Name: a semi-quantitative drug affinity responsive target stability (darts) assay for studying rapamycin/mtor interaction
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Description: Watch this Scientific Journal Video about A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction at JoVE.com

This work was supported partly by NIH research grants R01NS103931, R01AR062207, R01AR061484, and a DOD research grant W81XWH-16-1-0482.

1. Collect and lyse cells
<ol>
	<li>Grow 293T cells using Dulbecco&#39;s modified Eagle medium (DMEM) with 10% fetal bovine serum, 2 mM glutamine and 1% antibiotics. Incubate cultures at 37 &#176;C under 5% CO2. 
	NOTE: The growth state of the cells may affect the stability of subsequent experiments.</li>
	<li>Expand cells in culture until reaching 80&#8210;90% confluence.</li>
	<li>Mix 345 &#956;L of cell lysis reagent (see the Table of Materials) with 25 &#956;L of 20x protease inhibitor cocktail, 25 &#956;L of 1 M sodium fluoride, 50 &#956;L of 100 mM &#946;-glycerophosphate, 50 &#956;L of 50 mM sodium pyrophosphate, and 5 &#956;L of 200 mM sodium orthovanadate. Keep the lysis buffer chilled on ice. 
	NOTE: Other lysis buffers with various detergents (e.g., Triton X-100 or NP-40) can be used with DARTS as long as they are non-denaturing. Membrane proteins or nuclear proteins can be extracted by adding 0.4% Triton X-100 or 0.4% NP-40 to the cell lysate.</li>
	<li>Wash the cells twice with cold phosphate-buffered saline (PBS).</li>
	<li>Use a cell scraper to collect the cells in an appropriate amount of cell lysis buffer and transfer the lysing cells into a 1.5 mL tube. 
	NOTE: The number of cells needed for each DARTS experiment will vary based on how much protein can be extracted from various cell lines. In general, the protein concentration of the lysate used is between 4&#8210;6 &#956;g/&#956;L. One 10 cm plate of 293T cells at 85&#8210;90% confluency, lysed with 300 &#956;L of lysis buffer typically results in a lysate with a protein concentration of ~5 &#956;g/&#956;L.</li>
	<li>Mix the lysis buffer/lysing cells well and incubate the tube on ice for 10 min.</li>
	<li>Centrifuge the tube at 18,000 &#215; g for 10 min at 4 &#176;C.</li>
	<li>Transfer the supernatant into a new 1.5 mL tube and keep chilled on ice.</li>
	<li>Perform a BCA assay to approximate the protein concentration of lysates.</li>
</ol>
2. Incubate protein lysates with the small molecule
<ol>
	<li>Split 99 &#956;L of the lysates into two 1.5 mL tubes.</li>
	<li>Make a starting stock concentration of 10 mM small molecule. When performing DARTS, one may begin with a higher concentration of the small molecule (5&#8210;10x the EC50 value) to ensure optimal binding.
	<ol>
		<li>Add either 1 &#956;L of solvent that the small molecule is soluble in or 1 &#181;L of small molecule stock solutions to each aliquot of lysate. Incubate cell lysate with the solutions for 30&#8210;60 min at room temperature with shaking.</li>
	</ol>
	</li>
	<li>On ice, establish serial dilutions (1:200, 1:400, 1:800 and 1:1600) of freshly thawed pronase solution in 1x TNC (For 1 mL of 10x TNC buffer, mix 300 &#956;L of ultrapure water with 100 &#956;L of 5 M sodium chloride, 100 &#956;L of 1 M calcium chloride, and 500 &#956;L of 1 M Tris-HCl, pH 8.0).</li>
	<li>Examine a wide-breadth of pronase:protein ratios (e.g., spanning from 1:100 to 1:2000) to guarantee observability of the step-wise effect of pronase on target(s). 
	NOTE: To calculate pronase concentrations (example): 5 &#956;g/&#956;L protein concentration x 20 &#956;L sample = 100 &#956;g protein. For a pronase:protein ratio of 1:100 we need 0.5 &#956;g/&#956;L (100 &#956;g &#247; 100 &#247; 2 &#956;L) pronase solution. This experiment may have to be repeated several times to obtain a suitable range of pronase:protein ratios.</li>
</ol>
3. Perform proteolysis
NOTE: For proteolysis, steps are carried out at room temperature unless otherwise noted
<ol>
	<li>Following incubation with the small molecule, divide each aliquot into 20 &#956;L samples.</li>
	<li>Add 2 &#956;L of the range of pronase solutions in each sample at specific intervals (every 30 s). Use an equal volume of 1x TNC buffer to establish an undigested control sample.</li>
	<li>After 5&#8210;20 min, halt digestion via addition of 2 &#956;L of cold 20x protease inhibitor cocktail every 30 s. Mix well and incubate on ice for 10 min.</li>
	<li>Dilute samples with the appropriate volume of 5x SDS-PAGE loading buffer and boil at 95 &#176;C for 5 min.</li>
	<li>Carry out the next portion of the experiment or store the protein sample at &#8722;80 &#176;C.</li>
</ol>
4. Quantification and analysis
<ol>
	<li>After DARTS, perform Coomassie blue staining according to the previously published protocol22.</li>
	<li>The stained protein bands should be very clear after destaining. Pour off the used destain solution and add fresh 1% acetic acid solution to cover the gel. Put the gel under the light to observe the bands with significant differences between groups with (sample group) or without (control group) the small molecule.</li>
	<li>Cut the two corresponding bands from the gel with a sterile instrument and perform LC-MS/MS immediately. 
	NOTE: LC-MS/MS needs to be done as soon as possible because the proteins in the gel will degrade continuously.</li>
	<li>Digest the bands, extract peptides and perform LC-MS/MS analysis23.
	<ol>
		<li>To analyze LC-MS/MS data, first identify all the proteins in the individual band. Second, use peptide spectrum match (PSM) to represent the abundance of each protein. 
		NOTE: PSM provides the total number of identified peptide sequences for the protein, including those redundantly identified. In general, how often a peptide is identified/sequenced can be used as a rough estimate of how abundant the protein is in the sample. Proteins enriched in the sample group over the control group are proteins of interest.</li>
	</ol>
	</li>
	<li>Procure the primary antibodies of the selected proteins. Perform western blot to verify that the small molecule can bind directly to the potential target proteins24.
	<ol>
		<li>Load equal amounts of protein into the wells of an 8% SDS-PAGE gel, along with an appropriate molecular weight marker. Run the gel for 30 min at 80 V, then adjust the voltage to 120 V and continue running for 1&#8210;2 h.</li>
	</ol>
	</li>
	<li>Quantify the different target protein bands using image processing and analysis software (see the Table of Materials).</li>
	<li>Analyze the data and draw the curves.
	<ol>
		<li>Determination of the proteolytic curve for a target protein
		<ol>
			<li>The pronase:protein ratio is varied when performing proteolysis. Normalize data from image analysis by attributing the band intensity values corresponding to the undigested bands to 100%.</li>
			<li>Use nonlinear regression analysis of the statistical analysis and drawing software to plot a curve of normalized data for relative band intensity and pronase:protein ratios.</li>
			<li>First, open the software, select the type of table and graph as XY and allow for 3 replicate values in side-by-side subcolumns. Then enter the corresponding data in the space below x and y. Under x, input the numbers 0, 1, 2, 3, 4, 5 (as place holders corresponding pronase:protein ratios).</li>
			<li>In the y column, enter the normalized data for relative band intensities. Perform &#8220;Nonlinear regression&#8221; and implement a &#8220;Dose-response-Stimulation&#8221; using the &#8220;Log (agonist) versus response&#8212;Variable slope (four parameters)&#8221; equation.</li>
			<li>Convert the annotations of the X-axis in the curve to the corresponding pronase:protein ratios.</li>
		</ol>
		</li>
		<li>Determination of the dose-dependence curve for a target protein 
		NOTE: For dose-dependence analysis, the molarity of the small molecule is differed across samples. The stable pronase:protein ratio should be informed by analysis of proteolysis data. The pronase:protein ratio that showed maximal difference in target protein intensity during the proteolytic curve should be used for the dose-dependence experiment.
		<ol>
			<li>Quantify the different target protein bands using an image analysis software. As in generation of the dose-dependence curve, normalize data from image analysis by attributing the band intensity values corresponding to the least digested band to 100%.</li>
			<li>Apply the nonlinear regression analysis within the statistical analysis and drawing software to the normalized data. First, open the software, select the type of table and graph as XY, and enter 3 replicate values in side-by-side subcolumns.</li>
			<li>Then, enter the corresponding data in the space below x and y. For the &#8216;x&#8217; variable, input different concentrations of the small molecule; &#8216;y&#8217; includes the corresponding normalized data for relative band intensity.</li>
			<li>Transform x values using X = Log(X). Finally, employ nonlinear regression, and implement a dose-response stimulation using the &#8220;Log (agonist) versus response&#8212;Variable slope (four parameters)&#8221; equation.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

DARTS allows for identification of small molecule targets by exploiting the protective effect of protein binding against degradation. DARTS does not require any chemical modification or immobilization of the small molecule26. This allows small molecules to be used to determine their direct binding protein targets. Standard assessment criteria for the classical DARTS method include gel staining, mass spectrometry and western blotting12,13. ...

Identifying small molecule target proteins is essential to the mechanistic understanding and development of potential therapeutic drugs1,2,3. Affinity chromatography, as a classical method for identifying the target proteins of small molecules, has yielded good results4,5. However, this method has limitations, in that chemical modification of small molecules often results in reduced or altered binding specificity or affinity. To overcome these limitations, several new strategies have recently been developed and applied to identify the small molecule targets without chemical modification of the small molecules. These direct methods for target identification of label-free small molecules include drug affinity responsive target stability (DARTS)6, stability of proteins from rates of oxidation (SPROX)7, cellular thermal shift assay (CETSA)8,9, and thermal proteome profiling (TPP)10. These methods are highly advantageous because they use natural, unmodified small molecules and rely only on direct binding interactions to find target proteins11.
Among these new methods, DARTS is a comparatively simple methodology that can easily be adopted by most labs12,13. DARTS depends on the concept that ligand-bound proteins demonstrate modified susceptibility to enzymatic degradation relative to unbound proteins. The new target protein can be detected by examination of the altered band in SDS-PAGE gel through liquid chromatography-mass spectrometry (LC-MS/MS). This approach has been successfully implemented for identification of previously unknown targets of natural products and drugs14,15,16,17,18,19. It is also powerful as a means to screen or validate binding of compounds to a specific protein20,21. In this study, we present an improvement to the experiment by monitoring the changes in protein stability with small molecules and identifying protein-ligand binding affinities. We use mTOR- rapamycin interaction as an example to demonstrate our approach.

<table><tbody><tr><td>100X Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P8340</td><td>Dilute to 20X with ultrapure water</td></tr><tr><td>293T cell line</td><td>ATCC</td><td>CRL-3216</td><td>DMEM medium with 10% FBS</td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>A6283</td><td></td></tr><tr><td>BCA Protein Assay Kit</td><td>Thermo Fisher</td><td>23225</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C1016</td><td></td></tr><tr><td>Cell scraper</td><td>Thermo Fisher</td><td>179693</td><td></td></tr><tr><td>Coomassie Brilliant Blue R-250 Staining Solution</td><td>Bio-Rad</td><td>1610436</td><td></td></tr><tr><td>Dimethyl sulfoxide(DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software</td><td>Version 6.0</td><td>statistical analysis and drawing software</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>H1758</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td>Version 1.52</td><td>image processing and analysis software</td></tr><tr><td>M-PER Cell Lysis Reagent</td><td>Thermo Fisher</td><td>78501</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Corning</td><td>R21-040-CV</td><td></td></tr><tr><td>Pronase</td><td>Roche</td><td>PRON-RO</td><td>10 mg/ml</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium fluoride</td><td>Sigma-Aldrich</td><td>S7920</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>Sigma-Aldrich</td><td>450243</td><td></td></tr><tr><td>Sodium pyrophosphate</td><td>Sigma-Aldrich</td><td>221368</td><td></td></tr><tr><td>Trizma base</td><td>Sigma-Aldrich</td><td>T1503</td><td>adjust to pH 8.0</td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma-Aldrich</td><td>G9422</td><td></td></tr></tbody></table>

a semi-quantitative drug affinity responsive target stability (darts) assay for studying rapamycin/mtor interaction

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known small molecule-protein interactions and to find potential protein targets for natural products. Compared with other methods, DARTS uses native, unmodified, small molecules and is simple and easy to operate. In this study, we further enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The protein-ligand interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used the mTOR-rapamycin interaction as an exemplary case for establishment of our protocol. From the proteolytic curve we saw that the proteolysis of mTOR by pronase was inhibited by the presence of rapamycin. The dose-dependency curve allowed us to estimate the binding affinity of rapamycin and mTOR. This method is likely to be a powerful and simple method for accurately identifying novel target proteins and for the optimization of drug target engagement.

The flow chart of the experiment is outlined in Figure 1. The result of Coomassie blue staining is shown in Figure 2. Incubation with the small molecule confers protection against proteolysis. Three bands that appear to be protected by incubation with rapamycin over vehicle control are found. The expected results from proteolytic curve experiment are shown in Figure 3. As a proof-of-principle, we examined the well-studied protein mT...

Watch this Scientific Journal Video about A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction at JoVE.com

A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction

In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known ...

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JoVE Journal

Biochemistry

16.1K Views.  New York University Medical Center. Drug affiniteit responsieve doelstabiliteit, DARTS, is een robuuste methode voor de detectie van nieuwe kleine molecuul eiwit doelen. Het kan worden gebruikt om bekende kleine molecuul eiwit interacties te verifiëren en om potentiële eiwit doelen voor natuurlijke producten te vinden. In deze studie verbeteren we de data-analysemogelijkheden van het DARTS-experiment verder door de veranderingen in eiwitstabiliteit te monitoren en de affiniteit van eiwit-ligand interacties te schatten....

Video: A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

Developmental Biology

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Bioengineering

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Genetics

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

Cancer Research

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Immunology and Infection

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Biology

Flux-Based Assay for the Identification of Autophagy Modulators for Osteoarthritis

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.

This paper details monitoring autophagy flux to identify new molecules by cell-based imaging screening.

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify ...

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. In proliferating cells, macropinocytosis can deliver extracellular nutrients to the lysosome, processed into critical macromolecule building blocks. Recent studies have highlighted the dependence of multiple cancers on macropinocytosis, including breast, colorectal and pancreatic cancer. Ras mutations are thought to be the driver events behind macropinocytosis initiation, leading to the activation of cellular anabolic processes via the mTORC1 signaling pathway. Interestingly, mTORC1 can also be activated by macropinocytosis independently of Ras. Therefore, macropinocytosis represents a metabolic vulnerability that can be leveraged to target macropinocytic tumors by limiting their access to nutrients therapeutically.
In Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM), mTORC1-hyperactivation leads to enhanced macropinocytosis and metabolic reprogramming. Here, we describe a flow cytometry-based protocol to assess macropinocytosis in mammalian cells quantitatively. TSC2-deficient MEFs are employed, which exhibit aberrant activation of mTORC1 and have been shown to have increased macropinocytosis compared to TSC2-expressing cells. Cells treated with pharmacologic inhibitors of macropinocytosis are incubated with fluorescently labeled, lysine-fixable, 70 kDa dextran, or fluorescently labeled bovine serum albumin (BSA) assayed by flow cytometry. To date, robust image-based techniques have been developed to quantitatively assess macropinocytosis in tumor cells in vitro and in vivo. This analysis provides a quantitative assessment of macropinocytosis in multiple experimental conditions and complements existing image-based techniques.

This protocol provides experimental tools to evaluate macropinocytic uptake of nutrients (carbohydrate and protein) by mTORC1-hyperactive cells. Detailed steps to quantify the uptake of fluorescently labeled dextran and bovine serum albumin (BSA) are described.

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. ...

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

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