scholarly journals Systematic identification of recognition motifs for the hub protein LC8

2019 ◽  
Vol 2 (4) ◽  
pp. e201900366 ◽  
Author(s):  
Nathan Jespersen ◽  
Aidan Estelle ◽  
Nathan Waugh ◽  
Norman E Davey ◽  
Cecilia Blikstad ◽  
...  
Keyword(s):  
Phage Display Library ◽  
Titration Calorimetry ◽  
Cellular Regulation ◽  
Dynamic Binding ◽  
Binding Motifs ◽  
Dimer Interface ◽  
Binding Partners ◽  
Critical Residues ◽  
Recognition Motifs ◽  
Systematic Identification

Hub proteins participate in cellular regulation by dynamic binding of multiple proteins within interaction networks. The hub protein LC8 reversibly interacts with more than 100 partners through a flexible pocket at its dimer interface. To explore the diversity of the LC8 partner pool, we screened for LC8 binding partners using a proteomic phage display library composed of peptides from the human proteome, which had no bias toward a known LC8 motif. Of the identified hits, we validated binding of 29 peptides using isothermal titration calorimetry. Of the 29 peptides, 19 were entirely novel, and all had the canonical TQT motif anchor. A striking observation is that numerous peptides containing the TQT anchor do not bind LC8, indicating that residues outside of the anchor facilitate LC8 interactions. Using both LC8-binding and nonbinding peptides containing the motif anchor, we developed the “LC8Pred” algorithm that identifies critical residues flanking the anchor and parses random sequences to predict LC8-binding motifs with ∼78% accuracy. Our findings significantly expand the scope of the LC8 hub interactome.

scholarly journals Towards the sequence-specific multivalent molecular recognition of cyclodextrin oligomers

2014 ◽  
Vol 10 ◽  
pp. 2428-2440 ◽  
Author(s):  
Michael Kurlemann ◽  
Bart Jan Ravoo
Keyword(s):  
Molecular Recognition ◽  
Titration Calorimetry ◽  
Biological Processes ◽  
Sequence Specificity ◽  
New Materials ◽  
Guest Molecules ◽  
Binding Motifs ◽  
Specific Recognition ◽  
Recognition Motifs ◽  
Complementary Binding

Sequence-specific multivalent molecular recognition has been recognized to play a major role in biological processes. Furthermore, sequence-specific recognition motifs have been used in various artificial systems in the last years, e.g., to emulate biological processes or to build up new materials with highly specific recognition domains. In this article, we present the preparation of cyclodextrin (CD)-based strands and complementary and non-complementary strands modified with guest molecules and the investigation of their complexation behavior towards each other by isothermal titration calorimetry (ITC). As complementary binding motifs n-butyl and α-CD and adamantane and β-CD were selected. It was found that it is possible to realize sequence-specific molecular recognition by the use of host–guest chemistry, but the recognition motifs as well as the linkages have to be chosen very carefully. In the case of trivalent systems one adamantane moiety must be included to induce preferred formation of 1:1 adducts. Due to the too weak interaction between n-butyl and α-CD these systems have a negative chelate cooperativity and open adducts are preferentially formed. As soon as two adamantane moieties are present, the complementary systems have a positive chelate cooperativity and double-stranded structures are favored over open adducts. In this system the n-butyl moiety provides insufficient discrimination towards α- and β-CD and no sequence specificity is observed. By the combination of three adamantane moieties sequence specificity can be generated. Exclusively with the complementary CD sequence double-stranded structures are formed, with non-complementary strands aggregates of higher stoichiometry are generated.

scholarly journals Direct role for the Drosophila GIGYF protein in 4EHP-mediated mRNA repression

2019 ◽  
Vol 47 (13) ◽  
pp. 7035-7048 ◽  
Author(s):  
Vincenzo Ruscica ◽  
Praveen Bawankar ◽  
Daniel Peter ◽  
Sigrun Helms ◽  
Cátia Igreja ◽  
...  
Keyword(s):  
Effector Proteins ◽  
Translational Repression ◽  
Direct Role ◽  
Binding Motifs ◽  
Translational Repressor ◽  
Homologous Protein ◽  
Mrna Targets ◽  
Binding Partners ◽  
Specific Mrnas

Abstract The eIF4E-homologous protein (4EHP) is a translational repressor that competes with eIF4E for binding to the 5′-cap structure of specific mRNAs, to which it is recruited by protein factors such as the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins (GIGYF). Several experimental evidences suggest that GIGYF proteins are not merely facilitating 4EHP recruitment to transcripts but are actually required for the repressor activity of the complex. However, the underlying molecular mechanism is unknown. Here, we investigated the role of the uncharacterized Drosophila melanogaster (Dm) GIGYF protein in post-transcriptional mRNA regulation. We show that, when in complex with 4EHP, Dm GIGYF not only elicits translational repression but also promotes target mRNA decay via the recruitment of additional effector proteins. We identified the RNA helicase Me31B/DDX6, the decapping activator HPat and the CCR4–NOT deadenylase complex as binding partners of GIGYF proteins. Recruitment of Me31B and HPat via discrete binding motifs conserved among metazoan GIGYF proteins is required for downregulation of mRNA expression by the 4EHP–GIGYF complex. Our findings are consistent with a model in which GIGYF proteins additionally recruit decapping and deadenylation complexes to 4EHP-containing RNPs to induce translational repression and degradation of mRNA targets.

scholarly journals Binding partner- and force-promoted changes in αE-catenin conformation probed by native cysteine labeling

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ksenia Terekhova ◽  
Sabine Pokutta ◽  
Yee S. Kee ◽  
Jing Li ◽  
Emad Tajkhorshid ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Binding Protein ◽  
Binding Domain ◽  
Actin Binding ◽  
Titration Calorimetry ◽  
Allosteric Coupling ◽  
Actin Binding Protein ◽  
Binding Partners ◽  
Actin Binding Domain ◽  
Cysteine Labeling

Abstract Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and the F-actin binding protein αE-catenin. When subjected to mechanical force, the cadherin•catenin complex can tightly link to F-actin through αE-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in αE-catenin upon binding to the E-cadherin•β-catenin complex, vinculin and F-actin. A method to apply physiologically meaningful forces in solution revealed force-induced conformational changes in αE-catenin when bound to F-actin. Comparisons of wild-type αE-catenin and a mutant with enhanced vinculin affinity using cysteine labeling and isothermal titration calorimetry provide evidence for allosteric coupling of the N-terminal β-catenin-binding and the middle (M) vinculin-binding domain of αE-catenin. Cysteine labeling also revealed possible crosstalk between the actin-binding domain and the rest of the protein. The data provide insight into how binding partners and mechanical stress can regulate the conformation of full-length αE-catenin, and identify the M domain as a key transmitter of conformational changes.

scholarly journals Investigation of Phospholipase Cγ1 Interaction with SLP76 Using Molecular Modeling Methods for Identifying Novel Inhibitors

2019 ◽  
Vol 20 (19) ◽  
pp. 4721 ◽  
Author(s):  
Tripathi ◽  
Vetrivel ◽  
Téletchéa ◽  
Jean ◽  
Legembre ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Lupus Erythematosus ◽  
Md Simulations ◽  
Binding Energies ◽  
Binding Partners ◽  
Phospholipase Cγ1 ◽  
Biophysical Analysis ◽  
Critical Residues ◽  
The Common ◽  
First Time

The enzyme phospholipase C gamma 1 (PLCγ1) has been identified as a potential drug target of interest for various pathological conditions such as immune disorders, systemic lupus erythematosus, and cancers. Targeting its SH3 domain has been recognized as an efficient pharmacological approach for drug discovery against PLCγ1. Therefore, for the first time, a combination of various biophysical methods has been employed to shed light on the atomistic interactions between PLCγ1 and its known binding partners. Indeed, molecular modeling of PLCγ1 with SLP76 peptide and with previously reported inhibitors (ritonavir, anethole, daunorubicin, diflunisal, and rosiglitazone) facilitated the identification of the common critical residues (Gln805, Arg806, Asp808, Glu809, Asp825, Gly827, and Trp828) as well as the quantification of their interaction through binding energies calculations. These features are in agreement with previous experimental data. Such an in depth biophysical analysis of each complex provides an opportunity to identify new inhibitors through pharmacophore mapping, molecular docking and MD simulations. From such a systematic procedure, a total of seven compounds emerged as promising inhibitors, all characterized by a strong binding with PLCγ1 and a comparable or higher binding affinity to ritonavir (∆Gbind < −25 kcal/mol), one of the most potent inhibitor reported till now.

scholarly journals Interaction Maps of the Saccharomyces cerevisiae ESCRT-III Protein Snf7

2013 ◽  
Vol 12 (11) ◽  
pp. 1538-1546 ◽  
Author(s):  
Barbara Sciskala ◽  
Ralf Kölling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Interaction Network ◽  
Multivesicular Body ◽  
Binding Motifs ◽  
Content Type ◽  
Endosomal Membrane ◽  
Binding Partners ◽  
First Time ◽  
Complementary Binding

ABSTRACT The Saccharomyces cerevisiae ESCRT-III protein Snf7 is part of an intricate interaction network at the endosomal membrane. Interaction maps of Snf7 were established by measuring the degree of binding of individual binding partners to putative binding motifs along the Snf7 sequence by glutathione S -transferase (GST) pulldown. For each interaction partner, distinct binding profiles were obtained. The following observations were made. The ESCRT-III subunits Vps20 and Vps24 showed a complementary binding pattern, suggesting a model for the series of events in the ESCRT-III functional cycle. Vps4 bound to individual Snf7 motifs but not to full-length Snf7. This suggests that Vps4 does not bind to the closed conformation of Snf7. We also demonstrate for the first time that the ALIX/Bro1 homologue Rim20 binds to the α6 helix of Snf7. Analysis of a Snf7 α6 deletion mutant showed that the α6 helix is crucial for binding of Bro1 and Rim20 in vivo and is indispensable for the multivesicular body (MVB)-sorting and Rim-signaling functions of Snf7. The Snf7Δα6 protein still appeared to be incorporated into ESCRT-III complexes at the endosomal membrane, but disassembly of the complex seemed to be defective. In summary, our study argues against the view that the ESCRT cycle is governed by single one-to-one interactions between individual components and emphasizes the network character of the ESCRT interactions.

scholarly journals Analysis of peptide‐binding motifs for two disease associated HLA‐DR13 alleles using an M13 phage display library

Immunology ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 482-486 ◽  
Author(s):  
M. P. DAVENPORT ◽  
C. L. QUINN ◽  
P. VALSASNINI ◽  
F. SINIGAGLIA ◽  
A. V. S. HILL ◽  
...  
Keyword(s):  
Phage Display ◽  
Peptide Binding ◽  
Phage Display Library ◽  
Binding Motifs ◽  
M13 Phage

scholarly journals SIOMICS: a novel approach for systematic identification of motifs in ChIP-seq data

2013 ◽  
Vol 42 (5) ◽  
pp. e35-e35 ◽  
Author(s):  
Jun Ding ◽  
Haiyan Hu ◽  
Xiaoman Li
Keyword(s):  
Motif Discovery ◽  
De Novo ◽  
Data Sets ◽  
Random Data ◽  
Data Set ◽  
Binding Motifs ◽  
Gene Transcriptional Regulation ◽  
Novel Approach ◽  
De Novo Motif Discovery ◽  
Systematic Identification

Abstract The identification of transcription factor binding motifs is important for the study of gene transcriptional regulation. The chromatin immunoprecipitation (ChIP), followed by massive parallel sequencing (ChIP-seq) experiments, provides an unprecedented opportunity to discover binding motifs. Computational methods have been developed to identify motifs from ChIP-seq data, while at the same time encountering several problems. For example, existing methods are often not scalable to the large number of sequences obtained from ChIP-seq peak regions. Some methods heavily rely on well-annotated motifs even though the number of known motifs is limited. To simplify the problem, de novo motif discovery methods often neglect underrepresented motifs in ChIP-seq peak regions. To address these issues, we developed a novel approach called SIOMICS to de novo discover motifs from ChIP-seq data. Tested on 13 ChIP-seq data sets, SIOMICS identified motifs of many known and new cofactors. Tested on 13 simulated random data sets, SIOMICS discovered no motif in any data set. Compared with two recently developed methods for motif discovery, SIOMICS shows advantages in terms of speed, the number of known cofactor motifs predicted in experimental data sets and the number of false motifs predicted in random data sets. The SIOMICS software is freely available at http://eecs.ucf.edu/∼xiaoman/SIOMICS/SIOMICS.html.

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