scholarly journals Defining endogenous TACC3–chTOG–clathrin–GTSE1 interactions at the mitotic spindle using induced relocalization

2020 ◽  
pp. jcs.255794
Author(s):  
Ellis L. Ryan ◽  
James Shelford ◽  
Teresa Massam-Wu ◽  
Richard Bayliss ◽  
Stephen J. Royle
Keyword(s):  
Protein Interactions ◽  
Mitotic Spindle ◽  
Binding Sites ◽  
Gene Editing ◽  
Multiprotein Complex ◽  
Protein Protein Interactions ◽  
Core Complex ◽  
Microtubule Binding ◽  
Anti Cancer ◽  
Spindle Stability

A multiprotein complex containing TACC3, clathrin, and other proteins has been implicated in mitotic spindle stability. To disrupt this complex in an anti-cancer context, we need to understand its composition and how it interacts with microtubules. Induced relocalization of proteins in cells is a powerful way to analyze protein-protein interactions and additionally, monitor where and when these interactions occur. We used CRISPR/Cas9 gene-editing to add tandem FKBP-GFP tags to each complex member. The relocalization of endogenous tagged protein from the mitotic spindle to mitochondria and assessment of the effect on other proteins allowed us to establish that TACC3 and clathrin are core complex members and that chTOG and GTSE1 are ancillary to the complex, respectively binding to TACC3 and clathrin, but not each other. We also show that PIK3C2A, a clathrin-binding protein that was proposed to stabilize the TACC3–chTOG–clathrin–GTSE1 complex during mitosis, is not a member of the complex. This work establishes that targeting the TACC3–clathrin interface or their microtubule-binding sites are the two strategies most likely to disrupt spindle stability mediated by this multiprotein complex.

scholarly journals Defining endogenous TACC3–chTOG–clathrin–GTSE1 interactions at the mitotic spindle using induced relocalization

2020 ◽  
Author(s):  
Ellis L. Ryan ◽  
James Shelford ◽  
Teresa Massam-Wu ◽  
Richard Bayliss ◽  
Stephen J. Royle
Keyword(s):  
Protein Interactions ◽  
Membrane Trafficking ◽  
Mitotic Spindle ◽  
Binding Sites ◽  
Gene Editing ◽  
Multiprotein Complex ◽  
Protein Protein Interactions ◽  
Core Complex ◽  
Anti Cancer ◽  
Spindle Stability

A multiprotein complex containing TACC3, clathrin, and other proteins has been implicated in mitotic spindle stability. To disrupt this complex in an anti-cancer context, we need to understand the composition of the complex and the interactions between complex members and with microtubules. Induced relocalization of proteins in cells is a powerful way to analyze protein-protein interactions and additionally monitoring where and when these interactions occur. We used CRISPR/Cas9 gene-editing to add tandem FKBP-GFP tags to each complex member. The relocalization of endogenous tagged protein from the mitotic spindle to mitochondria and assessment of the effect on other proteins allowed us to establish that TACC3 and clathrin are core complex members and that chTOG and GTSE1 are ancillary to the complex, respectively binding to TACC3 and clathrin, but not each other. PIK3C2A, a membrane trafficking protein that binds clathrin, was previously proposed to also bind TACC3 and stabilize the TACC3–chTOG–clathrin–GTSE1 complex during mitosis. We show that PIK3C2A is not on the mitotic spindle and that knockout of this gene had no effect on the localization of the complex. We therefore conclude that PIK3C2A is not a member of the TACC3–chTOG–clathrin–GTSE1 complex. This work establishes that targeting the TACC3–clathrin interface or their microtubule-binding sites are the two strategies most likely to disrupt spindle stability mediated by this multiprotein complex.

scholarly journals Exploring Protein-Protein Interactions as Drug Targets for Anti-cancer Therapy with In Silico Workflows

2017 ◽  
pp. 221-236 ◽  
Author(s):  
Alexander Goncearenco ◽  
Minghui Li ◽  
Franco L. Simonetti ◽  
Benjamin A. Shoemaker ◽  
Anna R. Panchenko
Keyword(s):  
Cancer Therapy ◽  
Protein Interactions ◽  
In Silico ◽  
Drug Targets ◽  
Protein Protein Interactions ◽  
Anti Cancer

NMR for the design of functional mimetics of protein-protein interactions: one key is in the building of bridges

1998 ◽  
Vol 76 (2-3) ◽  
pp. 177-188 ◽  
Author(s):  
Jianxing Song ◽  
Feng Ni
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
Experimental Approach ◽  
Structural Information ◽  
Peptide Ligands ◽  
Protein Protein Interactions ◽  
Binding Residues ◽  
Related Proteins ◽  
Binding Inhibitors

Using the design of bivalent and bridge-binding inhibitors of thrombin as an example, we review an NMR-based experimental approach for the design of functional mimetics of protein-protein interactions. The strategy includes: (i) identification of binding residues in peptide ligands by differential resonance perturbation, (ii) determination of protein-bound structures of peptide ligands by use of transferred NOEs, (iii) minimization of larger protein and peptide ligands on the basis of NMR structural information, and (iv) linkage of two weakly binding mimetics to produce an inhibitor with enhanced affinity and specificity. This approach can be especially effective for the design of potent and selective functional mimetics of protein-protein interactions because it is less likely that the surfaces of two related proteins or enzymes share two identical binding sites or regions.Key words: NMR, protein-protein interactions, functional mimetics, bridge-binding inhibitors, thrombin.

scholarly journals Elucidating the druggable interface of protein−protein interactions using fragment docking and coevolutionary analysis

2016 ◽  
Vol 113 (50) ◽  
pp. E8051-E8058 ◽  
Author(s):  
Fang Bai ◽  
Faruck Morcos ◽  
Ryan R. Cheng ◽  
Hualiang Jiang ◽  
José N. Onuchic
Keyword(s):  
Drug Design ◽  
Protein Interactions ◽  
Binding Sites ◽  
Hot Spots ◽  
Therapeutic Agents ◽  
Molecular Probes ◽  
Therapeutic Drug ◽  
Protein Protein Interactions ◽  
Protein Surface ◽  
Fragment Docking

Protein−protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein−protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

scholarly journals Detection of Specific Protein–Protein Interactions in Nanocages by Engineering Bipartite FlAsH Binding Sites

2013 ◽  
Vol 135 (44) ◽  
pp. 16618-16624 ◽  
Author(s):  
Thomas A. Cornell ◽  
Jing Fu ◽  
Stephanie H. Newland ◽  
Brendan P. Orner
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
Specific Protein ◽  
Protein Protein Interactions

scholarly journals Hidden partners: Using cross-docking calculations to predict binding sites for proteins with multiple interactions

2018 ◽  
Author(s):  
Nathalie Lagarde ◽  
Alessandra Carbone ◽  
Sophie Sacquin-Mora
Keyword(s):  
Nucleic Acid ◽  
Binding Site ◽  
Protein Interactions ◽  
Binding Sites ◽  
Protein Protein Interactions ◽  
Protein Binding Sites ◽  
Cross Docking ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Docking Calculations

AbstractProtein-protein interactions control a large range of biological processes and their identification is essential to understand the underlying biological mechanisms. To complement experimental approaches, in silico methods are available to investigate protein-protein interactions. Cross-docking methods, in particular, can be used to predict protein binding sites. However, proteins can interact with numerous partners and can present multiple binding sites on their surface, which may alter the binding site prediction quality. We evaluate the binding site predictions obtained using complete cross-docking simulations of 358 proteins with two different scoring schemes accounting for multiple binding sites. Despite overall good binding site prediction performances, 68 cases were still associated with very low prediction quality, presenting individual area under the specificity-sensitivity ROC curve (AUC) values below the random AUC threshold of 0.5, since cross-docking calculations can lead to the identification of alternate protein binding sites (that are different from the reference experimental sites). For the large majority of these proteins, we show that the predicted alternate binding sites correspond to interaction sites with hidden partners, i.e. partners not included in the original cross-docking dataset. Among those new partners, we find proteins, but also nucleic acid molecules. Finally, for proteins with multiple binding sites on their surface, we investigated the structural determinants associated with the binding sites the most targeted by the docking partners.AbbreviationsANOVA: ANalysis Of Variance; AUC: Area Under the Curve; Best Interface: BI; CAPRI: Critical Assessment of Prediction of Interactions; CC-D: Complete Cross-Docking; DNA: DesoxyriboNucleic Acid; FDR: False Discovery Rate; FRIres(type): Fraction of each Residue type in the Interface; FP: False Positives; GI: Global Interface; HCMD: Help Cure Muscular Dystrophy; JET: Joint Evolutionary Tree; MAXDo: Molecular Association via Cross Docking; NAI: Nucleic Acid Interface; NPV: Negative Predicted Value; PDB: Protein Data Bank; PIP: Protein Interface Propensity; PiQSi: Protein Quaternary Structure investigation; PPIs: Protein-Protein Interactions; PPV: Positive Predicted Value; Prec.: Precision; PrimI: Primary Interface; RNA: RiboNucleic Acid; ROC: Receiver Operating Characteristic; SecI: Secondary Interface; Sen.: Sensitivity; Spe.: Specificity; TN: True Negatives; TP: True Positives; WCG: World Community Grid.

scholarly journals Elucidation of protein-protein interactions necessary for maintenance of the BCR-ABL signaling complex

2019 ◽  
Author(s):  
Tomas Gregor ◽  
Michaela Kunova Bosakova ◽  
Alexandru Nita ◽  
Sara P. Abraham ◽  
Bohumil Fafilek ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Protein Interactions ◽  
Kinase Inhibitors ◽  
Pleckstrin Homology Domain ◽  
Sequence Motif ◽  
Protein Protein Interactions ◽  
Core Complex ◽  
Knock Out ◽  
Signaling Complex ◽  
Intrinsically Disordered

AbstractApproximately 50% of chronic myeloid leukemia (CML) patients in deep remission experience a return of clinical CML after withdrawal of tyrosine kinase inhibitors (TKIs). This suggests signaling of inactive BCR-ABL, which allows for survival of cancer cells, leading to relapse. Understanding the dynamics of BCR-ABL signaling complex holds a key to the mechanism of BCR-ABL signaling. Here, we demonstrate that TKIs inhibit catalytic activity of BCR-ABL, but do not dissolve the BCR-ABL core signaling complex consisting of CrkL, SHC1, Grb2, SOS1, cCbl, and SHIP2. We show that CrkL binds to proline-rich regions located in C-terminal, intrinsically disordered region of BCR-ABL, that deletion of pleckstrin homology domain of BCR-ABL diminishes interaction with SHC1, and that BCR-ABL sequence motif located in disordered region around phosphorylated tyrosine 177 mediates binding of at least three core complex members, the Grb2, SOS1 and cCbl. Introduction of Y177F substitution blocks association with Grb2, SOS1 and cCbl. Further, we identified SHIP2 binding sites within the src-homology and tyrosine kinase domains of BCR-ABL. We found that BCR-ABL is unable to phosphorylate SHC1 in cells lacking SHIP2. Reintroducing SHIP2 into Ship2 knock-out cells restored SHC1 phosphorylation, which depended on inositol phosphatase activity of SHIP2. Our findings provide characterization of protein-protein interactions in the BCR-ABL signaling complex, and support the concept of targeting BCR-ABL signaling in CML by inhibition of its interactions with the members of the core complex.

scholarly journals Small-molecule binding sites to explore protein–protein interactions in the cancer proteome

2016 ◽  
Vol 12 (10) ◽  
pp. 3067-3087 ◽  
Author(s):  
David Xu ◽  
Shadia I. Jalal ◽  
George W. Sledge ◽  
Samy O. Meroueh
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Binding Sites ◽  
Cancer Genome ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Protein Protein Interactions ◽  
Poor Survival ◽  
Cancer Genome Atlas ◽  
Genome Atlas

The Cancer Genome Atlas (TCGA) offers an unprecedented opportunity to identify small-molecule binding sites on proteins with overexpressed mRNA levels that correlate with poor survival.

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