scholarly journals Functional analyses for site-specific phosphorylation of a target protein in cells

2007 ◽  
Author(s):  
Hidemasa Goto ◽  
Tohru Kiyono ◽  
Masaki Inagaki
Keyword(s):  
Target Protein ◽  
Site Specific ◽  
Functional Analyses ◽  
In Cells

O‐GlcNAc Engineering on a Target Protein in Cells with Nanobody‐OGT and Nanobody‐splitOGA

2021 ◽  
Vol 1 (5) ◽  
Author(s):  
Daniel H. Ramirez ◽  
Yun Ge ◽  
Christina M. Woo
Keyword(s):  
Target Protein ◽  
In Cells

scholarly journals Quantitative Chemoproteomics for Site-Specific Analysis of Protein Alkylation by 4-Hydroxy-2-Nonenal in Cells

2015 ◽  
Vol 87 (5) ◽  
pp. 2535-2541 ◽  
Author(s):  
Jing Yang ◽  
Keri A. Tallman ◽  
Ned A. Porter ◽  
Daniel C. Liebler
Keyword(s):  
Site Specific ◽  
Specific Analysis ◽  
In Cells

scholarly journals Activity-based ubiquitin-protein probes reveal target protein specificity of deubiquitinating enzymes

2018 ◽  
Vol 9 (40) ◽  
pp. 7859-7865 ◽  
Author(s):  
Ping Gong ◽  
Gregory A. Davidson ◽  
Weijun Gui ◽  
Kun Yang ◽  
William P. Bozza ◽  
...  
Keyword(s):  
Target Protein ◽  
Deubiquitinating Enzymes ◽  
Site Specific ◽  
Protein Specificity

Activity-based Ub-PCNA probes identify deubiquitinating enzymes (DUBs) specific for PCNA and demonstrate site-specific deubiquitination by DUBs.

scholarly journals Streptavidin interfacing as a general strategy to localize fluorescent membrane tension probes in cells

2019 ◽  
Vol 10 (1) ◽  
pp. 310-319 ◽  
Author(s):  
Antoine Goujon ◽  
Karolína Straková ◽  
Naomi Sakai ◽  
Stefan Matile
Keyword(s):  
General Strategy ◽  
Membrane Tension ◽  
Site Specific ◽  
In Cells

Site-specific labeling with biotinylated mechanophores is probed to address the next challenge toward the imaging of forces in cells.

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.

scholarly journals Site-Specific N- and O-Glycosylation Analysis of Human Plasma Fibronectin

2021 ◽  
Vol 9 ◽  
Author(s):  
Ding Liu ◽  
Shuaishuai Wang ◽  
Junping Zhang ◽  
Weidong Xiao ◽  
Carol H. Miao ◽  
...  
Keyword(s):  
Human Plasma ◽  
Collision Energy ◽  
T Antigen ◽  
Plasma Fibronectin ◽  
Site Specific ◽  
Adhesive Protein ◽  
Human Fibronectin ◽  
A Site ◽  
Functional Analyses ◽  
Labeling Method

Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the 18O-labeling method. O-glycopeptide enrichment and O-glycosite identification were achieved by an enzyme-assisted site-specific extraction method. An RP–LC–MS/MS system functionalized with collision-induced dissociation and stepped normalized collision energy (sNCE)-HCD tandem mass was applied to analyze the glycoforms of fibronectin. A total of 6 N-glycosites and 53 O-glycosites were identified, which were occupied by 38 N-glycoforms and 16 O-glycoforms, respectively. Furthermore, 77.31% of N-glycans were sialylated, and O-glycosylation was dominated by the sialyl-T antigen. These site-specific glycosylation patterns on human fibronectin can facilitate functional analyses of fibronectin and therapeutics development.

scholarly journals Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates myosin-II heavy chain

2012 ◽  
Vol 199 (4) ◽  
pp. 669-683 ◽  
Author(s):  
Matthew Raab ◽  
Joe Swift ◽  
P.C. Dave P. Dingal ◽  
Palak Shah ◽  
Jae-Won Shin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myosin Ii ◽  
Stress Fibers ◽  
Site Specific ◽  
Soft Matrix ◽  
3D Collagen ◽  
The Difference ◽  
Rigid Surfaces ◽  
Tissue Matrix ◽  
In Cells

On rigid surfaces, the cytoskeleton of migrating cells is polarized, but tissue matrix is normally soft. We show that nonmuscle MIIB (myosin-IIB) is unpolarized in cells on soft matrix in 2D and also within soft 3D collagen, with rearward polarization of MIIB emerging only as cells migrate from soft to stiff matrix. Durotaxis is the tendency of cells to crawl from soft to stiff matrix, and durotaxis of primary mesenchymal stem cells (MSCs) proved more sensitive to MIIB than to the more abundant and persistently unpolarized nonmuscle MIIA (myosin-IIA). However, MIIA has a key upstream role: in cells on soft matrix, MIIA appeared diffuse and mobile, whereas on stiff matrix, MIIA was strongly assembled in oriented stress fibers that MIIB then polarized. The difference was caused in part by elevated phospho-S1943–MIIA in MSCs on soft matrix, with site-specific mutants revealing the importance of phosphomoderated assembly of MIIA. Polarization is thus shown to be a highly regulated compass for mechanosensitive migration.

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