Name: studies of chaperone-cochaperone interactions using homogenous bead-based assay
Uploaded: 2021-07-21T13:00:00.000+00:00
Duration: 6 min 51 s
Description: Watch this Scientific Journal Video about Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay at JoVE.com

This study was supported by grants from Swedish Research Council (2018-02843), Brain Foundation (Fo 2019-0140), Foundation for Geriatric Diseases at Karolinska Institutet, Gunvor and Josef An&#233;rs Foundation, Magnus Bergvalls Foundation, Gun and Bertil Stohnes Foundation, Tore Nilssons Foundation for medical research, Margaretha af Ugglas foundation and the Foundation for Old Servants.

NOTE: An overview of this protocol is shown in Figure 2.
1. Expression and purification of GST-FKBP51 and GST-FKBP52 (Figure 2A)
<ol>
	<li>Plasmids 
	NOTE: Obtain cDNA clones for human FKBP51 (clone id: 5723416) and for human FKBP52 (clone id: 7474554) from IMAGE consortium.
	<ol>
		<li>Amplify the human FKBP51 DNA by PCR with primers (forward; 5`GGATCCATGACTACTGATGAAGGT-3`, reverse; 5`-CTCGAGCTATGCTTCTGTCTCCAC-3`) containing BamHI and XhoI overhangs and clone into pGEX6-1 vector at BamHI / XhoI restriction sites.</li>
		<li>Amplify the human FKBP52 DNA by PCR with primers (forward; 5`-GAATTCATGACAGCCGAGGAGATG-3`, reverse; 5`-CTCGAGCTATGCTTCTGTCTCCAC-3`) containing EcoRI and XhoI overhangs and clone into pGEX6-2 vector at EcoRI / XhoI restriction sites. 
		NOTE: PCR reaction set up and conditions are shown in Table 1 and Table 2.</li>
		<li>Verify the inserted sequence and transform the plasmids into the chemically competent E. coli&#160;according to the manufacture protocol.</li>
	</ol>
	</li>
	<li>Protein expression and purification
	<ol>
		<li>Add 25 g of Luria broth (LB) base in 1 L of distilled water to make the LB solution. Autoclave it at 121 &#176;C for 15 min. After cooling, add 50 &#181;g/mL ampicillin.</li>
		<li>Take a colony of bacteria expressing GST-FKBP51 or GST-FKBP52 and mix with 500 &#181;L of LB solution in a 1.5 mL tube. Vortex.</li>
		<li>Add the mixture of &#34;1.2.2&#34; into 1 L of LB solution in the Erlenmeyer flask covered with an aluminum foil. Incubate the Erlenmeyer flask in the shaker overnight at 37 &#176;C.</li>
		<li>Induce protein expression by adding 1 mM isopropyl-&#946;-D-thiogalactoside (IPTG) to the Erlenmeyer flask and continue the incubation for a further 2 h.</li>
		<li>To get cell pellets, centrifuge at 5,000 x g for 15 min. Remove the supernatant. 
		NOTE: The cell pellets can be stored at -20 &#176;C.</li>
		<li>Resuspend the cell pellets in 40 mL of PBS and sonicate 3 x 20 s on ice. Add 1 mM PMSF, 1 mM EDTA, and protease inhibitor cocktail (1 tablet) to prevent proteolysis.</li>
		<li>Centrifuge the suspension for 30 min 50,000 x g to remove cell debris and apply the supernatant onto 5 mL GST-trap column.</li>
		<li>After washing the column with 30 mL PBS, elute GST-FKBP51 and GST-FKBP52 with 5 mL of 10 mM glutathione in PBS.</li>
		<li>Concentrate proteins on 15 mL 10.000 MWCO centrifugation unit. To remove free glutathione, pass concentrates through PD-10 column equilibrated with 0.5x PBS and again concentrate on the filter centrifuge device.</li>
		<li>Collect protein-containing fractions. Verify the proteins in SDS-PAGE and adjust protein concentrations to 1 mg/mL. 
		&#8203;NOTE: Typical protein yield is 2-5 mg/L culture. The protein can be stored at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Coupling of Hsp90 C-terminal peptide to the acceptor beads (Figure 2B)
<ol>
	<li>Hsp90 peptide preparation
	<ol>
		<li>Synthesize ten amino acid peptide NH2-EDASRMEEVD-COOH corresponding to amino acids 714-724 of human Hsp90 beta isoform (UniProt ID: P08238) by a peptide synthesis service.</li>
		<li>Dilute the Hsp90 peptide in PBS to 1 mg/mL concentration.</li>
	</ol>
	</li>
	<li>Acceptor beads preparation
	<ol>
		<li>Dilute the unconjugated acceptor beads in PBS to 1 mg/mL concentration and transfer to a 1.5 mL tube.</li>
		<li>Perform the washing by centrifugation at 16,000 x g for 15 min. Carefully remove the supernatant.</li>
	</ol>
	</li>
	<li>Conjugation
	<ol>
		<li>Set the ratio between beads and peptide as 10:1. In the 1.5 mL tube containing 1 mg of acceptor bead pellet (prepared as described above), add 1 mL of PBS (pH 7.4), 0.1 mg of diluted peptide, 1.25 &#181;L of Tween-20, 10 &#181;L of a 400 mM solution of sodium cyanoborohydride (NaBH3CN) in water. 
		CAUTION: NaBH3CN is toxic; use a fume hood and gloves. NaBH3CN solution should be freshly prepared.</li>
		<li>Incubate for 24 h at 37 &#176;C with end-over-end agitation (10-20 rpm) on a rotary shaker.</li>
	</ol>
	</li>
	<li>Reaction quenching and beads washing
	<ol>
		<li>Add 20 &#181;L of 1 M Tris-HCl (pH 8.0) solution to the reaction to block unreacted sites. Incubate for 1 h at 37 &#176;C.</li>
		<li>Centrifuge at 16,000 x g (or maximum speed) for 15 min at 4 &#176;C. Remove the supernatant and resuspend the bead pellet in 1 mL of Tris-HCl solution (100 mM, pH 8.0).</li>
		<li>Repeat the washing step three times.</li>
		<li>After the last centrifugation, resuspend the beads at 1 mg/mL in storage buffer (1 mL of 0.5 &#215; PBS with 0.01% sodium azide as a preservative). Store the conjugated acceptor bead solution at 4 &#176;C light protected. 
		&#8203;CAUTION: Sodium azide is toxic; use a fume hood and gloves.</li>
	</ol>
	</li>
</ol>
3. The assay probing the interaction between GST-FKBP51 or GST-FKBP52 and Hsp90 C-terminal peptide, and inhibition with small molecular mass compounds (Figure 2C)
<ol>
	<li>GST-tagged proteins interacting with glutathione donor beads
	<ol>
		<li>Set up the reactions in 384-well plates.</li>
		<li>Prepare the solution containing 10 &#181;g/mL of the glutathione donor beads in 0.5x PBS, pH 7.4. 
		NOTE: After prolonged storage, beads settle down and need to be vortexed.</li>
		<li>Add GST-FKBP51 or GST-FKBP52 to a final concentration of 10 &#181;g/mL.</li>
		<li>Incubate in the dark at 25 &#176;C for 10 min. 
		NOTE: At this step, GST-tagged proteins will interact with glutathione attached to the beads. For each well, 22.5 &#181;L of this mixture will be used. The concentration of the binding partners must be determined empirically. Titrate GST-FKBP51 and GST-FKBP52 and choose the concentration that gives the best signal.</li>
	</ol>
	</li>
	<li>Compound addition
	<ol>
		<li>Make serial dilutions of test compounds in DMSO. 
		NOTE: The concentrations used are typically 10, 30, 100, 300, 1,000, and 3,000 &#181;M.</li>
		<li>Add 0.25 &#181;L of DMSO (negative control) or Hsp90 C-terminal peptide (positive control, 30 &#181;M) or compounds in DMSO to the corner of each well of the plate. Use triplicates for every compound concentration.</li>
		<li>Add 22.5 &#181;L of the solution containing glutathione donor beads with GST-tagged proteins to each well.</li>
		<li>Shake the plate gently with hand but thoroughly. Incubate in the dark at 25 &#176;C for 15 min. 
		&#8203;NOTE: During this time, compounds will interact with the TPR domain at the Hsp90 C-terminal peptide binding site.</li>
	</ol>
	</li>
	<li>Acceptor beads addition
	<ol>
		<li>Dilute the acceptor beads with attached Hsp90 C-terminal peptide to 100 &#181;g/mL in 0.5x PBS.</li>
		<li>Add 2.25 &#181;L of diluted acceptor beads to each well.</li>
		<li>Mix gently but thoroughly. Incubate in the dark at 25 &#176;C for 15 min. 
		NOTE: At this step, donor and acceptor beads are brought into proximity by the protein-peptide interactions. The final volume of the reaction mixture is 25 &#181;L. Therefore, the final concentrations of compounds are ranging from 0.1 to 30 &#181;M.</li>
	</ol>
	</li>
	<li>Plate reading 
	NOTE: Read the plate using a plate reader set in the relevant mode.
	<ol>
		<li>Turn on the instrument and open the software</li>
		<li>Choose the relevant protocol.</li>
		<li>Click Edit plate map and select the well being used in the plate for measurement.</li>
		<li>Click Next to continue and Run the selected protocol.</li>
		<li>After measurement, click Show Results to view results.</li>
		<li>Export the data.</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>Z&#39; factor and signal-to-background (S/B) ratio
	<ol>
		<li>Calculate the Z&#39; factor and S/B ratio for the assay using the following equation: 
		Z&#39;=1-(3&#963;pos+3&#963;neg)/&#9474;&#181;pos-&#181;neg&#9474;16 
		S/B=&#181;neg/&#181;pos 
		where, &#963; and &#181; represent the standard deviations and means of the positive (Hsp90 C-terminal peptide, 30 &#181;M) and negative (DMSO) controls, respectively. A Z&#39; factor &#62; 0.5 will ensure that the assay is robust enough for screening. To monitor the assay sensitivity, the S/B ratio has also been calculated.</li>
	</ol>
	</li>
	<li>Dose-response curve and IC 50 
	NOTE: Use nonlinear regression analysis to fit the data of Hsp90-cochaperones PPI inhibitors by software.
	<ol>
		<li>Create an XY data table in the Welcome dialog and select X Numbers, and Y Enter 3 (if triplicates) replicate values in side-by-side columns.</li>
		<li>Normalize the signal data of samples to the negative control group. Import concentration values to the X column and the signal values to the Y column.</li>
		<li>Click Analyze and choose Transform concentration (X) under Transform | Normalize. Choose Transform to Logarithms. 
		NOTE: This will transform the concentration to a log scale. If your starting concentration is zero, set it to a very small number that is effectively zero (e.g., 0.1 nM) not to lose those values since the logarithm of zero is not defined.</li>
		<li>Click Analyze and choose Nonlinear regression (curve fit) under XY analyses, open the Dose-Response-Inhibition and choose Log(inhibitor) vs. response -- Variable slope.</li>
		<li>Click OK to view the Results (containing IC50 value) and Graphs.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62762/62762fig2v2.jpg" /> 
Figure 2: Schematic of this protocol. (A) Expression and purification of GST-FKBP51 and GST-FKBP52. (B) Coupling of Hsp90 C-terminal peptide to the acceptor beads. (C) The assay probing the interaction between GST-FKBP51 or GST-FKBP52 and Hsp90 C-terminal peptide. Inhibition with small molecular mass compounds. Created with BioRender.com <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62762/62762fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Authors report no conflicts of interest.

Here we describe a protocol using the assay for screening small molecules inhibiting interactions between Hsp90 and TPR-motif co-chaperones, especially FKBP51 and FKBP52. Its high Z' score (&gt;0.8) demonstrates the robustness and reliability for a high-throughput format. Results can be obtained within one hour, and small amounts of beads, protein and compounds are required. Moreover, this protocol could easily be extended to any Hsp90/Hsp70 - TPR-motif co-chaperone interactions of interest. Several TPR-motif co-chaperones of Hsp90 have been implicated in various human disorders ranging from Alzheimer's disease to autoimmune diseases, cancer, etc. The protocol described here provides an in vitro robust and inexpensive assay for high-throughput screening of small molecules inhibiting chaperone-cochaperone interactions of high medical importance.
In addition, drugs targeting TPR domains and inhibiting interaction with Hsp90 must be assessed not only for their affinity towards the target but also for their selectivity towards other TPR-motif proteins. The human genome encodes &gt; 20 TPR motif proteins capable of interacting with the Hsp90 C-terminal peptide. This assay using glutathione beads and GST-tagged proteins allows assessment of the drug effect on multiple binding TPR partners. Our laboratory possesses a library of 20 human TPR proteins in their GST-tagged form and can obtain affinity and selectivity profiles for every small molecule tested.
It has been previously shown that PPI between Hsp90 and TPR co-chaperones is mediated by the C-terminal peptide of Hsp90; the deletion of Hsp90 C-terminus completely abolishes the binding of TPR-motif co-chaperones6. We have found that compounds efficient in our assay where the Hsp90 C-terminal peptide was used also were able to inhibit the interaction of full-length Hsp90 with GST-FKBP51/52 in a pull-down assay using glutathione sepharose beads (data not shown). Some of the selected compounds also show the binding affinity with FKBP51 by surface plasmon resonance technology, and their specific binding sites determined by co-crystallization are currently under investigation.
A critical step in our protocol is the order of addition; we first form a complex between a small molecule and its TPR target. If complexes between FKBP51 and Hsp90 C-terminal peptide have already been formed, longer incubation times and higher drug compound concentrations are required to break the PPIs. This is mostly due to the steric hindrance of bead limiting access of molecules to the site of interaction. 
It is possible to covalently couple TPR proteins and Hsp90 C-terminal peptides to donor and acceptor beads, respectively, and directly probe PPIs. However, it is not recommended to covalently attach both binding partners to the beads due to the reduced movement of molecules in solution affecting the kinetics of the interactions. This also increases the risk of steric hindrance due to the bead size. Therefore, we choose acceptor beads coupled with Hsp90 C-terminal peptide and glutathione donor beads that are interacting with GST-tagged proteins in our assay, where multiple TPR partners can be assessed.
In this assay, it is probable that some identified compounds can be false-positive because of their interference with the assay technology, such as quenching singlet oxygen, quenching light, and scattering light. False-positive results can also be induced by disrupting the binding of GST tag with glutathione donor beads. These false-positive results can be avoided when screening molecules both on Hsp90-FKBP51 and Hsp90-FKBP52 PPIs to identify selective inhibitors. Other methods detecting PPIs should also be applied to verify the selected inhibitors.
The limitation of our assay is that it cannot be used to detect the interaction between Hsp90 and TPR-motif co-chaperones in crude biological samples, such as cell lysates. After the high-throughput screening, the inhibitions of selected compounds on Hsp90 - TPR-motif co-chaperones PPI in biological samples need to be verified by other methods, such as co-immunoprecipitation and proximity ligation assay.

Molecular chaperones contribute to protein homeostasis, including protein folding, transport, and degradation. They regulate several cellular processes and are linked to numerous diseases such as cancer and neurodegenerative diseases1. Heat shock protein 90 (Hsp90) is one of the most important chaperones whose function is dependent on conformational changes driven by ATP hydrolysis and binding with client proteins mediated by its co-chaperones2. Despite an obvious potential of Hsp90 as the therapeutic target, fine-tuning its function represents a big challenge. There are several Hsp90 inhibitors targeting the N-terminal ATP binding region, which have been evaluated in clinical trials, but none of them has been approved for marketing3. Due to the lack of a well-defined ligand-binding pocket4, targeting the C-terminal region of Hsp90 has had limited success4. Recently, disruption of Hsp90-cochaperone interactions by small molecules has been investigated as an alternative strategy5. Targeting the Hsp90-cochaperone interactions would not elicit general cell stress response and provides the possibility to specifically regulate various 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-chaperones6. Of the 736 TPR motif-containing proteins annotated in the human protein database, ~20 different proteins interact with Hsp90 via this peptide7. Molecules competing for MEEVD peptide-binding would disrupt the interactions between Hsp90 and co-chaperones containing a TPR domain. The peptide binding site has similar tertiary structure but the overall homology between different TPR motif domains is relatively low7, providing an opportunity to identify molecules specifically capable of blocking interactions between Hsp90 and particular TPR-motif co-chaperones. Among these TPR-motif co-chaperones, FK506-binding protein (FKBP) 51 and FKBP52 are regulators of steroid hormone receptor (SHR) signaling and involved in several steroid hormone-dependent diseases including cancer, stress-related diseases, metabolic diseases, and Alzheimer&#39;s disease8. Although FKBP51 and FKBP52 share &#62; 80% sequence similarity, their functions differ: FKBP52 is a positive regulator of SHR activity, while FKBP51 is a negative regulator in most cases8. Therefore, identifying molecules, specifically blocking interactions between Hsp90 and FKBP51 or FKBP52, provides a promising therapeutic potential for related diseases.
Amplified Luminescent Proximity Homogenous Assay (AlphaScreen) was first developed in 1994 by Ullman EF et al.9. Now it is widely used to detect different types of biological interactions, such as peptide10, protein11, DNA12, RNA13, and sugar14. In this technique, there are two kinds of beads (diameter 200 nm), one is the donor bead and the other is the acceptor bead. The biomolecules are immobilized onto these beads; their biological interactions bring donor and acceptor beads into proximity. At 680 nm, a photosensitizer in the donor bead illuminates and converts oxygen to singlet oxygen. Because the singlet oxygen has a short lifetime, it can only diffuse up to 200 nm. If the acceptor bead is in proximity, its thioxene derivative reacts with the singlet oxygen generating chemiluminescence at 370 nm. This energy further activates fluorophores in the same acceptor bead to emit light at 520-620 nm15. If the biological interactions are disrupted, the acceptor bead and donor bead cannot reach proximity, resulting in the singlet oxygen decay and low produced signal.
Here we describe a protocol using this technique for screening small molecules inhibiting interactions between Hsp90 and TPR co-chaperones, especially FKBP51 and FKBP52.The 10 amino acid long peptides corresponding to Hsp90 extreme C-terminus are attached to acceptor beads. Purified GST-tagged TPR co-chaperones interact with glutathione-linked donor beads. When the interaction between Hsp90-derived peptides and TPR-motif co-chaperones brings the beads together, an amplified signal is produced (Figure 1A). If the screened small molecules can inhibit the interactions between Hsp90 and TPR-motif co-chaperones, this amplified signal will be decreased (Figure 1B). Their IC50 can be calculated by quantitative measurement. This protocol can be extended to any chaperone - TPR-motif co-chaperone interactions of interest and is of great importance in the development of novel molecules, specifically blocking the interaction between Hsp90 and FKBP51 or FKBP52.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62762/62762fig01.jpg" /> 
Figure 1: The basic principle of this assay. (A) Purified GST-FKBP51 interacts with glutathione-linked donor beads. The 10 amino acid long peptides corresponding to the extreme C-terminus of Hsp90 are attached to acceptor beads. The interaction between Hsp90-derived peptides and TPR domain of FKBP51 brings the donor and acceptor beads into proximity. At 680 nm, a photosensitizer in the donor bead illuminates and converts oxygen to singlet oxygen. The thioxene derivative on the acceptor bead reacts with the singlet oxygen and generates chemiluminescence at 370 nm. This energy further activates fluorophores in the same acceptor bead to emit light at 520-620 nm. (B) When small molecules inhibit the interactions between Hsp90 and FKBP51, the donor and acceptor beads cannot reach proximity. Then the singlet oxygen with short lifetime decays, and no detectable signal is produced. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62762/62762fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>384-well plates</td><td>Perkin Elmer</td><td>6008350</td><td>Assay volume 25 ml</td></tr><tr><td>Amicon 10.000 MWCO centrifugation unit</td><td>Millipore</td><td>UFC901008</td><td>Concentrate protein</td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A0166</td><td>Antibiotics</td></tr><tr><td>Bacteria shaker Unimax 1010</td><td>Heidolph</td><td></td><td>Culture bacteria</td></tr><tr><td>cDNA clones for human FKBP51</td><td>Source BioScience</td><td>clone id: 5723416</td><td>pCMV-SPORT6 vector</td></tr><tr><td>cDNA clones for human FKBP52</td><td>Source BioScience</td><td>clone id: 7474554</td><td>pCMV-SPORT6 vector</td></tr><tr><td>Chemically Competent E. coli</td><td>Invitrogen</td><td>C602003</td><td>One Shot BL21 Star (DE3)</td></tr><tr><td>Data analysis software</td><td>GraphPad Prism</td><td>9.0.0</td><td>Analysis data and make figures</td></tr><tr><td>Data analysis software</td><td>Excel</td><td></td><td>Analysis data</td></tr><tr><td>DMSO</td><td>Supelco</td><td>1.02952.1000</td><td>Dilute compounds</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-144</td><td>Prepare solution</td></tr><tr><td>EDTA</td><td>Calbiochem</td><td>344504</td><td>Prevent proteolysis during sonication</td></tr><tr><td>Glutathione</td><td>Sigma</td><td>G-4251</td><td>Elute GST-tagged proteins</td></tr><tr><td>Glutathione donor beads</td><td>Perkin Elmer</td><td>6765300</td><td>Donor bead</td></tr><tr><td>GST-trap column</td><td>Cytiva (GE Healthcare)</td><td>17528201</td><td>Purify GST-tagged proteins</td></tr><tr><td>Isopropyl-&amp;beta;-D-thiogalactoside</td><td>Thermo Fisher Scientific</td><td>R0392</td><td>Induce protein expression</td></tr><tr><td>LB Broth (Miller)</td><td>Sigma</td><td>L3522</td><td>Microbial growth medium</td></tr><tr><td>PCR instrument</td><td>BIO-RAD</td><td>S1000 Thermal Cycler</td><td>Amplification/PCR</td></tr><tr><td>PD-10 column</td><td>Cytiva (GE Healthcare)</td><td>17085101</td><td>Solution exchange</td></tr><tr><td>pGEX-6P-1 vector</td><td>Cytiva (GE Healthcare)</td><td>28954648</td><td>Plasmid</td></tr><tr><td>pGEX-6P-2 vector</td><td>Cytiva (GE Healthcare)</td><td>28954650</td><td>Plasmid</td></tr><tr><td>Plate reader</td><td>Perkin Elmer</td><td>EnSpire 2300 Multilabel Reader</td><td>Read alpha plate</td></tr><tr><td>Plate reader software</td><td>Perkin Elmer</td><td>EnSpire Manager</td><td>Plate reader software</td></tr><tr><td>Plate reader software protocol</td><td>Perkin Elmer</td><td>Alpha 384-well Low volume</td><td>Use this protocol to read plate</td></tr><tr><td>PMSF</td><td>Sigma</td><td>P7626</td><td>Prevent proteolysis during sonication</td></tr><tr><td>protease inhibitor cocktail</td><td>Sigma</td><td>S8830</td><td>Prevent proteolysis during sonication</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td>As a preservative</td></tr><tr><td>Sodium cyanoborohydride (NaBH3CN)</td><td>Sigma</td><td>156159</td><td>Activates matrix for coupling</td></tr><tr><td>Ten amino acid peptide NH2-EDASRMEEVD-COOH corresponding to amino acids 714-724 of human Hsp90 beta isoform</td><td>Peptide 2.0 inc</td><td></td><td>Synthesize Hsp90 C-terminal peptide</td></tr><tr><td>Test-Tube Rotator</td><td>LABINCO</td><td></td><td>Make end-over-end agitation</td></tr><tr><td>Tris-HCl</td><td>Sigma</td><td>10708976001</td><td>Block unreacted sites of acceptor beads</td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379</td><td>Prevent beads aggregation</td></tr><tr><td>Ultra centrifuge Avanti J-20 XP</td><td>Beckman Coulter</td><td></td><td>Centrifuge to get bacteria cell pellets</td></tr><tr><td>Ultrasonic cell disruptor</td><td>Microson</td><td></td><td>Sonicate cells to release protein</td></tr><tr><td>Unconjugated acceptor beads</td><td>Perkin Elmer</td><td>6762003</td><td>Acceptor beads</td></tr><tr><td>XCell SureLock Mini-Cell and XCell II Blot Module</td><td>Invitrogen</td><td>EI0002</td><td>SDS-PAGE</td></tr></tbody></table>

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.

In our assay, Z&#39; factor and S/B ratio are 0.82 and 13.35, respectively (Figure 3A), demonstrating that our assay is robust and reliable for high-throughput screening. We then used it to screen small molecular mass compounds. Figure 3B presents dose-dependent inhibition of chaperone-cochaperone interactions with a selected small molecule (D10). The dose-response curves for D10 are generated by nonlinear regression analysis, based on which the values of IC50 are calculated. D10 shows dose-dependent inhibition both on Hsp90 - GST-FKBP51 and Hsp90 - GST-FKBP52 PPIs. But the values of IC50 are different: its IC50 for Hsp90 - GST-FKBP51 interactions is 65 nM, whereas, for Hsp90 - GST-FKBP52 interactions, complete inhibition was not achieved with the highest compound concentration (100 &#181;M), its IC50 is estimated to be &#62; 30 &#181;M. These results provide evidence that selective inhibition of Hsp90-HKBP51 or Hsp90-FKBP52 PPIs with small molecules can be achieved (TPR domains of FKBP51 and FKBP52 have 60% sequence identity and &#62; 80% sequence similarity), and this assay can be applied for this screening.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62762/62762fig03.jpg" /> 
Figure 3: Assay analysis and results. (A) Z&#39; factor and signal-to-background (S/B) ratio of this assay. Data&#160;represent signals of (&#9675;) positive control (Hsp90 C-terminal peptide, 30 &#181;M) and (&#9679;) negative control (DMSO) from 48 wells. A Z&#39; value of 0.82 and an S/B ratio of 13.35 were calculated from these two populations of data. (B) The inhibition of selected compound (D10) on interactions of Hsp90 with FKBP51 (&#9679;) or FKBP52 (&#9675;) in this assay.&#160;D10 inhibits Hsp90-FKBP51 or Hsp90-FKBP52 dose-dependently. Its IC50 is 65 nM for Hsp90-FKBP51 interaction but above 30 &#181;M for Hsp90-FKBP52 interaction. Data are normalized to the control group and expressed as means &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62762/62762fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reaction component</td>
			<td>Volume (&#181;L)</td>
		</tr>
		<tr>
			<td>PCR buffer (5 x concentrate)</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Forward primer</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Reverse primer</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Plasmid</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>dNTP mix (10 mM each)</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>Phusion DNA polymerase</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>Water (DNA grade)</td>
			<td>12.5</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>20</td>
		</tr>
	</tbody>
</table>
Table 1: PCR reaction set up for human FKBP51 and FKBP52 DNA amplification.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stage</td>
			<td>Temperature (&#176;C)</td>
			<td>Time</td>
			<td>Cycles</td>
		</tr>
		<tr>
			<td>Initial denaturation</td>
			<td>94</td>
			<td>3 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Denaturation</td>
			<td>94</td>
			<td>30 sec</td>
			<td rowspan="3">35</td>
		</tr>
		<tr>
			<td>Annealing</td>
			<td>56</td>
			<td>30 sec</td>
		</tr>
		<tr>
			<td>Extension</td>
			<td>72</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>Final extension</td>
			<td>72</td>
			<td>5&#160; min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Note: Lid temperature is 105 &#176;C.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Thermocyler conditions for human FKBP51 and FKBP52 DNA amplification.

Watch this Scientific Journal Video about Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay at JoVE.com

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

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 ...

studies-chaperone-cochaperone-interactions-using-homogenous-bead

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.7K Views.  Karolinska Institutet. We bouwden een homogene op kralen gebaseerde test om chaperone-cochaperone-interacties te bestuderen. Met behulp van deze techniek screenen we kleine moleculen die interacties tussen Hsp90 en FKDP51 of FKBP52 remmen en identificeren we krachtige en selectieve remmers. Deze high throughput screening techniek is robuust en betrouwbaar.Resultaten kunnen binnen een uur worden verkregen en het vereist slechts kleine hoeveelheden kraleneiwit en verbindingen. Hsp90-interactie met deze cochape...

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

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Immunology and Infection

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

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 ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Biology

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Modified Yeast-One Hybrid Assay to Detect Heteromeric Protein Complex-DNA Interactions

Source: Tripathi, P. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56080">A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the modified yeast one-hybrid assay, which detects multiple proteins interacting with target DNA. The transformed yeast cells contain the target DNA sequence upstream of the reporter gene. Additionally, they express transcription factor proteins. When the proteins form complexes and bind to the target DNA, the reporter gene is expressed and can be detected enzymatically.

Source: Tripathi, P. et al., A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies. J. Vis. Exp. (2017)
In this ...

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

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