Browse
Submit Data
Databases
API
Help

Dataset Information

48 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

EndoBind detects endogenous protein-protein interactions in real time.

ABSTRACT: We present two high-throughput compatible methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer formation over an extended duration. Using examples of the Nrf2-KEAP1 and the CRAF-KRAS-G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of endogenous protein-protein interactions in real time.

SUBMITTER: Bill A

PROVIDER: S-EPMC8443649 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

EndoBind detects endogenous protein-protein interactions in real time.

Bill Anke A Espinola Sheryll S Guthy Daniel D Haling Jacob R JR Lanter Mylene M Lu Min M Marelli Anthony A Mendiola Angelica A Miraglia Loren L Taylor Brandon L BL Vargas Leonardo L Orth Anthony P AP King Frederick J FJ

Communications biology 20210915 1

We present two high-throughput compatible methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer formation over an extended duration. Using examples of the Nrf2-KEAP1 and the CRAF-KRAS-G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of endogenous protein-prot ...[more]

PMID: 34526658

Similar Datasets

Real-Time Transferrin-Based PET Detects MYC-Positive Prostate Cancer.

Project description:Noninvasive biomarkers that detect the activity of important oncogenic drivers could significantly improve cancer diagnosis and management of treatment. The goal of this study was to determine whether 68Ga-citrate (which avidly binds to circulating transferrin) can detect MYC-positive prostate cancer tumors, as the transferrin receptor is a direct MYC target gene. PET imaging paired with 68Ga-citrate and molecular analysis of preclinical models, human cell-free DNA (cfDNA), and clinical biopsies were conducted to determine whether 68Ga-citrate can detect MYC-positive prostate cancer. Importantly, 68Ga-citrate detected human prostate cancer models in a MYC-dependent fashion. In patients with castration-resistant prostate cancer, analysis of cfDNA revealed that all patients with 68Ga-citrate avid tumors had a gain of at least one MYC copy number. Moreover, biopsy of two PET avid metastases showed molecular or histologic features characteristic of MYC hyperactivity. These data demonstrate that 68Ga-citrate targets prostate cancer tumors with MYC hyperactivity. A larger prospective study is ongoing to demonstrate the specificity of 68Ga-citrate for tumors with hyperactive MYC.Implications: Noninvasive measurement of MYC activity with quantitative imaging modalities could substantially increase our understanding of the role of MYC signaling in clinical settings for which invasive techniques are challenging to implement or do not characterize the biology of all tumors in a patient. Moreover, measuring MYC activity noninvasively opens the opportunity to study changes in MYC signaling in patients under targeted therapeutic conditions thought to indirectly inhibit MYC. Mol Cancer Res; 15(9); 1221-9. ©2017 AACR.

| S-EPMC5581675 | biostudies-literature

Active metabolism unmasks functional protein-protein interactions in real time in-cell NMR.

Project description:Protein-protein interactions, PPIs, underlie most cellular processes, but many PPIs depend on a particular metabolic state that can only be observed in live, actively metabolizing cells. Real time in-cell NMR spectroscopy, RT-NMR, utilizes a bioreactor to maintain cells in an active metabolic state. Improvement in bioreactor technology maintains ATP levels at >95% for up to 24 hours, enabling protein overexpression and a previously undetected interaction between prokaryotic ubiquitin-like protein, Pup, and mycobacterial proteasomal ATPase, Mpa, to be detected. Singular value decomposition, SVD, of the NMR spectra collected over the course of Mpa overexpression easily identified the PPIs despite the large variation in background signals due to the highly active metabolome.

| S-EPMC7242440 | biostudies-literature

In-vivo real-time control of protein expression from endogenous and synthetic gene networks.

Project description:We describe an innovative experimental and computational approach to control the expression of a protein in a population of yeast cells. We designed a simple control algorithm to automatically regulate the administration of inducer molecules to the cells by comparing the actual protein expression level in the cell population with the desired expression level. We then built an automated platform based on a microfluidic device, a time-lapse microscopy apparatus, and a set of motorized syringes, all controlled by a computer. We tested the platform to force yeast cells to express a desired fixed, or time-varying, amount of a reporter protein over thousands of minutes. The computer automatically switched the type of sugar administered to the cells, its concentration and its duration, according to the control algorithm. Our approach can be used to control expression of any protein, fused to a fluorescent reporter, provided that an external molecule known to (indirectly) affect its promoter activity is available.

| S-EPMC4022480 | biostudies-literature

Capacitance-modulated transistor detects odorant binding protein chiral interactions.

Project description:Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.

| S-EPMC4309438 | biostudies-literature

Real-time measurement of protein-protein interactions at single-molecule resolution using a biological nanopore.

Project description:Protein-protein interactions (PPIs) are essential for many cellular processes. However, transient PPIs are difficult to measure at high throughput or in complex biological fluids using existing methods. We engineered a genetically encoded sensor for real-time sampling of transient PPIs at single-molecule resolution. Our sensor comprises a truncated outer membrane protein pore, a flexible tether, a protein receptor and a peptide adaptor. When a protein ligand present in solution binds to the receptor, reversible capture and release events of the receptor can be measured as current transitions between two open substates of the pore. Notably, the binding and release of the receptor by a protein ligand can be unambiguously discriminated in a complex sample containing fetal bovine serum. Our selective nanopore sensor could be applied for single-molecule protein detection, could form the basis for a nanoproteomics platform or might be adapted to build tools for protein profiling and biomarker discovery.

| S-EPMC6557705 | biostudies-literature

Real-Time Insights into Biological Events: In-Cell Processes and Protein-Ligand Interactions.

Project description:FlowNMR has the aim of continuously monitoring processes that occur in conditions that are not compatible with being carried out within a closed tube. However, it is sample intensive and not suitable for samples, such as proteins or living cells, that are often available in limited volumes and possibly low concentrations. We here propose a dialysis-based modification of a commercial flowNMR setup that allows for recycling the medium while confining the sample (proteins and cells) within the active volume of the tube. This approach is demonstrated in the specific cases of in-cell NMR and protein-based ligand studies.

| S-EPMC6350048 | biostudies-literature

A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions.

Project description:The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions. We apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, we explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. We further explore the effect of calmodulin on creatine kinase activity and show that it is increased by the interaction between the two proteins. These findings demonstrate the potential of quantitative microfluidic techniques to characterize binding equilibria between biomolecules under native solution conditions.

| S-EPMC4939750 | biostudies-literature

High-sensitivity real-time imaging of dual protein-protein interactions in living subjects using multicolor luciferases.

Project description:Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1-Smad4 and Smad2-Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects.

| S-EPMC2697115 | biostudies-literature

Real-Time In-Cell Nuclear Magnetic Resonance: Ribosome-Targeted Antibiotics Modulate Quinary Protein Interactions.

Project description:How ribosome antibiotics affect a wide range of biochemical pathways is not well understood; changes in RNA-mediated protein quinary interactions and consequent activity inside the crowded cytosol may provide one possible mechanism. We developed real-time (RT) in-cell nuclear magnetic resonance (NMR) spectroscopy to monitor temporal changes in protein quinary structure, for ≥24 h, in response to external and internal stimuli. RT in-cell NMR consists of a bioreactor containing gel-encapsulated cells inside a 5 mm NMR tube, a gravity siphon for continuous exchange of medium, and a horizontal drip irrigation system to supply nutrients to the cells during the experiment. We showed that adding antibiotics that bind to the small ribosomal subunit results in more extensive quinary interactions between thioredoxin and mRNA. The results substantiate the idea that RNA-mediated modulation of quinary protein interactions may provide the physical basis for ribosome inhibition and other regulatory pathways.

| S-EPMC5801172 | biostudies-literature

SILAC-based quantitative MS approach for real-time recording protein-mediated cell-cell interactions.

Project description:In tumor microenvironment, interactions among multiple cell types are critical for cancer progression. To understand the molecular mechanisms of these complex interplays, the secreted protein analysis between malignant cancer cells and the surrounding nonmalignant stroma is a good viewpoint to investigate cell-cell interactions. Here, we developed two stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry (MS)/MS approaches termed spike-in SILAC and triple-SILAC to quantify changes of protein secretion level in a cell co-cultured system. Within the co-culture system of CT26 and Ana-1 cells, the spike-in SILAC and triple-SILAC MS approaches are sensitive to quantitatively measure protein secretion changes. Three representative quantified proteins (Galectin-1, Cathepsin L1 and Thrombospondin-1) by two SILAC-based MS methods were further validated by Western blotting, and the coming result matched well with SILACs'. We further applied these two SILACs to human cell lines, NCM460 and HT29 co-culture system, for evaluating the feasibility, which confirmed the spike-in and triple SILAC were capable of monitoring the changed secreted proteins of human cell lines. Considering these two strategies in time consuming, sample complexity and proteome coverage, the triple-SILAC way shows more efficiency and economy for real-time recording secreted protein levels in tumor microenvironment.

| S-EPMC5981645 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data