A crosslinked and ribosylated actin trimer does not interact productively with myosin

2019 ◽  
Vol 97 (2) ◽  
pp. 140-147 ◽  
Author(s):  
Navneet Sidhu ◽  
John F. Dawson
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Binding Proteins ◽  
Binding Protein ◽  
Dnase I ◽  
Protein Protein Interactions ◽  
Adp Ribosylation ◽  
Myosin Binding ◽  
Pointed End

A purified F-actin-derived actin trimer that interacts with end-binding proteins did not activate or bind the side-binding protein myosin under rigor conditions. Remodeling of the actin trimer by the binding of gelsolin did not rescue myosin binding, nor did the use of different means of inhibiting the polymerization of the trimer. Our results demonstrate that ADP-ribosylation on all actin subunits of an F-actin-derived trimer inhibits myosin binding and that the binding of DNase-I to the pointed end subunits of a crosslinked trimer also remodels the myosin binding site. Taken together, this work highlights the need for a careful balance between modification of actin subunits and maintaining protein–protein interactions to produce a physiologically relevant short F-actin complex.

scholarly journals Androgen signaling uses a writer and a reader of ADP-ribosylation to regulate protein complex assembly

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chun-Song Yang ◽  
Kasey Jividen ◽  
Teddy Kamata ◽  
Natalia Dworak ◽  
Luke Oostdyk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Prostate Cancer Cells ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Complex Assembly ◽  
Adp Ribosylation ◽  
Signaling Axis ◽  
Regulate Protein ◽  
Protein Complex Assembly

AbstractAndrogen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity.

Identification of a C/EBP-Rel complex in avian lymphoid cells

1994 ◽  
Vol 14 (10) ◽  
pp. 6635-6646
Author(s):  
J A Diehl ◽  
M Hannink
Keyword(s):  
Gene Expression ◽  
Affinity Chromatography ◽  
Protein Interactions ◽  
Binding Proteins ◽  
Regulation Of Gene Expression ◽  
Lymphoid Cells ◽  
Cytokine Gene Expression ◽  
Protein Protein Interactions ◽  
Dna Complex ◽  
Dna Affinity Chromatography

Protein-protein interactions between the CCAAT box enhancer-binding proteins (C/EBP) and the Rel family of transcription factors have been implicated in the regulation of cytokine gene expression. We have used sequence-specific DNA affinity chromatography to purify a complex from avian T cells that binds to a consensus C/EBP motif. Our results provide evidence that Rel-related proteins are components of the C/EBP-DNA complex as a result of protein-protein interactions with the C/EBP proteins. A polyclonal antiserum raised against the Rel homology domain of v-Rel and antisera raised against two human RelA-derived peptides specifically induced a supershift of the C/EBP-DNA complex in mobility shift assays using the affinity-purified C/EBP. In addition, several kappa B-binding proteins copurified with the avian C/EBP complex through two rounds of sequence-specific DNA affinity chromatography. The kappa B-binding proteins are distinct from the C/EBP proteins that directly contact DNA containing the C/EBP binding site. The identification of a protein complex that binds specifically to a consensus C/EBP site and contains both C/EBP and Rel family members suggests a novel mechanism for regulation of gene expression by Rel family proteins.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals Bcl-2 and FKBP12 bind to IP3 and ryanodine receptors at overlapping sites: the complexity of protein–protein interactions for channel regulation

2015 ◽  
Vol 43 (3) ◽  
pp. 396-404 ◽  
Author(s):  
Tim Vervliet ◽  
Jan B. Parys ◽  
Geert Bultynck
Keyword(s):  
Protein Interactions ◽  
Hippocampal Neurons ◽  
Binding Protein ◽  
Ryanodine Receptors ◽  
B Cell Lymphoma ◽  
Experimental Models ◽  
Amino Acid Sequences ◽  
Protein Protein Interactions ◽  
Fk506 Binding Protein ◽  
Fk506 Binding Proteins

The 12- and 12.6-kDa FK506-binding proteins, FKBP12 (12-kDa FK506-binding protein) and FKBP12.6 (12.6-kDa FK506-binding protein), have been implicated in the binding to and the regulation of ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs), both tetrameric intracellular Ca2+-release channels. Whereas the amino acid sequences responsible for FKBP12 binding to RyRs are conserved in IP3Rs, FKBP12 binding to IP3Rs has been questioned and could not be observed in various experimental models. Nevertheless, conservation of these residues in the different IP3R isoforms and during evolution suggested that they could harbour an important regulatory site critical for IP3R-channel function. Recently, it has become clear that in IP3Rs, this site was targeted by B-cell lymphoma 2 (Bcl-2) via its Bcl-2 homology (BH)4 domain, thereby dampening IP3R-mediated Ca2+ flux and preventing pro-apoptotic Ca2+ signalling. Furthermore, vice versa, the presence of the corresponding site in RyRs implied that Bcl-2 proteins could associate with and regulate RyR channels. Recently, the existence of endogenous RyR–Bcl-2 complexes has been identified in primary hippocampal neurons. Like for IP3Rs, binding of Bcl-2 to RyRs also involved its BH4 domain and suppressed RyR-mediated Ca2+ release. We therefore propose that the originally identified FKBP12-binding site in IP3Rs is a region critical for controlling IP3R-mediated Ca2+ flux by recruiting Bcl-2 rather than FKBP12. Although we hypothesize that anti-apoptotic Bcl-2 proteins, but not FKBP12, are the main physiological inhibitors of IP3Rs, we cannot exclude that Bcl-2 could help engaging FKBP12 (or other FKBP isoforms) to the IP3R, potentially via calcineurin.

scholarly journals Use of periplasmic target protein capture for phage display engineering of tight-binding protein–protein interactions

2011 ◽  
Vol 24 (11) ◽  
pp. 819-828 ◽  
Author(s):  
Bartlomiej G. Fryszczyn ◽  
Nicholas G. Brown ◽  
Wanzhi Huang ◽  
Miriam A. Balderas ◽  
Timothy Palzkill
Keyword(s):  
Phage Display ◽  
Protein Interactions ◽  
Binding Protein ◽  
Tight Binding ◽  
Target Protein ◽  
Protein Protein Interactions ◽  
Protein Capture

scholarly journals Mapping the binding site of thymosin β4 on actin by competition with G-actin binding proteins indicates negative co-operativity between binding sites located on opposite subdomains of actin

1997 ◽  
Vol 327 (3) ◽  
pp. 787-793 ◽  
Author(s):  
Edda BALLWEBER ◽  
Ewald HANNAPPEL ◽  
Thomas HUFF ◽  
Hans Georg MANNHERZ
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Ternary Complex ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Critical Concentration ◽  
Dnase I ◽  
Actin Binding ◽  
Thymosin Β4 ◽  
Actin Binding Proteins

The β-thymosins are small monomeric (G-)actin-binding proteins of 5 kDa that are supposed to act intracellularly as actin-sequestering factors stabilizing the cytoplasmic monomeric pool of actin. The binding region of thymosin β4 was determined by analysing the binding of thymosin β4 to actin complexed with DNase I, gelsolin or gelsolin segment 1. Binding was analysed by determining the increase in the critical concentration of actin polymerization by native gel electrophoresis or chemical cross-linking. The formation of a ternary complex including thymosin β4 should indicate that the actin-binding proteins attach to different sites on actin. Competition would be indicative of binding to identical or overlapping sites on actin or of a negative co-operative linkage between the two binding sites. Competition of thymosin β4 for actin binding was observed in the presence of intact gelsolin or the N-terminal gelsolin fragment, segment 1, indicating that thymosin β4 binds to a site close to or identical with the gelsolin segment 1-binding site. The ternary complex of actin-DNase I-thymosin β4 was obtained only when using the chemically cross-linked actin-thymosin β4 complex, indicating that thymosin β4 is dissociated by the binding of DNase I to actin. It is suggested that the dissociation of thymosin β4 by DNase I binding to actin is caused by negative co-operativity between their spatially separated binding sites on actin. A similar negative co-operativity was observed between DNase I and gelsolin segment 1 binding to actin. The results therefore indicate that the respective binding sites for DNase I and segment 1 on subdomains 1 and 2 of actin are linked in a negative co-operative manner.

scholarly journals Binding Site Prediction for Protein-Protein Interactions and Novel Motif Discovery using Re-occurring Polypeptide Sequences

2011 ◽  
Vol 12 (1) ◽  
pp. 225 ◽  
Author(s):  
Adam Amos-Binks ◽  
Catalin Patulea ◽  
Sylvain Pitre ◽  
Andrew Schoenrock ◽  
Yuan Gui ◽  
...  
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Motif Discovery ◽  
Protein Protein Interactions ◽  
Site Prediction ◽  
Binding Site Prediction ◽  
Polypeptide Sequences

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.

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