Solid-state nanopore analysis on conformation change of p53TAD–MDM2 fusion protein induced by protein–protein interaction

Nanoscale ◽  
2018 ◽  
Vol 10 (36) ◽  
pp. 17227-17235 ◽  
Author(s):  
Hongsik Chae ◽  
Dong-Kyu Kwak ◽  
Mi-Kyung Lee ◽  
Seung-Wook Chi ◽  
Ki-Bum Kim
Keyword(s):  
Solid State ◽  
Fusion Protein ◽  
Single Molecule ◽  
Protein Interaction ◽  
Conformation Change ◽  
Protein Protein Interaction ◽  
Translocation Event

Nutlin-3-induced conformation change of p53TAD–MDM2 fusion protein were probed by single molecule translocation event using solid-state nanopores.

scholarly journals Correction: Solid-state nanopore analysis on conformation change of p53TAD–MDM2 fusion protein induced by protein–protein interaction

Nanoscale ◽  
2018 ◽  
Vol 10 (38) ◽  
pp. 18423-18423
Author(s):  
Hongsik Chae ◽  
Dong-Kyu Kwak ◽  
Mi-Kyung Lee ◽  
Seung-Wook Chi ◽  
Ki-Bum Kim
Keyword(s):  
Solid State ◽  
Fusion Protein ◽  
Protein Interaction ◽  
Conformation Change ◽  
Protein Protein Interaction

Correction for ‘Solid-state nanopore analysis on conformation change of p53TAD–MDM2 fusion protein induced by protein–protein interaction’ by Hongsik Chae et al., Nanoscale, 2018, DOI: 10.1039/c8nr06423g.

Probing the Small-Molecule Inhibition of an Anticancer Therapeutic Protein-Protein Interaction Using a Solid-State Nanopore

2016 ◽  
Vol 128 (19) ◽  
pp. 5807-5811 ◽  
Author(s):  
Dong-Kyu Kwak ◽  
Hongsik Chae ◽  
Mi-Kyung Lee ◽  
Ji-Hyang Ha ◽  
Gaurav Goyal ◽  
...  
Keyword(s):  
Solid State ◽  
Small Molecule ◽  
Protein Interaction ◽  
Therapeutic Protein ◽  
Protein Protein Interaction ◽  
Small Molecule Inhibition

Microfluidic device for coupling isotachophoretic sample focusing with nanopore single-molecule sensing

Nanoscale ◽  
2020 ◽  
Vol 12 (34) ◽  
pp. 17805-17811
Author(s):  
Joshua D. Spitzberg ◽  
Xander F. van Kooten ◽  
Moran Bercovici ◽  
Amit Meller
Keyword(s):  
Solid State ◽  
Microfluidic Device ◽  
Single Molecule ◽  
Event Rate ◽  
Translocation Event

Electrokinetic focusing of analytes to a solid-state nanopore enhances the translocation event rate, enabling rapid nanopore sensing at low analyte concentrations.

Development of Small Molecule Inhibitors of the AML1-ETO and CBFβ-SMMHC Oncoproteins.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1591-1591
Author(s):  
Jolanta E. Grembecka ◽  
Kristin Graf ◽  
Yali Kong ◽  
Michael Douvas ◽  
Tomasz Cierpicki ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Cell Line ◽  
Small Molecule ◽  
Protein Interaction ◽  
Small Molecule Inhibitors ◽  
Leukemia Cell Line ◽  
Runt Domain ◽  
Protein Protein Interaction ◽  
Lead Compounds ◽  
Binding Interface

Abstract Core binding factor (CBF) is a heterodimeric transcription factor composed of RUNX1 (CBFα) and CBFβ subunits which are essential for normal blood cell development. CBFβ functions to increase the DNA-binding of the RUNX1 subunit 20–40 fold and to protect the RUNX1 subunit against ubiqitination and proteasome degradation, making this protein-protein interaction critical for CBF function. Two of the most common translocations involving the subunits of CBF are the inv(16) and the t(8;21) which produce the chimeric proteins CBFβ-SMMHC and AML1-ETO, respectively, which are associated with the development of Acute Myeloid Leukemia (AML). The AML1-ETO fusion protein is a dominant inhibitor of wildtype RUNX1-CBFβ activity in vivo and causes a blockage in normal hematopoiesis, predisposing for the development of leukemia. The interaction between CBFβ and AML1-ETO is critical for its function, therefore treatments targeting AML1-ETO and blocking its interaction with CBFβ are highly likely to be therapeutically beneficial. The CBFβ-SMMHC fusion protein causes dysregulation of CBF function by means of anomalously tight binding to RUNX1. Since binding to RUNX1 is required for the dysfunction associated with CBFβ-SMMHC, this interaction represents an excellent target for inhibition as a potential therapeutic strategy. We have initiated efforts to develop small molecule inhibitors of the RUNX1-CBFβ interaction as possible therapeutics for the treatment of the associated leukemias. Both virtual screening searches, focused on the X-ray structures of RUNX1 Runt domain and CBFβ, and high-throughput screening of NCI (National Cancer Institute) and Maybridge fragment libraries were used to identify initial lead compounds interacting with these proteins and blocking heterodimerization of CBF. Compounds were tested experimentally by FRET (Fluorescence Resonance Energy Transfer) and ELISA for their inhibition of RUNX1-CBFβ interaction. This resulted in a number of initial lead compounds targeting either the Runt domain or CBFβ and inhibiting this protein-protein interaction. Based on the docking mode selected lead compounds were further optimized using medicinal chemistry approaches to increase their affinity and determine the structure-activity relationships (SAR). This resulted in several compounds with low micromolar affinity (IC50 < 10 μM) which effectively block the heterodimerization of CBF in vitro and in a cell-based assay. Interestingly, compounds targeting CBFβ bind to a site displaced from the binding interface for RUNX1 as shown by the NMR-based docking, i.e. these compounds function as allosteric inhibitors of this protein-protein interaction. The most potent compounds were tested either in the Kasumi-1 leukemia cell line harboring t(8;21) translocation or in the ME-1 cell line with inv(16), resulting in a blockage of proliferation, induction of apoptosis and differentiation of these cells. These compounds represent the first small molecule inhibitors targeting CBF and inhibiting this interaction. They represent good starting points for the development of therapeutically useful inhibitors. Several approaches are being explored to modify these compounds to achieve selectivity towards AML1-ETO or CBFβ-SMMHC oncoproteins versus wild type proteins.

scholarly journals Single-molecule dynamics and genome-wide transcriptomics reveal that NF-κB (p65)-DNA binding times can be decoupled from transcriptional activation

2018 ◽  
Author(s):  
Andrea Callegari ◽  
Christian Sieben ◽  
Alexander Benke ◽  
David M. Suter ◽  
Beat Fierz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Single Molecule ◽  
Protein Interaction ◽  
Transcriptional Activation ◽  
Dna Binding Domain ◽  
Protein Protein Interaction ◽  
Genome Wide ◽  
Binding Time ◽  
Transcriptional Output

AbstractTranscription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an “enhanceosome,” nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.Author SummaryTo control transcription of a certain gene or a group of genes, both eukaryotes and prokaryotes express specialized proteins, transcription factors (TFs). During gene activation, TFs bind gene promotor sequences to recruit the transcriptional machinery including DNA polymerase II. TFs are often multi-subunit proteins containing a DNA-binding domain (DBD) as well as a protein-protein interaction interface. It was suggested that the duration of a TF-DNA binding event 1) depends on these two subunits and 2) dictates the outcome, i.e. the amount of mRNA produced from an activated gene. We set out to address these hypotheses using the transcriptional activator NF-κB (p65) as well as a number of mutants affecting different functional subunits. Using a combination of live-cell microscopy and RNA sequencing, we show that p65 DNA-binding time indeed correlates with the transcriptional output, but that this relationship depends on, and hence can be uncoupled by altering, the protein-protein interaction capacity. Our results suggest that, while p65 DNA binding times are dominated by the DBD, a transcriptional output can only be achieved with a functional protein-protein interaction subunit.

scholarly journals Split-HaloTag® Imaging Assay for Sophisticated Microscopy of Protein-Protein Interactions in planta

2020 ◽  
Author(s):  
Rieke Minner-Meinen ◽  
Jan-Niklas Weber ◽  
Andreas Albrecht ◽  
Rainer Matis ◽  
Maria Behnecke ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interaction ◽  
Fluorescence Intensity ◽  
Protein Interactions ◽  
Specific Interaction ◽  
Plant Cells ◽  
In Planta ◽  
Protein Protein Interactions ◽  
Filamentous Actin ◽  
Protein Protein Interaction

AbstractAn ever-increasing number of protein complexes participating in metabolic pathways and of multi-protein intracellular networks have been identified in plant cells. Split-GFP based protein-protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualisation of protein complex localisation. However, fluorescence proteins entail several drawbacks for sophisticated microscopy: high photobleaching rate during long-term observations, correlation of fluorescence intensity to expression level or blinking behaviour. With the HaloTag® system these drawbacks can be overcome asthis reporter is able to form covalent irreversible bonds with several synthetic photostable fluorescence ligands,which can be used in variable concentrations for optimal fluorescence intensity. Therefore, we established the new Split-HaloTag® imaging assay to enable sophisticated fluorescence imaging of protein-protein interactions. This is demonstrated using an well-characterised interaction as an example for protein-protein interaction at cellular structures: the molybdenum cofactor biosynthesis complex anchoring to filamentous actin. Additionally, a specific interaction was visualised with advanced subdiffractional polarisation microscopy in a more distinctive manner as example for sophisticated imaging. Therefore, this assay is a promising new tool for continuative 3D-imaging, single molecule tracking and super-resolution microscopy of protein-protein interactions in plant cells.

scholarly journals Single-molecule analysis of interaction between p53TAD and MDM2 using aerolysin nanopores

2021 ◽  
Vol 12 (16) ◽  
pp. 5883-5891
Author(s):  
Sohee Oh ◽  
Mi-Kyung Lee ◽  
Seung-Wook Chi
Keyword(s):  
Small Molecule ◽  
Single Molecule ◽  
Protein Interaction ◽  
Protein Protein Interaction ◽  
Single Molecule Analysis

Using aerolysin nanopores, we probed protein–protein interaction (PPI) between p53TAD and MDM2 and its modulation by small-molecule PPI inhibitors and p53TAD phosphorylation.

Visualization of a protein-protein interaction at a single-molecule level by atomic force microscopy

2013 ◽  
Vol 406 (5) ◽  
pp. 1411-1421 ◽  
Author(s):  
Klaus Bonazza ◽  
Hanspeter Rottensteiner ◽  
Birgit K. Seyfried ◽  
Gerald Schrenk ◽  
Günter Allmaier ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Protein Interaction ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Protein Protein Interaction ◽  
Atomic Force
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