scholarly journals Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Huy Quoc Nguyen ◽  
Jagoree Roy ◽  
Björn Harink ◽  
Nikhil P Damle ◽  
Naomi R Latorraca ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Signaling Networks ◽  
Energy Landscapes ◽  
Post Translational Modifications ◽  
Short Linear Motifs ◽  
Quantitative Mapping ◽  
Wide Range ◽  
Binding Peptides ◽  
Linear Motifs ◽  
Peptide Affinity

Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and perturbing signaling networks but is challenging due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with a wide range of affinities. The quantitative biophysical data provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.

scholarly journals Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

2018 ◽  
Author(s):  
Huy Nguyen ◽  
Jagoree Roy ◽  
Björn Harink ◽  
Nikhil Damle ◽  
Brian Baxter ◽  
...  
Keyword(s):  
Computational Modeling ◽  
Protein Phosphatase ◽  
Therapeutic Potential ◽  
Signaling Networks ◽  
Post Translational Modifications ◽  
Inhibitory Peptides ◽  
Quantitative Measurements ◽  
Quantitative Mapping ◽  
Linear Motifs ◽  
Peptide Affinity

AbstractTransient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and therapeutically perturbing signaling networks, but is challenging due to their weak affinities. We present a powerful technology, MRBLE-pep, that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using computational modeling and MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We establish that flanking residues and post- translational modifications critically contribute to PxIxIT-CN affinity, and discover CN-inhibitory peptides with unprecedented affinity and therapeutic potential. The quantitative measurements provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.

scholarly journals Inhibitory activities of short linear motifs underlie Hox interactome specificity in vivo

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Manon Baëza ◽  
Séverine Viala ◽  
Marjorie Heim ◽  
Amélie Dard ◽  
Bruno Hudry ◽  
...  
Keyword(s):  
Cell Fate ◽  
Drosophila Embryo ◽  
Traditional Role ◽  
Hox Proteins ◽  
Short Linear Motifs ◽  
Wide Range ◽  
Mouse Species ◽  
Inhibitory Activities ◽  
Linear Motifs

Hox proteins are well-established developmental regulators that coordinate cell fate and morphogenesis throughout embryogenesis. In contrast, our knowledge of their specific molecular modes of action is limited to the interaction with few cofactors. Here, we show that Hox proteins are able to interact with a wide range of transcription factors in the live Drosophila embryo. In this context, specificity relies on a versatile usage of conserved short linear motifs (SLiMs), which, surprisingly, often restrains the interaction potential of Hox proteins. This novel buffering activity of SLiMs was observed in different tissues and found in Hox proteins from cnidarian to mouse species. Although these interactions remain to be analysed in the context of endogenous Hox regulatory activities, our observations challenge the traditional role assigned to SLiMs and provide an alternative concept to explain how Hox interactome specificity could be achieved during the embryonic development.

scholarly journals Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

2019 ◽  
Author(s):  
Callie P. Wigington ◽  
Jagoree Roy ◽  
Nikhil P. Damle ◽  
Vikash K. Yadav ◽  
Cecilia Blikstad ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Nuclear Pore ◽  
Binding Affinities ◽  
Protein Protein Interactions ◽  
Short Linear Motifs ◽  
Binding Peptides ◽  
Linear Motifs ◽  
Calcineurin Phosphatase

SummaryShort linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins – structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

scholarly journals Orchestrating serine/threonine phosphorylation and elucidating downstream effects by short linear motifs

2022 ◽  
Vol 479 (1) ◽  
pp. 1-22
Author(s):  
Johanna Kliche ◽  
Ylva Ivarsson
Keyword(s):  
Complex Formation ◽  
Protein Interactions ◽  
Binding Sites ◽  
Protein Protein Interactions ◽  
Post Translational Modifications ◽  
Short Linear Motifs ◽  
Protein Complex Formation ◽  
Downstream Effects ◽  
Linear Motifs ◽  
Threonine Kinases

Cellular function is based on protein–protein interactions. A large proportion of these interactions involves the binding of short linear motifs (SLiMs) by folded globular domains. These interactions are regulated by post-translational modifications, such as phosphorylation, that create and break motif binding sites or tune the affinity of the interactions. In addition, motif-based interactions are involved in targeting serine/threonine kinases and phosphatases to their substrate and contribute to the specificity of the enzymatic actions regulating which sites are phosphorylated. Here, we review how SLiM-based interactions assist in determining the specificity of serine/threonine kinases and phosphatases, and how phosphorylation, in turn, affects motif-based interactions. We provide examples of SLiM-based interactions that are turned on/off, or are tuned by serine/threonine phosphorylation and exemplify how this affects SLiM-based protein complex formation.

scholarly journals Peptide array–based interactomics

2021 ◽  
Author(s):  
Daniel Perez Hernandez ◽  
Gunnar Dittmar
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Large Scale ◽  
New Technologies ◽  
Membrane Surface ◽  
Protein Protein Interactions ◽  
Post Translational Modifications ◽  
Short Linear Motifs ◽  
Binding Partners ◽  
Linear Motifs

AbstractThe analysis of protein-protein interactions (PPIs) is essential for the understanding of cellular signaling. Besides probing PPIs with immunoprecipitation-based techniques, peptide pull-downs are an alternative tool specifically useful to study interactome changes induced by post-translational modifications. Peptides for pull-downs can be chemically synthesized and thus offer the possibility to include amino acid exchanges and post-translational modifications (PTMs) in the pull-down reaction. The combination of peptide pull-down and analysis of the binding partners with mass spectrometry offers the direct measurement of interactome changes induced by PTMs or by amino acid exchanges in the interaction site. The possibility of large-scale peptide synthesis on a membrane surface opened the possibility to systematically analyze interactome changes for mutations of many proteins at the same time. Short linear motifs (SLiMs) are amino acid patterns that can mediate protein binding. A significant number of SLiMs are located in regions of proteins, which are lacking a secondary structure, making the interaction motifs readily available for binding reactions. Peptides are particularly well suited to study protein interactions, which are based on SLiM-mediated binding. New technologies using arrayed peptides for interaction studies are able to identify SLIM-based interaction and identify the interaction motifs. Graphical abstract

Biogenesis and activity regulation of protein phosphatase 1

2017 ◽  
Vol 45 (1) ◽  
pp. 89-99 ◽  
Author(s):  
Iris Verbinnen ◽  
Monica Ferreira ◽  
Mathieu Bollen
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Interacting Proteins ◽  
Subcellular Targeting ◽  
Substantial Fraction ◽  
Short Linear Motifs ◽  
Specific Subset ◽  
Linear Motifs ◽  
And Function ◽  
And Control

Protein phosphatase 1 (PP1) is expressed in all eukaryotic cells and catalyzes a substantial fraction of phosphoserine/threonine dephosphorylation reactions. It forms stable complexes with PP1-interacting proteins (PIPs) that guide the phosphatase throughout its life cycle and control its fate and function. The diversity of PIPs is huge (≈200 in vertebrates), and most of them combine short linear motifs to form large and unique interaction interfaces with PP1. Many PIPs have separate domains for PP1 anchoring, PP1 regulation, substrate recruitment and subcellular targeting, which enable them to direct associated PP1 to a specific subset of substrates and mediate acute activity control. Hence, PP1 functions as the catalytic subunit of a large number of multimeric holoenzymes, each with its own subset of substrates and mechanism(s) of regulation.

scholarly journals Short Linear Motifs Characterizing Snake Venom and Mammalian Phospholipases A2

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 290
Author(s):  
Caterina Peggion ◽  
Fiorella Tonello
Keyword(s):  
Snake Venom ◽  
Protein Interactions ◽  
Biological Properties ◽  
Protein Protein Interactions ◽  
Sequence Alignments ◽  
Toxic Activity ◽  
Short Linear Motifs ◽  
Phospholipases A2 ◽  
Group I ◽  
Linear Motifs

Snake venom phospholipases A2 (PLA2s) have sequences and structures very similar to those of mammalian group I and II secretory PLA2s, but they possess many toxic properties, ranging from the inhibition of coagulation to the blockage of nerve transmission, and the induction of muscle necrosis. The biological properties of these proteins are not only due to their enzymatic activity, but also to protein–protein interactions which are still unidentified. Here, we compare sequence alignments of snake venom and mammalian PLA2s, grouped according to their structure and biological activity, looking for differences that can justify their different behavior. This bioinformatics analysis has evidenced three distinct regions, two central and one C-terminal, having amino acid compositions that distinguish the different categories of PLA2s. In these regions, we identified short linear motifs (SLiMs), peptide modules involved in protein–protein interactions, conserved in mammalian and not in snake venom PLA2s, or vice versa. The different content in the SLiMs of snake venom with respect to mammalian PLA2s may result in the formation of protein membrane complexes having a toxic activity, or in the formation of complexes whose activity cannot be blocked due to the lack of switches in the toxic PLA2s, as the motif recognized by the prolyl isomerase Pin1.

Visualising intrinsic disorder and conformational variation in protein ensembles

2014 ◽  
Vol 169 ◽  
pp. 179-193 ◽  
Author(s):  
Julian Heinrich ◽  
Michael Krone ◽  
Seán I. O'Donoghue ◽  
Daniel Weiskopf
Keyword(s):  
Intrinsic Disorder ◽  
Molecular Graphics ◽  
Post Translational Modifications ◽  
3D Structures ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Wide Range ◽  
Conformational Variations ◽  
Protein Ensembles ◽  
Traditional Approaches

Intrinsically disordered regions (IDRs) in proteins are still not well understood, but are increasingly recognised as important in key biological functions, as well as in diseases. IDRs often confound experimental structure determination—however, they are present in many of the available 3D structures, where they exhibit a wide range of conformations, from ill-defined and highly flexible to well-defined upon binding to partner molecules, or upon post-translational modifications. Analysing such large conformational variations across ensembles of 3D structures can be complex and difficult; our goal in this paper is to improve this situation by augmenting traditional approaches (molecular graphics and principal components) with methods from human–computer interaction and information visualisation, especially parallel coordinates. We present a new tool integrating these approaches, and demonstrate how it can dissect ensembles to reveal functional insights into conformational variation and intrinsic disorder.

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