

We designed and synthesized amphiphilic glycopeptides with glucose or galactose at the C-terminals. We observed the protein-induced structural changes of the amphiphilic glycopeptide assembly in the lipid bilayer membrane using transmission electron microscopy (TEM) and Fourier transform infrared reflection-absorption spectra (FTIR-RAS) measurements. The glycopeptides re-arranged to form a bundle that acted as an ion channel due to the interaction among the target protein and the terminal sugar groups of the glycopeptides. The bundle in the lipid bilayer membrane was fixed on a gold-deposited quartz crystal microbalance (QCM) electrode by the membrane fusion method. The protein-induced re-arrangement of the terminal sugar groups formed a binding site that acted as a receptor, and the re-binding of the target protein to the binding site induced the closing of the channel. We monitored the detection of target proteins by the changes of the electrochemical properties of the membrane. The response current of the membrane induced by the target protein recognition was expressed by an equivalent circuit consisting of resistors and capacitors when a triangular voltage was applied. We used peanut lectin (PNA) and concanavalin A (ConA) as target proteins. The sensing membrane induced by PNA shows the specific response to PNA, and the ConA-induced membrane responded selectively to ConA. Furthermore, PNA-induced sensing membranes showed relatively low recognition ability for lectin from Ricinus Agglutinin (RCA120) and mushroom lectin (ABA), which have galactose binding sites. The protein-induced self-organization formed the spatial arrangement of the sugar chains specific to the binding site of the target protein. These findings demonstrate the possibility of fabricating a sensing device with multi-recognition ability that can recognize proteins even if the structure is unknown, by the protein-induced self-organization process.

A novel mesoionic carbene based highly fluorescent Pd(ii) complex as an endoplasmic reticulum tracker in live cells

2018 ◽  
Vol 47 (44) ◽  
pp. 15646-15650 ◽  
Author(s):  
Sanjay K. Verma ◽  
Pratibha Kumari ◽  
Shagufi Naz Ansari ◽  
Mohd Ovais Ansari ◽  
Dondinath Deori ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescence Recovery ◽  
Live Cells

Synthesis of new organometallic MIC based mononuclear Pd(ii) complex 1, specifically target ER of live cells and have fluorescence recovery after photobleaching (FRAP) property.

scholarly journals Conserved Mode of Interaction between Yeast Bro1 Family V Domains and YP(X) n L Motif-Containing Target Proteins

2015 ◽  
Vol 14 (10) ◽  
pp. 976-982 ◽  
Author(s):  
Yoko Kimura ◽  
Mirai Tanigawa ◽  
Junko Kawawaki ◽  
Kenji Takagi ◽  
Tsunehiro Mizushima ◽  
...  
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Mammalian Cells ◽  
Tyrosine Phosphatase ◽  
Binding Specificity ◽  
Target Protein ◽  
Target Proteins ◽  
Protein Tyrosine ◽  
Hydrophobic Groove ◽  
Domain Protein ◽  
Common Architecture

ABSTRACT Yeast Bro1 and Rim20 belong to a family of proteins which possess a common architecture of Bro1 and V domains. Alix and His domain protein tyrosine phosphatase (HD-PTP), mammalian Bro1 family proteins, bind YP(X) n L ( n = 1 to 3) motifs in their target proteins through their V domains. In Alix, the Phe residue, which is located in the hydrophobic groove of the V domain, is critical for binding to the YP(X) n L motif. Although the overall sequences are not highly conserved between mammalian and yeast V domains, we show that the conserved Phe residue in the yeast Bro1 V domain is important for binding to its YP(X) n L-containing target protein, Rfu1. Furthermore, we show that Rim20 binds to its target protein Rim101 through the interaction between the V domain of Rim20 and the YPIKL motif of Rim101. The mutation of either the critical Phe residue in the Rim20 V domain or the YPIKL motif of Rim101 affected the Rim20-mediated processing of Rim101. These results suggest that the interactions between V domains and YP(X) n L motif-containing proteins are conserved from yeast to mammalian cells. Moreover, the specificities of each V domain to their target protein suggest that unidentified elements determine the binding specificity.

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

scholarly journals A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking

2017 ◽  
Vol 30 (12) ◽  
pp. 771-780 ◽  
Author(s):  
M Hinrichsen ◽  
M Lenz ◽  
J M Edwards ◽  
O K Miller ◽  
S G J Mochrie ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Single Cells ◽  
Covalent Bond ◽  
Subcellular Location ◽  
Turnover Time ◽  
Target Protein ◽  
Live Cells ◽  
General Applicability ◽  
Novel Approach ◽  
And Function

AbstractWe present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein’s turnover time from such data.

scholarly journals Selective protein O-GlcNAcylation in cells by a proximity-directed O-GlcNAc transferase

2019 ◽  
Author(s):  
Daniel H. Ramirez ◽  
Chanat Aonbangkhen ◽  
Hung-Yi Wu ◽  
Jeffrey A. Naftaly ◽  
Stephanie Tang ◽  
...  
Keyword(s):  
Target Protein ◽  
Protein Signaling ◽  
Substrate Selection ◽  
Loss Of Function ◽  
Chemical Proteomics ◽  
Post Translational Modifications ◽  
Target Proteins ◽  
Glcnac Transferase ◽  
Selective Increase ◽  
In Cells

AbstractO-Linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Yet, the study of post-translational modifications like O-GlcNAc has been limited by the lack of strategies to induce O-GlcNAcylation on a target protein in cells. Here, we report a generalizable genetic strategy to induce O-GlcNAc to specific target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT(4), a truncated form of OGT, yielded a nanobody-OGT(4) construct that selectively delivered O-GlcNAc to the target protein (e.g., JunB, cJun, Nup62) and reduced alteration of global O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the selective increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for glycosylation in HEK293T cells. Thus, the use of nanobodies to redirect OGT substrate selection is a versatile strategy to induce glycosylation of desired target proteins in cells that will facilitate discovery of O-GlcNAc functions and provide a mechanism to engineer O-GlcNAc signaling. The proximity-directed OGT approach for protein-selective O-GlcNAcylation is readily translated to additional protein targets and nanobodies that may constitute a generalizable strategy to control post-translational modifications in cells.Significance StatementNature uses post-translational modifications (PTMs) like glycosylation as a mechanism to alter protein signaling and function. However, the study of these modified proteins in cells is confined to loss-of-function strategies, such as mutagenic elimination of the modification site. Here, we report a generalizable strategy for induction of O-GlcNAc to a protein target in cells. The O-GlcNAc modification is installed by O-GlcNAc transferase (OGT) to thousands of nucleocytoplasmic proteins. Fusion of a nanobody to OGT enables the selective increase of O-GlcNAc levels on a series of target proteins. The described approach will facilitate direct studies of O-GlcNAc and its regulatory enzymes and drive new approaches to engineer protein signaling via a strategy that may be conceptually translatable to additional PTMs.

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.

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.

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