scholarly journals Listening to the Whispers in Neuroimmune Crosstalk: A Comprehensive Workflow to Investigate Neurotrophin Receptor p75NTR Under Endogenous, Low Abundance Conditions

2021 ◽  
Vol 12 ◽  
Author(s):  
Benjamin W. Dorschner ◽  
Ralf Wiedemuth ◽  
Ann-Christin Funke ◽  
Marc Gentzel ◽  
Mary-Louise Rogers ◽  
...  
Keyword(s):  
Cell Line ◽  
Protein Interactions ◽  
Plasmacytoid Dendritic Cell ◽  
Specific Interaction ◽  
Receptor Expression ◽  
Neurotrophin Receptor ◽  
Protein Protein Interactions ◽  
Inflammatory Conditions ◽  
Interaction Partners ◽  
Antibody Validation

Inflammatory conditions are critically influenced by neuroimmune crosstalk. Cytokines and neurotrophic factors shape the responses of both nervous and immune systems. Although much progress has been made, most findings to date are based on expression of recombinant (tagged) proteins. The examination of receptor interactions by immunoprecipitation (IP) at endogenous levels provides further insight into the more subtle regulations of immune responses. Here, we present a comprehensive workflow and an optimized IP protocol that provide step-by-step instructions to investigate neurotrophin receptor p75NTR at endogenous, low abundance levels: from lysate preparation and confirmation of receptor expression to antibody validation and successful detection of protein-protein interactions. We employ human melanoma cell line A375 to validate specific antibodies and IP conditions, and apply these methods to explore p75NTR interactions in human leukemic plasmacytoid dendritic cell line PMDC05 detecting 14-3-3ϵ:p75NTR interaction in this cell type. With p75NTR as an exemplary protein, our approach provides a strategy to detect specific interaction partners even under endogenous, low abundance expression conditions.

scholarly journals Inferring interaction partners from protein sequences

2016 ◽  
Author(s):  
Anne-Florence Bitbol ◽  
Robert S. Dwyer ◽  
Lucy J. Colwell ◽  
Ned S. Wingreen
Keyword(s):  
Protein Interactions ◽  
Sequence Data ◽  
Protein Complexes ◽  
A Priori ◽  
Specific Interaction ◽  
Three Dimensional ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Entropy Model ◽  
Interaction Partners

Specific protein-protein interactions are crucial in the cell, both to ensure the formation and stability of multi-protein complexes, and to enable signal transduction in various pathways. Functional interactions between proteins result in coevolution between the interaction partners. Hence, the sequences of interacting partners are correlated. Here we exploit these correlations to accurately identify which proteins are specific interaction partners from sequence data alone. Our general approach, which employs a pairwise maximum entropy model to infer direct couplings between residues, has been successfully used to predict the three-dimensional structures of proteins from sequences. Building on this approach, we introduce an iterative algorithm to predict specific interaction partners from among the members of two protein families. We assess the algorithm's performance on histidine kinases and response regulators from bacterial two-component signaling systems. The algorithm proves successful without any a priori knowledge of interaction partners, yielding a striking 0.93 true positive fraction on our complete dataset, and we uncover the origin of this surprising success. Finally, we discuss how our method could be used to predict novel protein-protein interactions.

Prediction of Protein-Protein Interactions Between Human Host and Two Mycobacterial Organisms

2013 ◽  
pp. 175-187
Author(s):  
Oruganty Krishnadev ◽  
Shveta Bisht ◽  
Narayanaswamy Srinivasan
Keyword(s):  
Protein Interactions ◽  
Serine Proteases ◽  
Cellular Membrane ◽  
Human Pathogens ◽  
Protein Protein Interactions ◽  
Human Host ◽  
Interaction Partners ◽  
Human Proteins ◽  
Mycobacterial Antigens ◽  
Ras Signalling

The genomes of many human pathogens have been sequenced but the protein-protein interactions across a pathogen and human are still poorly understood. The authors apply a simple homology-based method to predict protein-protein interactions between human host and two mycobacterial organisms viz., M.tuberculosis and M.leprae. They focused on secreted proteins of pathogens and cellular membrane proteins to restrict to uncovering biologically significant and feasible interactions. Predicted interactions include five mycobacterial proteins of yet unknown function, thus suggesting a role for these proteins in pathogenesis. The authors predict interaction partners for secreted mycobacterial antigens such as MPT70, serine proteases and other proteins interacting with human proteins, such as toll-like receptors, ras signalling proteins and immune maintenance proteins, that are implicated in pathogenesis. These results suggest that the list of predicted interactions is suitable for further analysis and forms a useful step in the understanding of pathogenesis of these mycobacterial organisms.

scholarly journals Peptide-based Interaction Proteomics

2020 ◽  
Vol 19 (7) ◽  
pp. 1070-1075 ◽  
Author(s):  
Katrina Meyer ◽  
Matthias Selbach
Keyword(s):  
Protein Interactions ◽  
Synthetic Peptides ◽  
Protein Protein Interactions ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Interaction Partners ◽  
Linear Motifs ◽  
Cellulose Membranes ◽  
Interaction Proteomics ◽  
Proteins Interactions

Protein-protein interactions are often mediated by short linear motifs (SLiMs) that are located in intrinsically disordered regions (IDRs) of proteins. Interactions mediated by SLiMs are notoriously difficult to study, and many functionally relevant interactions likely remain to be uncovered. Recently, pull-downs with synthetic peptides in combination with quantitative mass spectrometry emerged as a powerful screening approach to study protein-protein interactions mediated by SLiMs. Specifically, arrays of synthetic peptides immobilized on cellulose membranes provide a scalable means to identify the interaction partners of many peptides in parallel. In this minireview we briefly highlight the relevance of SLiMs for protein-protein interactions, outline existing screening technologies, discuss unique advantages of peptide-based interaction screens and provide practical suggestions for setting up such peptide-based screens.

scholarly journals The Effect of Proline cis-trans Isomerization on the Folding of the C-Terminal SH2 Domain from p85

2019 ◽  
Vol 21 (1) ◽  
pp. 125
Author(s):  
Francesca Troilo ◽  
Francesca Malagrinò ◽  
Lorenzo Visconti ◽  
Angelo Toto ◽  
Stefano Gianni
Keyword(s):  
Protein Interactions ◽  
Specific Interaction ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Sh2 Domains ◽  
Trans Isomerization ◽  
A Site ◽  
Kinetics Of

SH2 domains are protein domains that modulate protein–protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the βE and βF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.

scholarly journals NMR Spectroscopy of supramolecular chemistry on protein surfaces

2020 ◽  
Vol 16 ◽  
pp. 2505-2522
Author(s):  
Peter Bayer ◽  
Anja Matena ◽  
Christine Beuck
Keyword(s):  
Nmr Spectroscopy ◽  
Supramolecular Chemistry ◽  
Molecular Interactions ◽  
Protein Interactions ◽  
Atomic Resolution ◽  
Protein Protein Interactions ◽  
Protein Surfaces ◽  
Interaction Partners ◽  
Binding Pockets ◽  
Nmr Methods

As one of the few analytical methods that offer atomic resolution, NMR spectroscopy is a valuable tool to study the interaction of proteins with their interaction partners, both biomolecules and synthetic ligands. In recent years, the focus in chemistry has kept expanding from targeting small binding pockets in proteins to recognizing patches on protein surfaces, mostly via supramolecular chemistry, with the goal to modulate protein–protein interactions. Here we present NMR methods that have been applied to characterize these molecular interactions and discuss the challenges of this endeavor.

scholarly journals Subcellular Targeting of Plant Sucrose Transporters Is Affected by Their Oligomeric State

Plants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 158 ◽  
Author(s):  
Varsha Garg ◽  
Aleksandra Hackel ◽  
Christina Kühn
Keyword(s):  
Protein Interactions ◽  
Subcellular Distribution ◽  
Sucrose Transporter ◽  
Membrane Microdomains ◽  
Protein Protein Interactions ◽  
Subcellular Targeting ◽  
Oligomeric State ◽  
Sucrose Transporters ◽  
Interaction Partners ◽  
The Impact

Post-translational regulation of sucrose transporters represents one possibility to adapt transporter activity in a very short time frame. This can occur either via phosphorylation/dephosphorylation, oligomerization, protein–protein interactions, endocytosis/exocytosis, or degradation. It is also known that StSUT1 can change its compartmentalization at the plasma membrane and concentrate in membrane microdomains in response to changing redox conditions. A systematic screen for protein–protein-interactions of plant sucrose transporters revealed that the interactome of all three known sucrose transporters from the Solanaceous species Solanum tuberosum and Solanum lycopersicum represents a specific subset of interaction partners, suggesting different functions for the three different sucrose transporters. Here, we focus on factors that affect the subcellular distribution of the transporters. It was already known that sucrose transporters are able to form homo- as well as heterodimers. Here, we reveal the consequences of homo- and heterodimer formation and the fact that the responses of individual sucrose transporters will respond differently. Sucrose transporter SlSUT2 is mainly found in intracellular vesicles and several of its interaction partners are involved in vesicle traffic and subcellular targeting. The impact of interaction partners such as SNARE/VAMP proteins on the localization of SlSUT2 protein will be investigated, as well as the impact of inhibitors, excess of substrate, or divalent cations which are known to inhibit SUT1-mediated sucrose transport in yeast cells. Thereby we are able to identify factors regulating sucrose transporter activity via a change of their subcellular distribution.

scholarly journals Gene phylogenies and protein–protein interactions: possible artifacts resulting from shared protein interaction partners

2004 ◽  
Vol 231 (2) ◽  
pp. 197-202 ◽  
Author(s):  
Paulo R.A. Campos ◽  
Viviane M. de Oliveira ◽  
Günter P. Wagner ◽  
Peter F. Stadler
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Interaction Partners

scholarly journals Mechanism of Mediator Recruitment by Tandem Gcn4 Activation Domains and Three Gal11 Activator-Binding Domains

2010 ◽  
Vol 30 (10) ◽  
pp. 2376-2390 ◽  
Author(s):  
Eric Herbig ◽  
Linda Warfield ◽  
Lisa Fish ◽  
James Fishburn ◽  
Bruce A. Knutson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Specific Interaction ◽  
Protein Protein Interactions ◽  
Activation Domain ◽  
Activation Domains ◽  
Binding Domains ◽  
Cross Links ◽  
Kix Domain ◽  
The Individual ◽  
Was Gene

ABSTRACT Targets of the tandem Gcn4 acidic activation domains in transcription preinitiation complexes were identified by site-specific cross-linking. The individual Gcn4 activation domains cross-link to three common targets, Gal11/Med15, Taf12, and Tra1, which are subunits of four conserved coactivator complexes, Mediator, SAGA, TFIID, and NuA4. The Gcn4 N-terminal activation domain also cross-links to the Mediator subunit Sin4/Med16. The contribution of the two Gcn4 activation domains to transcription was gene specific and varied from synergistic to less than additive. Gcn4-dependent genes had a requirement for Gal11 ranging from 10-fold dependence to complete Gal11 independence, while the Gcn4-Taf12 interaction did not significantly contribute to the expression of any gene studied. Complementary methods identified three conserved Gal11 activator-binding domains that bind each Gcn4 activation domain with micromolar affinity. These Gal11 activator-binding domains contribute additively to transcription activation and Mediator recruitment at Gcn4- and Gal11-dependent genes. Although we found that the conserved Gal11 KIX domain contributes to Gal11 function, we found no evidence of specific Gcn4-KIX interaction and conclude that the Gal11 KIX domain does not function by specific interaction with Gcn4. Our combined results show gene-specific coactivator requirements, a surprising redundancy in activator-target interactions, and an activator-coactivator interaction mediated by multiple low-affinity protein-protein interactions.

scholarly journals Cell cycle regulation of mitochondrial protein import revealed by genome-scale pooled bimolecular fluorescence complementation screening

2019 ◽  
Author(s):  
Kim Blakely ◽  
Patricia Mero ◽  
Roland Arnold ◽  
Ayesha Saleem ◽  
Christine Misquitta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
High Throughput ◽  
Protein Interactions ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Protein Protein Interactions ◽  
Mitochondrial Protein Import ◽  
Interaction Partners ◽  
Genome Scale ◽  
Bimolecular Fluorescence

ABSTRACTA central focus of systems biology is the functional mapping of protein-protein interactions under physiological conditions. Here we describe MaGiCaL-BiFC, a lentivirus-based bimolecular fluorescence protein-fragment complementation approach for the high-throughput, genome-scale identification of protein-protein interactions in mammalian cells. After developing and validating this methodology using known protein-protein interaction pairs, we constructed genome-scale pooled BiFC libraries using the human ORFeome cDNA collection. These pooled libraries, containing ∼ 12,000 unique human cDNAs, were used to screen for candidate interaction partners of the mitochondrial transmembrane protein TOMM22. Following infection of cells with the TOMM22 bait and the pooled cDNA libraries, cells harboring candidate TOMM22 interacting proteins were isolated from the cell pool via fluorescence activated cell sorting, and identified via microarray analysis. This approach identified several known interaction partners of TOMM22, as well as novel physical and functional partners that link the mitochondrial network to proteins involved in diverse cellular processes. Notably, protein kinase CK2 was identified as a novel physical interaction partner of human TOMM22. We found that this association occurs preferentially during mitosis and involves direct phosphorylation of TOMM22, an event that may lead to attenuation of mitochondrial protein import. Together, this data contributes to the growing body of evidence suggesting eloquent coordination between cell cycle progression and mitochondrial physiology. Importantly, through high-throughput screening and focused validation, our study demonstrates the power of the MaGiCaL-BiFC approach to uncover novel functional protein-protein interactions, including those involving proteins with membrane-spanning domains, or of a transient nature, all within their native cellular environment.

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