A reversible Renilla luciferase protein complementation assay for rapid identification of protein–protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus

Abstract Direct drug targeting of mutated proteins in cancer is not always possible and efficacy can be nullified by compensating protein–protein interactions (PPIs). Here, we establish an in silico pipeline to identify specific PPI sub-networks containing mutated proteins as potential targets, which we apply to mutation data of four different leukaemias. Our method is based on extracting cyclic interactions of a small number of proteins topologically and functionally linked in the Protein–Protein Interaction Network (PPIN), which we call short loop network motifs (SLM). We uncover a new property of PPINs named ‘short loop commonality’ to measure indirect PPIs occurring via common SLM interactions. This detects ‘modules’ of PPI networks enriched with annotated biological functions of proteins containing mutation hotspots, exemplified by FLT3 and other receptor tyrosine kinase proteins. We further identify functional dependency or mutual exclusivity of short loop commonality pairs in large-scale cellular CRISPR–Cas9 knockout screening data. Our pipeline provides a new strategy for identifying new therapeutic targets for drug discovery.

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Frequent assembly of chimeric complexes in the protein interaction network of an interspecies yeast hybrid

Molecular Biology and Evolution ◽

10.1093/molbev/msaa298 ◽

2020 ◽

Author(s):

Rohan Dandage ◽

Caroline M Berger ◽

Isabelle Gagnon-Arsenault ◽

Kyung-Mee Moon ◽

Richard Greg Stacey ◽

...

Keyword(s):

Protein Interactions ◽

Molecular Level ◽

Yeast Species ◽

Protein Complexes ◽

Mitochondrial Protein ◽

Chimeric Protein ◽

Interaction Network ◽

Protein Protein Interactions ◽

Extreme Phenotypes ◽

Yeast Hybrid

Abstract Hybrids between species often show extreme phenotypes, including some that take place at the molecular level. In this study, we investigated the phenotypes of an interspecies diploid hybrid in terms of protein-protein interactions inferred from protein correlation profiling. We used two yeast species, Saccharomyces cerevisiae and Saccharomyces uvarum, which are interfertile, but yet have proteins diverged enough to be differentiated using mass spectrometry. Most of the protein-protein interactions are similar between hybrid and parents, and are consistent with the assembly of chimeric complexes, which we validated using an orthogonal approach for the prefoldin complex. We also identified instances of altered protein-protein interactions in the hybrid, for instance in complexes related to proteostasis and in mitochondrial protein complexes. Overall, this study uncovers the likely frequent occurrence of chimeric protein complexes with few exceptions, which may result from incompatibilities or imbalances between the parental proteins.

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Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

Nuclear Receptor Signaling ◽

10.1621/nrs.04021 ◽

2006 ◽

Vol 4 (1) ◽

pp. nrs.04021 ◽

Cited By ~ 14

Author(s):

Kristen L. Koterba ◽

Brian G. Rowan

Keyword(s):

Energy Transfer ◽

Protein Interactions ◽

Fusion Proteins ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Renilla Luciferase ◽

Receptor Protein ◽

Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

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PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data

BioMed Research International ◽

10.1155/2016/2891918 ◽

2016 ◽

Vol 2016 ◽

pp. 1-13

Author(s):

Stefan Kalkhof ◽

Stefan Schildbach ◽

Conny Blumert ◽

Friedemann Horn ◽

Martin von Bergen ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Interactions ◽

Isotope Labeling ◽

Affinity Purification ◽

Interaction Network ◽

Mass Spectrometry Data ◽

Protein Protein Interactions ◽

Protein Protein Interaction ◽

Binding Behavior ◽

Bona Fide

The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6.

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Detecting Protein-Protein Interactions Using Renilla Luciferase Fusion Proteins

BioTechniques ◽

10.2144/02335st05 ◽

2002 ◽

Vol 33 (5) ◽

pp. 1044-1050 ◽

Cited By ~ 3

Author(s):

Peter D. Burbelo ◽

Adam E. Kisailus ◽

Jeremy W. Peck

Keyword(s):

Protein Interactions ◽

Fusion Proteins ◽

Renilla Luciferase ◽

Protein Protein Interactions

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Optimizing Luciferase Protein Fragment Complementation for Bioluminescent Imaging of Protein–Protein Interactions in Live Cells and Animals

Methods in Enzymology - Imaging in Biological Research, Part A ◽

10.1016/s0076-6879(04)85019-5 ◽

2004 ◽

pp. 349-360 ◽

Cited By ~ 35

Author(s):

Kathryn E Luker ◽

David Piwnica-Worms

Keyword(s):

Protein Interactions ◽

Live Cells ◽

Bioluminescent Imaging ◽

Protein Protein Interactions ◽

Protein Fragment ◽

Protein Fragment Complementation ◽

Luciferase Protein ◽

Fragment Complementation

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Analysis of Protein‐Protein Interactions by Split Luciferase Complementation Assay

Current Protocols in Toxicology ◽

10.1002/cptx.90 ◽

2019 ◽

Vol 82 (1) ◽

Cited By ~ 3

Author(s):

Yuekun Lang ◽

Zhong Li ◽

Hongmin Li

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions ◽

Complementation Assay ◽

Split Luciferase Complementation ◽

Split Luciferase

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Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art

International Journal of Molecular Sciences ◽

10.3390/ijms20122958 ◽

2019 ◽

Vol 20 (12) ◽

pp. 2958 ◽

Cited By ~ 6

Author(s):

Wouters ◽

Vasudevan ◽

Crans ◽

Saini ◽

Stove

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions ◽

Vitro Assay ◽

Current State ◽

Target Disease ◽

Gpcr Dimer ◽

Overexpression System ◽

Protein Complementation

G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR–GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein–protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR–GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR–GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays.

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