scholarly journals Time-gated detection of protein-protein interactions with transcriptional readout

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Min Woo Kim ◽  
Wenjing Wang ◽  
Mateo I Sanchez ◽  
Robert Coukos ◽  
Mark von Zastrow ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
High Specificity ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
High Background ◽  
Specificity And Sensitivity ◽  
Time Courses ◽  
Rapid Kinetics ◽  
Two Hybrid

Transcriptional assays, such as yeast two-hybrid and TANGO, that convert transient protein-protein interactions (PPIs) into stable expression of transgenes are powerful tools for PPI discovery, screens, and analysis of cell populations. However, such assays often have high background and lose information about PPI dynamics. We have developed SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics), in which proteolytic release of a membrane-tethered transcription factor (TF) requires both a PPI to deliver a protease proximal to its cleavage peptide and blue light to uncage the cleavage site. SPARK was used to detect 12 different PPIs in mammalian cells, with 5 min temporal resolution and signal ratios up to 37. By shifting the light window, we could reconstruct PPI time-courses. Combined with FACS, SPARK enabled 51 fold enrichment of PPI-positive over PPI-negative cells. Due to its high specificity and sensitivity, SPARK has the potential to advance PPI analysis and discovery.

scholarly journals Time-gated detection of protein-protein interactions with transcriptional readout

2017 ◽  
Author(s):  
Min Woo Kim ◽  
Wenjing Wang ◽  
Mateo I. Sanchez ◽  
Robert Coukos ◽  
Mark Von Zastrow ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Time Window ◽  
High Specificity ◽  
Large Cell ◽  
Protein Protein Interactions ◽  
Signal Ratio ◽  
High Background ◽  
Tev Protease ◽  
Fast Light

AbstractTranscriptional assays such as yeast two hybrid, split ubiquitin, and Tango that convert transient protein-protein interactions (PPIs) in cells into stable expression of transgenes are powerful tools for PPI discovery, high-throughput screens, and analysis of large cell populations. However, these assays frequently suffer from high background and they lose all information about PPI dynamics. To address these limitations, we developed a light-gated transcriptional assay for PPI detection called PPI-FLARE (PPI-Fast Light- and Activity-Regulated Expression). PPI-FLARE requires both a PPI to deliver TEV protease proximal to its cleavage peptide, and externally-applied blue light to uncage the cleavage peptide, in order to release a membrane-tethered transcription factor (TF) for translocation to the nucleus. We used PPI-FLARE to detect the ligand-induced association of 12 different PPIs in living mammalian cells, with a temporal resolution of 5 minutes and a ±ligand signal ratio up to 37. By systematically shifting the light irradiation window, we could reconstruct PPI time-courses, distinguishing between GPCRs that engage in transient versus sustained interactions with the cytosolic effector arrestin. When combined with FACS, PPI-FLARE enabled >100-fold enrichment of cells experiencing a specific GPCR-arrestin PPI during a short 10-minute light window over cells missing that PPI during the same time window. Due to its high specificity, sensitivity, and generality, PPI-FLARE should be a broadly useful tool for PPI analysis and discovery.

scholarly journals Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Stephanie Berger ◽  
Erik Procko ◽  
Daciana Margineantu ◽  
Erinna F Lee ◽  
Betty W Shen ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Human Cancer ◽  
High Specificity ◽  
Specific Protein ◽  
Biological Processes ◽  
Protein Protein Interactions ◽  
Human Cancer Cell Lines ◽  
Bcl2 Family ◽  
Bcl2 Protein

Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.

Catalysis of disulphide bond formation in the endoplasmic reticulum

2004 ◽  
Vol 32 (5) ◽  
pp. 663-667 ◽  
Author(s):  
L. Ellgaard
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Protein Misfolding ◽  
Mammalian Cells ◽  
Specific Protein ◽  
Surface Proteins ◽  
Redox Conditions ◽  
Protein Protein Interactions ◽  
Disulphide Bonds ◽  
Correct Pairing

Disulphide bonds are critical for the maturation and stability of secretory and cell-surface proteins. In eukaryotic cells, disulphide bonds are introduced in the ER (endoplasmic reticulum), where the redox conditions are optimal to support their formation. Yet, the correct pairing of cysteine residues is not simple and often requires the assistance of redox-active proteins. The enzymes of the thiol-disulphide oxidoreductase family catalyse oxidation, reduction and isomerization, and thereby play important roles for the folding of many proteins. To allow all three redox reactions to take place concurrently in the same compartment, specific protein–protein interactions regulate the function of individual enzymes, while a careful balance of the ER redox environment is maintained. At the same time, the system must be capable of responding to changes in the cellular conditions, caused, for instance, by oxidative stress and protein misfolding. This review presents recent progress in understanding how ER redox conditions are regulated and how protein disulphides are formed in the ER of mammalian cells.

scholarly journals Optimizing the fragment complementation of APEX2 for detection of specific protein-protein interactions in live cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Miaomiao Xue ◽  
Junjie Hou ◽  
Linlin Wang ◽  
Dongwan Cheng ◽  
Jingze Lu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
High Specificity ◽  
Mass Spectrometry Analysis ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Hek 293 ◽  
Spectrometry Analysis ◽  
Specificity And Sensitivity ◽  
Fragment Complementation

Abstract Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. Recently, an ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. In this study, we tested whether APEX2 could be employed for PFC. By screening split APEX2 pairs attached to FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin binding (FRB) domain, which interact with each other only in the presence of rapamycin, we successfully obtained an optimized pair for visualizing the interaction between FRB and FKBP12 with high specificity and sensitivity in live cells. The robustness of this APEX2 pair was confirmed by its application toward detecting the STIM1 and Orial1 homodimers in HEK-293 cells. With a subsequent mass spectrometry analysis, we obtained five different biotinylated sites that were localized to the known interaction region on STIM1 and were only detected when the homodimer formed. These results suggest that our PFC pair of APEX2 provides a potential tool for detecting PPIs and identifying binding regions with high specificity in live cells.

scholarly journals The COP9 signalosome: at the interface between signal transduction and ubiquitin-dependent proteolysis

2002 ◽  
Vol 115 (3) ◽  
pp. 467-473 ◽  
Author(s):  
Dawadschargal Bech-Otschir ◽  
Michael Seeger ◽  
Wolfgang Dubiel
Keyword(s):  
Cell Cycle ◽  
Signal Transduction ◽  
Transcription Factors ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Specific Protein ◽  
26S Proteasome ◽  
Cop9 Signalosome ◽  
Protein Protein Interactions ◽  
Ubiquitin System

Recently the COP9 signalosome (CSN) has become a focus of interest for many researchers, because of its function at the interface between signal transduction and ubiquitin-dependent proteolysis. It is required for the proper progression of the cell cycle in Schizosaccharomyces pombe and is essential for development in plants and Drosophila. However, its function in mammalian cells remains obscure. Although the CSN shares structural similarities with the 26S proteasome lid complex (LID), its functions seem to be different from that of the LID. A variety of CSN-specific protein-protein interactions have been described in mammalian cells. However,it is currently unclear how many reflect true functions of the complex. Two activities associated with the CSN have been identified so far: a protein kinase and a deneddylase. The CSN-associated kinase phosphorylates transcription factors, which determines their stability towards the ubiquitin system. The associated deneddylase regulates the activity of specific SCF E3 ubiquitin ligases. The CSN thus appears to be a platform connecting signalling with proteolysis.

scholarly journals The Fluorescent Two-Hybrid Assay to Screen for Protein–Protein Interaction Inhibitors in Live Cells

2014 ◽  
Vol 19 (4) ◽  
pp. 516-525 ◽  
Author(s):  
Larisa Yurlova ◽  
Maarten Derks ◽  
Andrea Buchfellner ◽  
Ian Hickson ◽  
Marc Janssen ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Stapled Peptides ◽  
Protein Protein Interaction ◽  
Real Time Visualization ◽  
Two Hybrid

Protein–protein interactions (PPIs) are attractive but challenging targets for drug discovery. To overcome numerous limitations of the currently available cell-based PPI assays, we have recently established a fully reversible microscopy-assisted fluorescent two-hybrid (F2H) assay. The F2H assay offers a fast and straightforward readout: an interaction-dependent co-localization of two distinguishable fluorescent signals at a defined spot in the nucleus of mammalian cells. We developed two reversible F2H assays for the interactions between the tumor suppressor p53 and its negative regulators, Mdm2 and Mdm4. We then performed a pilot F2H screen with a subset of compounds, including small molecules (such as Nutlin-3) and stapled peptides. We identified five cell-penetrating compounds as potent p53–Mdm2 inhibitors. However, none exhibited intracellular activity on p53–Mdm4. Live cell data generated by the F2H assays enable the characterization of stapled peptides based on their ability to penetrate cells and disrupt p53–Mdm2 interaction as well as p53–Mdm4 interaction. Here, we show that the F2H assays enable side-by-side analysis of substances’ dual Mdm2–Mdm4 activity. In addition, they are suitable for testing various types of compounds (e.g., small molecules and peptidic inhibitors) and concurrently provide initial data on cellular toxicity. Furthermore, F2H assays readily allow real-time visualization of PPI dynamics in living cells.

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