This study was supported by the Korea Research Fellowship (KRF) program, postdoctoral fellow of the National Research Foundation of Korea (NRF), funded by the ministry of science &#38; ICT (NRF-2019H1D3A1A01102952). The authors are thankful to KIST intramural grant and Ministry of Oceans and Fisheries grant number 2MRB130 for providing financial assistance for this project.

1. Lab-scale malachite green assay
<ol>
	<li>Preparation of assay buffer
	<ol>
		<li>Prepare the assay buffer, as per the composition and preparation presented in Table 1.</li>
	</ol>
	</li>
	<li>Preparation of phosphate standards
	<ol>
		<li>Use 1 mM phosphate standard, provided in the malachite green assay phosphate assay kit (stored at 4 &#176;C).</li>
		<li>Pipette 40 &#181;L of 1 mM phosphate standard in 960 &#181;L of ultra-pure water to obtain 40 &#181;M phosphate solution (premix solution). Add the premix solution with ultra-pure water, as per manufacturer&#39;s instructions, to form a serial dilution of phosphate from 0 to 40 &#181;M.</li>
	</ol>
	</li>
	<li>Preparation of yeast Hsp90
	<ol>
		<li>Use yeast Hsp90 with a glycerol stock concentration of 0.66 mg/mL. Thaw on ice just before use.</li>
		<li>Use 8 &#181;L (5.98 &#181;g, 0.80 &#181;M) of yeast Hsp90 for measurable ATPase activity. Check that the optical density difference after the addition of malachite green reagent between the blank and positive control is at least 0.5 and less than 1.0. Here, the absorbance difference between the blank (without Hsp90) and positive control (with Hsp90) was found to be 0.510.</li>
	</ol>
	</li>
	<li>Preparation of ATP
	<ol>
		<li>Transfer 1 mg of ATP to a vial containing 453.39 &#181;L of ultra-pure water to obtain 4 mM of stock solution. Perform weight calculations based on anhydrous ATP (molecular weight [m.w.] = 551.14). Prepare ATP fresh, as it can spontaneously hydrolyze over time.</li>
		<li>Add 4 &#181;L of ATP stock solution to each well to give a final well concentration of 0.2 mM (final total assay well volume of 80 &#181;L). Add ATP as the last component to the reaction, using a multichannel pipette so that all the reactions start simultaneously.</li>
	</ol>
	</li>
	<li>Preparation of geldanamycin (GA)
	<ol>
		<li>Prepare a 10 mM stock of GA by dissolving 1 mg in 178.37 &#181;L of DMSO (m.w. = 560.64). Using the stock solution, prepare 4 mM, 0.8 mM, 0.16 mM, 0.032 mM, 0.0064 mM, and 0.00128 mM solution in DMSO by serial dilution.</li>
		<li>Add 2 &#181;L of these stock solutions to each well to obtain a final well concentration of 0.1 mM, 0.02 mM, 0.004 mM, 0.0008 mM, 0.00016 mM, and 0.000032 mM, respectively.</li>
		<li>Prepare the wells as described in Table 2 and Table 3, with the wells containing the blank (buffer + water + DMSO) and negative control (Hsp90 in buffer + water + DMSO). The wells containing GA (Hsp90 in buffer + water + GA) serve as the positive control.</li>
	</ol>
	</li>
	<li>Preparation of well plates and order of addition
	<ol>
		<li>Prepare 96-well plates with the layout presented in Table 2. For compounds that show absorbance at 620 nm, prepare a separate well for recording the absorbance at this wavelength. Do not prepare a separate well for GA, as GA does not show absorbance at 620 nm for the concentration used in the assay.</li>
		<li>Prepare each constituent with the total volume of each constituent, as presented in Table 3.</li>
		<li>Set up assay wells by adding ultra-pure water to each well. Following this, add the required amount of assay buffer and a compound solution (for example, GA solution).</li>
		<li>Then, add Hsp90 to the appropriate wells, and shake the plates for 2 min on a plate shaker at room temperature.</li>
		<li>Add 4 &#181;L of ATP solution using a multi-channel pipette. Wrap the plate in aluminum foil and shake for 2 min on a plate shaker (200 rpm) at room temperature. Incubate the plate at 37 &#176;C for 3 h.</li>
	</ol>
	</li>
	<li>Preparation and addition of malachite green reagent
	<ol>
		<li>The malachite green reagent consists of reagents A (ammonium molybdate in 3M HCl) and B (malachite green and polyvinyl alcohol). Bring the reagent to room temperature before use. Mix reagents A and B at a 100:1 ratio (1,000 &#181;L:10 &#181;L). Prepare this within 3 h before use, since it is unstable and starts decomposing after 3 h.</li>
		<li>After 3 h of step 1.6.5, stop the reaction by adding 20 &#181;L of malachite green reagent, added in the same order as ATP, using a multi-channel pipette.</li>
		<li>After a 15 min incubation at room temperature, measure the absorbance of the plate at 620 nm in a plate reader.</li>
	</ol>
	</li>
</ol>
2. High-throughput screening of Hsp90 inhibitors by malachite green assay
NOTE: The protocol for high-throughput screening is similar to the lab-scale methodology. The final well volume in each case is 80 &#181;L. However, there is a slight difference in the order of the addition of reagents. In the lab-scale based method, there are five stages of solution addition (34 &#181;L of water, 32 &#181;L of buffer, 2 &#181;L compound in DMSO, 8 &#181;L of Hsp90, and finally 4 &#181;L of ATP solution). In contrast, with HTS there are three stages of addition (40 &#181;L of buffer solution containing yeast Hsp90, 2 &#181;L of DMSO in 18 &#181;L of water containing the compounds, and finally 20 &#181;L of ATP dissolved in water). The minimum amount of solution that can be pipetted accurately in the HTS setup is 20 &#181;L. Hence, a difference in pipetting between lab and HTS scales is observed.
<ol>
	<li>Preparation of assay buffer (pH 7.4)
	<ol>
		<li>Use the same buffer in HTS as for the lab-scale malachite green assay (Table 1).</li>
	</ol>
	</li>
	<li>Preparation of yeast Hsp90
	<ol>
		<li>Use protein with a glycerol stock concentration of 0.66 mg/mL. Thaw on ice just before use.</li>
		<li>Use 8 &#181;L (5.98 &#181;g, 0.80 &#181;M) of yeast Hsp90 in each well, which provides measurable ATPase activity.</li>
		<li>Dilute the protein (8 &#181;L) in buffer (32 &#181;L) for each well. The final total working volume for each well is 40 &#181;L in a total assay volume of 80 &#181;L.</li>
	</ol>
	</li>
	<li>Preparation of 0.8 mM ATP
	<ol>
		<li>Dissolve 1 mg of ATP in 2,267 &#181;L of distilled water to obtain a 0.8 mM solution. The total working volume for each well is 20 &#181;L in a total assay volume of 80 &#181;L.</li>
	</ol>
	</li>
	<li>Preparation of geldanamycin (GA)/test compounds
	<ol>
		<li>Prepare a 0.8 mM stock of GA in DMSO, as in step 1.5. Dilute 10 &#181;L of the GA stock with 90 &#181;L of water to give a final concentration of 80 &#181;M.</li>
		<li>Use 20 &#181;L of the 80 &#181;M GA solution in appropriate assay wells (final total assay well volume = 80 &#181;L), giving a final well concentration of 20 &#181;M. This serves as a positive control.</li>
		<li>For test compounds, prepare solutions in DMSO as per their desired concentration for evaluation.</li>
		<li>Prepare 10 &#181;L of DMSO in 90 &#181;L of water for addition to blank (buffer + water + DMSO) and negative control wells (Hsp90 in buffer + water + DMSO). Prepare the test compounds in a similar fashion at a 100 &#181;M final well concentration.</li>
	</ol>
	</li>
	<li>Design of assay plates and order of addition
	<ol>
		<li>Use four main plates, one compound plate, and four addition plates containing stocks of reagents to set up the assay (Figure 2 and Table 4).</li>
		<li>In addition, plate A, add 90 &#181;L of assay buffer in each well; in addition plate B, add 90 &#181;L of buffer with Hsp90 in each well; in addition plates C and D, add 90 &#181;L of ATP and 90 &#181;L of malachite green reagent, respectively, in each well (Table 5 and Table 6).</li>
		<li>From addition plate A and plate B, transfer 40 &#181;L of solution from each well to main plate 1 and 2, and 3 and 4, respectively, using an automated multichannel dispensing system. Here, the Biomek(R) FXP laboratory automation workstation was used (Figure 2 and Table 6).</li>
		<li>From each well of the compound plate, transfer 20 &#181;L of the solution to main plates 1, 2, 3, and 4 (Figure 2). Shake the plates for 1 min in a shaker (200 rpm).</li>
		<li>From each well of the addition plate C (ATP), transfer 20 &#181;L of solution to main plates 1, 2, 3, and 4 (Figure 2). Shake the plates for 1 min in a shaker (200 rpm).</li>
		<li>Incubate the plates for 3 h at 37 &#176;C.</li>
		<li>Prepare the malachite green reagent as per step 1.7. After 3 h, transfer 20 &#181;L of malachite green reagent from addition plate D into the wells of main plates 1 ,2, 3 and 4 (Figure 2).</li>
		<li>After a 15 min incubation at room temperature, measure the absorbance of the plate is at 620 nm in a plate reader.</li>
	</ol>
	</li>
</ol>

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There are no competing financial interests.

Hsp90 is a significant target for the discovery of novel anticancer drug molecules. Since its druggability was established in 199410, 18 molecules have reached clinical trials. At present, seven molecules are in various phases of clinical trials, either alone or in combination22. All such small molecules are N-terminal ATP binding inhibitors. The other means of inhibiting the chaperone (C-terminal inhibitors, middle domain inhibitors) have not proceeded as fast as N-termina...

Heat shock protein 90 (Hsp90) is a molecular chaperone that maintains the stability of proteins responsible for the development and progression of cancer. In addition, proteins responsible for the development of resistance to antineoplastic agents are also clients of Hsp901. Hsp90 is overexpressed ubiquitously in all cancer cell types (&#62;90% of cellular proteins), compared to normal cells where it may constitute less than 2% of total proteins. Moreover, the Hsp90 of cancer cells resides in a complex with co-chaperones, whereas in a normal cell it is present predominantly in a free, un-complexed state2,3. In recent years, several Hsp90 inhibitors have been demonstrated to possess senolytic effects in in vitro and in vivo studies, where they have significantly improved the life span of mice4,5,6. All the aforementioned findings substantiate the fact that Hsp90 inhibitors could be effective in multiple cancer types, with fewer adverse effects and reduced chances of developing resistance. The chaperoning function of Hsp90 is accomplished by binding ATP at the N-terminal domain of Hsp90 and hydrolyzing it into ADP and free phosphate7. Small molecules that competitively bind to the ATP binding pocket of Hsp90 were found to successfully suppress the chaperoning effect of the protein. To date, this remains the best strategy for Hsp90 inhibition, which is supported by the fact that such inhibitors have reached clinical trials8. One of them, Pimitespib, was approved in Japan for the treatment of gastrointestinal stromal tumor (GIST) in June 20229. This is the first Hsp90 inhibitor approved since the druggability of the chaperone was established in 199410.
The malachite green assay is a simple, sensitive, fast, and inexpensive procedure for the detection of inorganic phosphate, suitable for automation and high-throughput screening (HTS) of compounds against its desired target11. The assay has been successfully employed for the screening of Hsp90 inhibitors in small lab-scale setups, as well as in a HTS12,13,14,15,16,17. The assay uses a colorimetric method that determines the free inorganic phosphate formed due to the ATPase activity of Hsp90. The basis of this quantification is the formation of a phosphomolybdate complex between free phosphate and molybdenum, which subsequently reacts with malachite green to generate a green color (Figure 1). This rapid color formation is measured on a spectrophotometer, or on a plate reader, between 600-660 nm18,19.
In the present protocol, the procedure for carrying out a malachite green assay with yeast Hsp90 and subsequent identification of inhibitors against the chaperone is described. The natural product molecule, geldanamycin (GA), with which the druggability of Hsp90 was first established, was taken as an authentic inhibitor10. HTS has become an integral part of the current drug discovery program, owing to the availability of a large number of molecules for testing. This technique has gained more significance in the past 2 years because of the urgent need for repurposing drugs for treating Covid-19 infection20,21. Therefore, a detailed outline for the HTS of molecules against yeast Hsp90 protein by adopting the malachite green assay method is presented.

<table><tbody><tr><td>1M Magnesium chloride solution in water</td><td>Sigma-Aldrich</td><td>63069-100ml</td><td></td></tr><tr><td>1M Potassium chloride solution in water</td><td>Sigma-Aldrich</td><td>60142-100ml</td><td></td></tr><tr><td>96-well plate</td><td>SPL Life Sciences</td><td>Not applicable</td><td></td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate disodium salt hydrate</td><td>Sigma-Aldrich</td><td>A7699-5G</td><td></td></tr><tr><td>Biomek FX laboratory automation workstation</td><td>Beckman Coulter</td><td>Not applicable</td><td></td></tr><tr><td>Compounds 3-96</td><td>Not applicable</td><td>Not applicable</td><td>Histidine tagged yeast Hsp90 was obtained from Dr. Chrisostomos Prodromou, School of Life Sciences, University of Sussex, United Kingdom, and protein was expressed in KIST Gangneung Institute of Natural Products. Details cannot be disclosed due to patent infringement issues.</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Geldanamycin, 99% (HPLC), powder</td><td>AK Scientific, Inc.</td><td>V2064</td><td></td></tr><tr><td>Invitroge UltraPure DNase/RNase-Free Distilled Water</td><td>ThermoFisher Scientific</td><td>10977015</td><td></td></tr><tr><td>Malachite Green Phosphate Assay&amp;nbsp; Assay kit</td><td>Sigma-Aldrich</td><td>MAK307-1KT</td><td></td></tr><tr><td>Multi-Detection Microplate Reader Synergy HT</td><td>Biotek Instruments, Inc.</td><td>Not applicable</td><td></td></tr><tr><td>Synergy HT multi-plate reader</td><td>Biotek Instruments, Inc.</td><td>Not applicable</td><td></td></tr><tr><td>Trizma hydrochloride buffer solution, pH7.4</td><td>Sigma-Aldrich</td><td>93313-1L</td><td></td></tr><tr><td>Yeast Hsp90</td><td>Not applicable</td><td>Not applicable</td><td>School of Life Sciences, University of Sussex, United Kingdom and protein was expressed in KIST Gangneung Institute of Natural Products. Primary Accession number: P02829</td></tr></tbody></table>

malachite green assay for the discovery of heat-shock protein 90 inhibitors

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is dependent on its ability to hydrolyze adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and free phosphate. The ATPase activity of Hsp90 is linked to its chaperoning function; ATP binds to the N-terminal domain of the Hsp90, and disrupting its binding was found to be the most successful strategy in suppressing Hsp90 function. The ATPase activity can be measured by a colorimetric malachite green assay, which determines the amount of free phosphate formed by ATP hydrolysis. Here, a procedure for determining the ATPase activity of yeast Hsp90 by using the malachite green phosphate assay kit is described. Further, detailed instructions for the discovery of Hsp90 inhibitors by taking geldanamycin as an authentic inhibitor is provided. Finally, the application of this assay protocol through the high-throughput screening (HTS) of inhibitor molecules against yeast Hsp90 is discussed.

The results of the assay are interpreted in terms of absorbance due to free phosphate ion concentration. The absorbance by free phosphate due to ATP hydrolysis by the yeast Hsp90 at 620 nm is considered as 100% ATPase activity, or zero percentage protein inhibition. The inhibition of protein leads to the cessation of ATP hydrolysis (less free phosphate). which is reflected in terms of decreased absorbance at 620 nm.
Results of lab-scale malachite green assay 
The standard g...

Watch this Scientific Journal Video about Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors at JoVE.com

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

The malachite green assay protocol is a simple and cost-effective method to discover heat shock protein 90 (Hsp90) suppressors, as well as other inhibitor compounds against ATP-dependent enzymes.

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is ...

malachite-green-assay-for-discovery-heat-shock-protein-90

Research

JoVE Journal

Biochemistry

5.1K Views.  Korea Institute of Science and Technology (KIST). The malachite green assay protocol is a simple and cost-effective method to discover heat shock protein 90 (Hsp90) suppressors, as well as other inhibitor compounds against ATP-dependent enzymes.

Video: Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

Genetics

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2&#160;is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...

Cancer Research

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...

Assessment of Hsp70 Chaperone Activity Using Escherichia coli dnaK756 Cells

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70&#160;genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756&#160;cells are unable to support &#955; bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756&#160;cells grow adequately at temperatures ranging from 30 &#176;C to 37 &#176;C, but they die at elevated temperatures (&#62;40 &#176;C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

Author Spotlight: Exploring Heat Shock Proteins in Malaria and Tuberculosis Infections

This protocol demonstrates the chaperone activity of heat shock protein 70 (Hsp70). E. coli dnaK756 cells serve as a model for the assay as they harbor a native, functionally impaired Hsp70, making them susceptible to heat stress. The heterologous introduction of functional Hsp70 rescues the growth deficiency of the cells.

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. ...

High-Throughput Thermal Shift Assays for Protein-Ligand Interactions

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding. 
Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.

Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays

Here, we present the thermal shift assay, a high-throughput, fluorescence-based technique used to investigate the binding of small molecules to proteins of interest.

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although ...

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Biology

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

Summary

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Chapters in this video

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

Intracellular Refolding Assay

A Protocol for the Identification of Protein-protein Interactions Based on ¹⁵N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae