scholarly journals MAPPIT: a versatile tool to study cytokine receptor signalling

2008 ◽  
Vol 36 (6) ◽  
pp. 1448-1451 ◽  
Author(s):  
Irma Lemmens ◽  
Sam Lievens ◽  
Jan Tavernier
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Cytokine Receptor ◽  
Bait Protein ◽  
Protein Protein Interaction ◽  
Receptor Signalling ◽  
Versatile Tool ◽  
Two Hybrid ◽  
Interaction Trap ◽  
Mammalian Protein

MAPPIT (mammalian protein–protein interaction trap) is a cytokine receptor-based two-hybrid method that operates in intact mammalian cells. A bait is fused C-terminally to a STAT (signal transducer and activator of transcription) recruitment-deficient receptor, whereas the prey is linked to functional STAT-binding sites. When bait and prey interact a ligand-dependent complementation of the STAT recruitment deficiency occurs, leading to activation of a STAT-responsive reporter. MAPPIT is very well suited to study protein interactions involving activated cytokine receptors as the technique allows modification of the bait protein in a physiologically optimal environment.

scholarly journals Straightforward Protein-Protein Interaction Interface Mapping via Random Mutagenesis and Mammalian Protein Protein Interaction Trap (MAPPIT)

2019 ◽  
Vol 20 (9) ◽  
pp. 2058 ◽  
Author(s):  
Laurens Vyncke ◽  
Delphine Masschaele ◽  
Jan Tavernier ◽  
Frank Peelman
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Random Mutagenesis ◽  
Interaction Mechanism ◽  
Protein Protein Interaction ◽  
Interaction Sites ◽  
Multiple Partners ◽  
Interaction Trap ◽  
Mammalian Protein

The MAPPIT (mammalian protein protein interaction trap) method allows high-throughput detection of protein interactions by very simple co-transfection of three plasmids in HEK293T cells, followed by a luciferase readout. MAPPIT detects a large percentage of all protein interactions, including those requiring posttranslational modifications and endogenous or exogenous ligands. Here, we present a straightforward method that allows detailed mapping of interaction interfaces via MAPPIT. The method provides insight into the interaction mechanism and reveals how this is affected by disease-associated mutations. By combining error-prone polymerase chain reaction (PCR) for random mutagenesis, 96-well DNA prepping, Sanger sequencing, and MAPPIT via 384-well transfections, we test the effects of a large number of mutations of a selected protein on its protein interactions. The entire screen takes less than three months and interactions with multiple partners can be studied in parallel. The effect of mutations on the MAPPIT readout is mapped on the protein structure, allowing unbiased identification of all putative interaction sites. We have thus far analysed 6 proteins and mapped their interfaces for 16 different interaction partners. Our method is broadly applicable as the required tools are simple and widely available.

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.

scholarly journals Mammalian Protein-Protein Interaction Trap (MAPPIT) Analysis of STAT5, CIS, and SOCS2 Interactions with the Growth Hormone Receptor

2007 ◽  
Vol 21 (11) ◽  
pp. 2821-2831 ◽  
Author(s):  
Isabel Uyttendaele ◽  
Irma Lemmens ◽  
Annick Verhee ◽  
Anne-Sophie De Smet ◽  
Joël Vandekerckhove ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Binding Sites ◽  
Growth Hormone Receptor ◽  
Functional Divergence ◽  
Critical Role ◽  
Sh2 Domain ◽  
Cytokine Signaling ◽  
Protein Protein Interaction ◽  
Interaction Trap ◽  
Mammalian Protein

Abstract Binding of GH to its receptor induces rapid phosphorylation of conserved tyrosine motifs that function as recruitment sites for downstream signaling molecules. Using mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, we mapped the binding sites in the GH receptor for signal transducer and activator of transcription 5 (STAT5) a and b and for the negative regulators of cytokine signaling cytokine-inducible Src-homology 2 (SH2)-containing protein (CIS) and suppressor of cytokine signaling 2 (SOCS2). Y534, Y566, and Y627 are the major recruitment sites for STAT5. A non-overlapping recruitment pattern is observed for SOCS2 and CIS with positions Y487 and Y595 as major binding sites, ruling out SOCS-mediated inhibition of STAT5 activation by competition for shared binding sites. More detailed analysis revealed that CIS binding to the Y595, but not to the Y487 motif, depends on both its SH2 domain and the C-terminal part of its SOCS box, with a critical role for the CIS Y253 residue. This functional divergence of the two CIS/SOCS2 recruitment sites is also observed upon substitution of the Y+1 residue by leucine, turning the Y487, but not the Y595 motif into a functional STAT5 recruitment site.

Visual detection of binary, ternary, and quaternary protein interactions in fission yeast by Pil1 co-tethering assay

2021 ◽  
Author(s):  
Zhong-Qiu Yu ◽  
Xiao-Man Liu ◽  
Dan Zhao ◽  
Dan-Dan Xu ◽  
Li-Lin Du
Keyword(s):  
Fission Yeast ◽  
Protein Interactions ◽  
Visual Detection ◽  
Protein Protein Interactions ◽  
Phosphatidylinositol 3 Kinase ◽  
Basic Form ◽  
Bait Protein ◽  
Cellular Processes ◽  
Prey Protein ◽  
Versatile Tool

Protein-protein interactions are vital for executing nearly all cellular processes. To facilitate the detection of protein-protein interactions in living cells of the fission yeast Schizosaccharomyces pombe, here we present an efficient and convenient method termed the Pil1 co-tethering assay. In its basic form, we tether a bait protein to mCherry-tagged Pil1, which forms cortical filamentary structures, and examine whether a GFP-tagged prey protein colocalizes with the bait. We demonstrate that this assay is capable of detecting pairwise protein-protein interactions of cytosolic proteins and nuclear proteins. Furthermore, we show that this assay can be used for detecting not only binary protein-protein interactions, but also ternary and quaternary protein-protein interactions. Using this assay, we systematically characterized the protein-protein interactions in the Atg1 complex and in the phosphatidylinositol 3-kinase (PtdIns3K) complexes and found that Atg38 is incorporated into the PtdIns3K complex I via an Atg38-Vps34 interaction. Our data show that this assay is a useful and versatile tool and should be added to the routine toolbox of fission yeast researchers.

scholarly journals Mass spectrometry-based methods for analysing the mitochondrial interactome in mammalian cells

2019 ◽  
Vol 167 (3) ◽  
pp. 225-231 ◽  
Author(s):  
Takumi Koshiba ◽  
Hidetaka Kosako
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Living Cells ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Protein Protein Interaction ◽  
Membrane Protein Complexes ◽  
Protein Protein Interaction Networks

Abstract Protein–protein interactions are essential biologic processes that occur at inter- and intracellular levels. To gain insight into the various complex cellular functions of these interactions, it is necessary to assess them under physiologic conditions. Recent advances in various proteomic technologies allow to investigate protein–protein interaction networks in living cells. The combination of proximity-dependent labelling and chemical cross-linking will greatly enhance our understanding of multi-protein complexes that are difficult to prepare, such as organelle-bound membrane proteins. In this review, we describe our current understanding of mass spectrometry-based proteomics mapping methods for elucidating organelle-bound membrane protein complexes in living cells, with a focus on protein–protein interactions in mitochondrial subcellular compartments.

On the structure of protein–protein interaction networks

2003 ◽  
Vol 31 (6) ◽  
pp. 1491-1496 ◽  
Author(s):  
A. Thomas ◽  
R. Cannings ◽  
N.A.M. Monk ◽  
C. Cannings
Keyword(s):  
Power Law ◽  
Protein Interactions ◽  
Large Scale ◽  
Underlying Structure ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Human Proteins ◽  
Approximate Power ◽  
Two Hybrid

We present a simple model for the underlying structure of protein–protein pairwise interaction graphs that is based on the way in which proteins attach to each other in experiments such as yeast two-hybrid assays. We show that data on the interactions of human proteins lend support to this model. The frequency of the number of connections per protein under this model does not follow a power law, in contrast to the reported behaviour of data from large-scale yeast two-hybrid screens of yeast protein–protein interactions. Sampling sub-graphs from the underlying graphs generated with our model, in a way analogous to the sampling performed in large-scale yeast two-hybrid searches, gives degree distributions that differ subtly from the power law and that fit the observed data better than the power law itself. Our results show that the observation of approximate power law behaviour in a sampled sub-graph does not imply that the underlying graph follows a power law.

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