Multiplexed Imaging of Posttranslational Modifications of Endogenous Proteins in Live Cells

2021 ◽  
pp. 31-41
Author(s):  
Yuko Sato ◽  
Hiroshi Kimura
Keyword(s):  
Posttranslational Modifications ◽  
Live Cells ◽  
Multiplexed Imaging ◽  
Endogenous Proteins

scholarly journals Deciphering and engineering chromodomain-methyllysine peptide recognition

2018 ◽  
Vol 4 (11) ◽  
pp. eaau1447 ◽  
Author(s):  
Ryan Hard ◽  
Nan Li ◽  
Wei He ◽  
Brian Ross ◽  
Gary C. H. Mo ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Large Scale ◽  
Regulation Of Gene Expression ◽  
Live Cells ◽  
Lysine Methylation ◽  
Dynamic Regulation ◽  
Peptide Recognition ◽  
Protein Functions ◽  
Domain Peptide

Posttranslational modifications (PTMs) play critical roles in regulating protein functions and mediating protein-protein interactions. An important PTM is lysine methylation that orchestrates chromatin modifications and regulates functions of non-histone proteins. Methyllysine peptides are bound by modular domains, of which chromodomains are representative. Here, we conducted the first large-scale study of chromodomains in the human proteome interacting with both histone and non-histone methyllysine peptides. We observed significant degenerate binding between chromodomains and histone peptides, i.e., different histone sites can be recognized by the same set of chromodomains, and different chromodomains can share similar binding profiles to individual histone sites. Such degenerate binding is not dictated by amino acid sequence or PTM motif but rather rooted in the physiochemical properties defined by the PTMs on the histone peptides. This molecular mechanism is confirmed by the accurate prediction of the binding specificity using a computational model that captures the structural and energetic patterns of the domain-peptide interaction. To further illustrate the power and accuracy of our model, we used it to effectively engineer an exceptionally strong H3K9me3-binding chromodomain and to label H3K9me3 in live cells. This study presents a systematic approach to deciphering domain-peptide recognition and reveals a general principle by which histone modifications are interpreted by reader proteins, leading to dynamic regulation of gene expression and other biological processes.

scholarly journals Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

2014 ◽  
Vol 25 (22) ◽  
pp. 3610-3618 ◽  
Author(s):  
Robert Mahen ◽  
Birgit Koch ◽  
Malte Wachsmuth ◽  
Antonio Z. Politi ◽  
Alexis Perez-Gonzalez ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Quantitative Imaging ◽  
Live Cells ◽  
Single Molecule Spectroscopy ◽  
Mitotic Kinase ◽  
Endogenous Proteins

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

Recognition-driven chemical labeling of endogenous proteins in multi-molecular crowding in live cells

2017 ◽  
Vol 53 (88) ◽  
pp. 11972-11983 ◽  
Author(s):  
Kazuma Amaike ◽  
Tomonori Tamura ◽  
Itaru Hamachi
Keyword(s):  
Protein Labeling ◽  
Live Cells ◽  
Molecular Crowding ◽  
Chemical Labeling ◽  
Endogenous Protein ◽  
Bona Fide ◽  
Endogenous Proteins

Endogenous protein labeling is one of the most invaluable methods for studying the bona fide functions of proteins in live cells.

scholarly journals Profiling novel pharmacology of receptor complexes using Receptor-HIT

2021 ◽  
Vol 49 (4) ◽  
pp. 1555-1565
Author(s):  
Elizabeth K.M. Johnstone ◽  
Kevin D.G. Pfleger
Keyword(s):  
Tyrosine Kinases ◽  
Drug Targets ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Reporter System ◽  
Receptor Complexes ◽  
Receptor Heteromers ◽  
Endogenous Proteins ◽  
The Individual

Many receptors are able to undergo heteromerisation, leading to the formation of receptor complexes that may have pharmacological profiles distinct from those of the individual receptors. As a consequence of this, receptor heteromers can be classed as new drug targets, with the potential for achieving greater specificity and selectivity over targeting their constituent receptors. We have developed the Receptor-Heteromer Investigation Technology (Receptor-HIT), which enables the detection of receptor heteromers using a proximity-based reporter system such as bioluminescence resonance energy transfer (BRET). Receptor-HIT detects heteromers in live cells and in real time, by utilising ligand-induced signals that arise from altered interactions with specific biomolecules, such as ligands or proteins. Furthermore, monitoring the interaction between the receptors and the specific biomolecules generates functional information about the heteromer that can be pharmacologically quantified. This review will discuss various applications of Receptor-HIT, including its use with different classes of receptors (e.g. G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and others), its use to monitor receptor interactions both intracellularly and extracellularly, and also its use with genome-edited endogenous proteins.

Activity-Dependent Probes Containing Epsilon-N-Thioacyllysine and -Epsilon-N-Acyl-(Delta-Aza)lysine Residues

2019 ◽  
Author(s):  
Michael Bæk, ◽  
Pablo Martín-Gago ◽  
Jonas S. Laursen ◽  
Julie L. H. Madsen ◽  
Saswati Chakladar ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Protein Function ◽  
Splice Variants ◽  
Peptide Sequence ◽  
Fluorescence Measurements ◽  
Lysine Residues ◽  
Covalent Adducts ◽  
Endogenous Proteins ◽  
Activity Dependent ◽  
Hydrolysis Of

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. Here, we describe development of peptide activity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of epsilon-<i>N</i>-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the epsilon-<i>N</i>-glutaryllysine and epsilon<i>-N</i>-myristoyllysine analogues, were successfully applied to enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.

Activity-Dependent Probes Containing Epsilon-N-Thioacyllysine and -Epsilon-N-Acyl-(Delta-Aza)lysine Residues

2019 ◽  
Author(s):  
Michael Bæk, ◽  
Pablo Martín-Gago ◽  
Jonas S. Laursen ◽  
Julie L. H. Madsen ◽  
Saswati Chakladar ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Protein Function ◽  
Splice Variants ◽  
Peptide Sequence ◽  
Fluorescence Measurements ◽  
Lysine Residues ◽  
Covalent Adducts ◽  
Endogenous Proteins ◽  
Activity Dependent ◽  
Hydrolysis Of

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. Here, we describe development of peptide activity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of epsilon-<i>N</i>-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the epsilon-<i>N</i>-glutaryllysine and epsilon<i>-N</i>-myristoyllysine analogues, were successfully applied to enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.

scholarly journals Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells

2015 ◽  
Vol 16 (12) ◽  
pp. 14695-14716 ◽  
Author(s):  
Sean Warren ◽  
Anca Margineanu ◽  
Matilda Katan ◽  
Chris Dunsby ◽  
Paul French
Keyword(s):  
Live Cells ◽  
Multiplexed Imaging

scholarly journals Highly multiplexed imaging of biosensors in live cells

2020 ◽  
Author(s):  
Jr-Ming Yang ◽  
Wei-Yu Chi ◽  
Jessica Liang ◽  
Pablo Iglesias ◽  
Chuan-Hsiang Huang
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Spectral Space ◽  
Fluorescent Biosensors ◽  
Multiplexed Imaging ◽  
Mixed Populations

AbstractFluorescent biosensors allow for real-time monitoring of biochemical activities in cells, but their multiplexing capacity is severely limited by the availability of spectral space. We overcome this problem by developing a set of barcoding proteins that are spectrally separable from commonly used FRET (fluorescence resonance energy transfer)-based and single-fluorophore biosensors. Mixed populations of barcoded cells expressing different biosensors can be concurrently imaged and computationally unmixed to achieve highly multiplexed tracking of biochemical activities in live cells.

scholarly journals Design of genetically encoded sensors to detect nucleosome ubiquitination in live cells

2021 ◽  
Vol 220 (4) ◽  
Author(s):  
Carolina dos Santos Passos ◽  
Yun-Seok Choi ◽  
Christopher D. Snow ◽  
Tingting Yao ◽  
Robert E. Cohen
Keyword(s):  
Posttranslational Modifications ◽  
Fluorescent Proteins ◽  
Live Cells ◽  
Transcriptional Elongation ◽  
Binding Domains ◽  
Kd Values ◽  
Binding Peptides ◽  
Genetically Encoded Sensors ◽  
And Function ◽  
Nucleosome Binding

Histone posttranslational modifications (PTMs) are dynamic, context-dependent signals that modulate chromatin structure and function. Ubiquitin (Ub) conjugation to different lysines of histones H2A and H2B is used to regulate diverse processes such as gene silencing, transcriptional elongation, and DNA repair. Despite considerable progress made to elucidate the players and mechanisms involved in histone ubiquitination, there remains a lack of tools to monitor these PTMs, especially in live cells. To address this, we combined an avidity-based strategy with in silico approaches to design sensors for specifically ubiquitinated nucleosomes. By linking Ub-binding domains to nucleosome-binding peptides, we engineered proteins that target H2AK13/15Ub and H2BK120Ub with Kd values from 10−8 to 10−6 M; when fused to fluorescent proteins, they work as PTM sensors in cells. The H2AK13/15Ub-specific sensor, employed to monitor signaling from endogenous DNA damage through the cell cycle, identified and differentiated roles for 53BP1 and BARD1 as mediators of this histone PTM.

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