scholarly journals Photocatalytic Proximity Labelling of MCL-1 by a BH3 Ligand

2019 ◽  
Author(s):  
Hester Beard ◽  
Rachel George ◽  
Andrew Wilson ◽  
Robin Bon
Keyword(s):  
Visible Light ◽  
Dynamic Analysis ◽  
Protein Interaction ◽  
Affinity Purification ◽  
Cysteine Residue ◽  
Tandem Ms ◽  
Dependent Manner ◽  
Protein Protein Interaction ◽  
Apoptotic Protein ◽  
Family Protein

Ligand-directed protein labelling can be used to introduce diverse chemical functionalities onto proteins without the need for incorporation of genetically encoded tags. Here we report a method for the rapid and efficient labelling of a protein using a ruthenium-bipyridyl (Ru(II)(bpy)3) modified peptide designed to mimic an interacting BH3 ligand within a BCL-2 family protein-protein interaction (PPI). Using sub-stoichiometric quantities of (Ru(II)(bpy)3)-modified NOXA-B and irradiation with visible light for 1 minute, the anti-apoptotic protein MCL-1 was photolabelled in a ligand-dependent manner with a variety of functional tags, as determined by in-gel fluorescence, affinity purification, and ESIMS analysis. In contrast with previous reports on Ru(II)(bpy)3-catalysed photolabelling, tandem MS experiments revealed that the dominant labelling occurred on a cysteine residue of MCL-1. Labelling of MCL-1 occurred selectively in mixtures with other proteins, including the structurally related BCL-2 member, BCL-xL. These results improve methodology for proximity-induced photolabelling of proteins, demonstrate the approach is applicable to interfaces that mediate PPIs, and pave the way towards future use of ligand-directed proximity labelling for dynamic analysis of the localisation and interactome of BCL-2 family proteins.<br>

scholarly journals Photocatalytic Proximity Labelling of MCL-1 by a BH3 Ligand

2019 ◽  
Author(s):  
Hester Beard ◽  
Rachel George ◽  
Andrew Wilson ◽  
Robin Bon
Keyword(s):  
Visible Light ◽  
Dynamic Analysis ◽  
Protein Interaction ◽  
Affinity Purification ◽  
Cysteine Residue ◽  
Tandem Ms ◽  
Dependent Manner ◽  
Protein Protein Interaction ◽  
Apoptotic Protein ◽  
Family Protein

Ligand-directed protein labelling can be used to introduce diverse chemical functionalities onto proteins without the need for incorporation of genetically encoded tags. Here we report a method for the rapid and efficient labelling of a protein using a ruthenium-bipyridyl (Ru(II)(bpy)3) modified peptide designed to mimic an interacting BH3 ligand within a BCL-2 family protein-protein interaction (PPI). Using sub-stoichiometric quantities of (Ru(II)(bpy)3)-modified NOXA-B and irradiation with visible light for 1 minute, the anti-apoptotic protein MCL-1 was photolabelled in a ligand-dependent manner with a variety of functional tags, as determined by in-gel fluorescence, affinity purification, and ESIMS analysis. In contrast with previous reports on Ru(II)(bpy)3-catalysed photolabelling, tandem MS experiments revealed that the dominant labelling occurred on a cysteine residue of MCL-1. Labelling of MCL-1 occurred selectively in mixtures with other proteins, including the structurally related BCL-2 member, BCL-xL. These results improve methodology for proximity-induced photolabelling of proteins, demonstrate the approach is applicable to interfaces that mediate PPIs, and pave the way towards future use of ligand-directed proximity labelling for dynamic analysis of the localisation and interactome of BCL-2 family proteins.<br>

scholarly journals Photocatalytic proximity labelling of MCL-1 by a BH3 ligand

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Hester A. Beard ◽  
Jacob R. Hauser ◽  
Martin Walko ◽  
Rachel M. George ◽  
Andrew J. Wilson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Visible Light ◽  
Dynamic Analysis ◽  
Protein Interactions ◽  
Cysteine Residue ◽  
Tandem Mass ◽  
Protein Protein Interactions ◽  
Apoptotic Protein ◽  
Family Protein

AbstractLigand-directed protein labelling allows the introduction of diverse chemical functionalities onto proteins without the need for genetically encoded tags. Here we report a method for the rapid labelling of a protein using a ruthenium-bipyridyl (Ru(II)(bpy)3)-modified peptide designed to mimic an interacting BH3 ligand within a BCL-2 family protein-protein interactions. Using sub-stoichiometric quantities of (Ru(II)(bpy)3)-modified NOXA-B and irradiation with visible light for 1 min, the anti-apoptotic protein MCL-1 can be photolabelled with a variety of functional tags. In contrast with previous reports on Ru(II)(bpy)3-mediated photolabelling, tandem mass spectrometry experiments reveal that the labelling site is a cysteine residue of MCL-1. MCL-1 can be labelled selectively in mixtures with other proteins, including the structurally related BCL-2 member, BCL-xL. These results demonstrate that proximity-induced photolabelling is applicable to interfaces that mediate protein-protein interactions, and pave the way towards future use of ligand-directed proximity labelling for dynamic analysis of the interactome of BCL-2 family proteins.

Development of Allosteric Inhibitors of the Interaction of AML1 and CBFβ for the Treatment of Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 653-653 ◽  
Author(s):  
Michael G. Douvas ◽  
L. Roudaiya ◽  
J. Grembecka ◽  
N. Speck ◽  
J.H. Bushweller
Keyword(s):  
Small Molecule ◽  
Protein Interaction ◽  
Cellular Proliferation ◽  
Dependent Manner ◽  
Runt Domain ◽  
Cd11b Expression ◽  
Hek 293 ◽  
Protein Protein Interaction ◽  
293 Cells ◽  
Dose Dependent

Abstract The protein-protein interaction between the subunits of the nuclear transcription factor core binding factor, RUNX1 (CBFα) and CBFβ, plays a critical role in hematopoiesis. Chromosomal rearrangements that target CBF genes are among the most common mutations in leukemia, including t(8;21) found in approximately 12% of cases of AML and resulting in the protein AML1-ETO. We describe the development of small molecule inhibitors of the interaction between RUNX1 and CBFβ. We used virtual screening to identify lead compounds. These leads were used to generate a library of compounds to explore the structure-activity relationships and optimize activity, resulting in the identification of low micromolar IC50 inhibitors of this protein-protein interaction. We confirmed by FACS FRET that inhibition also occurs in mammalian cells. HEK-293 cells were transfected with Cerulean-Runt domain and Venus-CBFβ. FRET emission from cells was monitored on a flow cytometer to assess binding of CBFβ and the Runt domain. Results with drug were compared to FRET between Runt and wild-type CBFβ and FRET between Runt and a mutant CBFβ with 2 point mutations abrogating binding to Runt. The small molecule inhibitor KG-3-275 inhibited CBFβ - Runt domain binding in a dose dependent manner (Table 1). Treatment of the t(8;21) leukemia cell lines Kasumi-1 and SKNO-1 results in inhibition of proliferation. SKNO-1 cells are inhibited in a dose-dependent manner by KG-3-275 but not by the weak inhibitor KG-1-253 (Fig 1). KG-3-275 does not inhibit the growth of renal tubular and hepatocellular cell lines (Fig 1). Kasumi-1 and SKNO-1 undergo apoptosis in a dose-dependent manner upon treatment with KG-3-275 (Table 2). Finally, KG-3-275 shows synergy with ATRA in increasing differentiation of Kasumi-1 cells as measured by CD11b expression, consistent with recent published results establishing a link between AML1-ETO and repression of RAR signaling (Fig 2). These data indicate drugs inhibiting CBF interactions hold promise as targeted agents in the treatment of CBF leukemias. Fluorescence Resonance Energy Transfer Geometric Mean in HEK-293 Cells Cerulean-Runt domain + Venus-CBFb 4.07 +/− 0.1 Cerulean-RD + Venus-CBFb61/104 1.85 +/− .09 C-RD + V-CBFb + KG-3-275 200 mcm 3.05 +/− .15 C-RD + V-CBFb + KG-3-275 100 mcm 3.25 +/− 0.1 C-RD + V-CBFb + KG-3-275 50 mcm 3.45 +/− .25 C-RD + V-CBFb + KG-3-275 25 mcm 3.6 +/− 0.2 % Cells Annexin V/PI Negative at 72 Hrs Kasumi-1 0.25% DMSO 91.2 +/− 0.9 KG-3-275 25 mcm 91.2 +/− 1.2 KG-3-275 50 mcm 85.9 +/− 2.4 KG-3-275 100 mcm 66.7 +/− 6.7 SKNO-1 0.25% DMSO 77.6 +/− 3.4 KG-3-275 25 mcm 77.6 +/− 2.3 KG-3-275 50 mcm 63.7 +/− 3.6 KG-3-275 100 mcm 13.7 +/− 6.8 Cellular Proliferation as measured by MTTAssay at 72 Hrs Cellular Proliferation as measured by MTTAssay at 72 Hrs Kasumi-1 Differentiation Measured by CD11b expression at 72 Hrs Kasumi-1 Differentiation Measured by CD11b expression at 72 Hrs

Functional interference between the ubiquitous and constitutive octamer transcription factor 1 (OTF-1) and the glucocorticoid receptor by direct protein-protein interaction involving the homeo subdomain of OTF-1

1992 ◽  
Vol 12 (11) ◽  
pp. 4960-4969
Author(s):  
E Kutoh ◽  
P E Strömstedt ◽  
L Poellinger
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Protein Interaction ◽  
Binding Activity ◽  
Dependent Manner ◽  
Protein Protein Interaction ◽  
Dna Binding Activity ◽  
Functional Interference

The ubiquitous and constitutive octamer transcription factor OTF-1 (Oct 1) is the target of positive regulation by the potent herpes simplex virus trans-activator VP16, which forms a complex with the homeodomain of OTF-1. Here we present evidence that the glucocorticoid receptor can negatively regulate OTF-1 function by a mechanism that is independent of DNA binding. In vivo-expressed glucocorticoid receptor inhibited in a hormone-dependent manner activation of a minimal promoter construct carrying a functional octamer site. Moreover, expression of the receptor in vivo resulted in hormone-dependent repression of OTF-1-dependent DNA-binding activity in nuclear extract. In vitro, the DNA-binding activity of partially purified OTF-1 was repressed following incubation with purified glucocorticoid receptor. Cross-linking and immunoprecipitation experiments indicated that the functional interference may be due to a strong association between these two proteins in solution. Finally, preliminary evidence indicates that the homeo subdomain of OTF-1 that directs formation of a complex with VP16 may also be critical for interaction with the glucocorticoid receptor. Thus, OTF-1 is a target for both positive and negative regulation by protein-protein interaction. Moreover, the functional interference between OTF-1 and the glucocorticoid receptor represents a novel regulatory mechanism in the cross-coupling of signal transduction pathways of nuclear receptors and constitutive transcription factors.

scholarly journals Protein-Protein Interaction Detection: Methods and Analysis

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
V. Srinivasa Rao ◽  
K. Srinivas ◽  
G. N. Sujini ◽  
G. N. Sunand Kumar
Keyword(s):  
Protein Interaction ◽  
Protein Function ◽  
In Silico ◽  
Affinity Purification ◽  
Phylogenetic Profile ◽  
Detection Methods ◽  
Protein Protein Interaction ◽  
Interaction Detection

Protein-protein interaction plays key role in predicting the protein function of target protein and drug ability of molecules. The majority of genes and proteins realize resulting phenotype functions as a set of interactions. The in vitro and in vivo methods like affinity purification, Y2H (yeast 2 hybrid), TAP (tandem affinity purification), and so forth have their own limitations like cost, time, and so forth, and the resultant data sets are noisy and have more false positives to annotate the function of drug molecules. Thus, in silico methods which include sequence-based approaches, structure-based approaches, chromosome proximity, gene fusion, in silico 2 hybrid, phylogenetic tree, phylogenetic profile, and gene expression-based approaches were developed. Elucidation of protein interaction networks also contributes greatly to the analysis of signal transduction pathways. Recent developments have also led to the construction of networks having all the protein-protein interactions using computational methods for signaling pathways and protein complex identification in specific diseases.

scholarly journals Protein-protein-interaction Network Organization of the Hypusine Modification System

2012 ◽  
Vol 11 (11) ◽  
pp. 1289-1305 ◽  
Author(s):  
Henning Sievert ◽  
Simone Venz ◽  
Oscar Platas-Barradas ◽  
Vishnu M. Dhople ◽  
Martin Schaletzky ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Large Scale ◽  
Affinity Purification ◽  
Interaction Network ◽  
Initiation Factor ◽  
Post Translational Modification ◽  
Comprehensive Overview ◽  
Data Set ◽  
Modification System ◽  
Protein Protein Interaction

Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: “bioreactor-TAP-MS/MS.” By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions.

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