This research was supported by the Minerva Foundation, the HFSP Organization, and a European Research Council Grant (Starting Grant 338265).

1. Synthesis of the &#39;Chemical Transducer&#39;
<ol>
	<li>Preliminary Preparations
	<ol>
		<li>Prepare 2 M triethylammonium acetate (TEAA) buffer by mixing 278 ml of triethylamine with 114 ml of acetic acid and 400 ml of ultrapure water. Adjust the pH to 7 and add water to a final volume of 1 L. Keep it in a dark bottle. 
		Note: This solution is stable for years.</li>
		<li>Prepare a 5 mM ascorbic acid solution by dissolving 18 mg of ascorbic acid in 20 ml of ultrapure water. Use a fresh solution; the solution is stable for one day.</li>
		<li>Prepare a 10 mM Cu(II)/Tris(benzyltriazolylmethyl)amine (TBTA) solution by dissolving 25 mg of copper(II) sulfate pentahydrate in 10 ml of ultrapure water and 58 mg of TBTA in 11 ml dimethyl sulfoxide (DMSO). Mix the two solutions. Keep it at room temperature and protect it from light.</li>
	</ol>
	</li>
	<li>Conjugation Procedure
	<ol>
		<li>Dissolve 100 nmol of the modified oligonucleotide (ODN-1) in 80 &#181;l of fresh ultrapure water. Add 20 &#181;l of 2 M TEAA, pH = 7. Add 80 &#181;l of a freshly made solution of ascorbic acid (5 mM in water).</li>
		<li>Dissolve 1.5 &#181;mol (574.5 &#181;g) of azido-modified ethacrynic acid in 180 &#181;l of DMSO and add it to the solution. Degas the solution using Argon for 60 sec and quickly add 40 &#181;l from the Cu(II)/TBTA solution (10 mM in 55% (v/v) DMSO/water).</li>
		<li>Purge again with Argon, close tightly, and stir overnight.</li>
		<li>Monitor the progress of the reaction and purify the conjugate by RP-HPLC (mobile phase: A) 5% Acetonitrile, 5% TEAA, 90% ultrapure water; B) 65% Acetonitrile, 5% TEAA, 30% ultrapure water).24</li>
	</ol>
	</li>
</ol>
2. Controlling GST Activity by PDGF
<ol>
	<li>Preliminary Preparations
	<ol>
		<li>Prepare 50 ml of the Assay buffer by mixing 33.9 ml of phosphate buffered saline (PBSx1) with 16.1 ml of ultrapure water to achieve an 8 mM final phosphate concentration and add 23.8 mg of MgCl2 to achieve a 5 mM final concentration.</li>
		<li>Prepare a stock solution of GST M1-1 by dissolving the protein in a buffer containing 50 mM Tris pH 7.5, 50 mM NaCl, 1 mM dithiothreitol (DTT), and 5 mM ethylenediaminetetraacetic acid (EDTA) to a final concentration of 30 &#181;M. Divide this solution into small aliquots and store at -80 &#730;C. Dilute freshly, according to section 2.1.5.1, in the Assay buffer and keep on ice. 
		Note: The solution will be stable for about 5 hr, or until there is a reduction in the enzyme activity.</li>
		<li>Prepare the substrate according to the following instructions:
		<ol>
			<li>Dissolve 10 mg of reduced Glutathione (GSH) in 325 &#181;l of ultrapure water to a final concentration of 100 mM stock solution. Dilute 21 &#181;l from this stock solution in 979 &#181;l of Assay buffer for a final concentration of 2.1 mM working solution.</li>
			<li>Dissolve 10 mg of 2,4-dinitrochlorobenzene (CDNB) in 492 &#181;l of ethanol to a final concentration of 100 mM stock solution. Dilute 43.2 &#181;l from the stock solution in 956.8 &#181;l of an Assay buffer for a final concentration of 4.32 mM working solution.</li>
		</ol>
		</li>
		<li>In a 96-well plate, put each substrate in a separate line (12 wells). Insert at least 60 &#181;l into each well to allow fast and easy withdrawal of the solution. Cover the plate with an aluminum sheet for light protection.</li>
		<li>Prepare the following stock solutions in the Assay buffer:
		<ol>
			<li>Dilute GST M1-1 by 50 to a final concentration of 0.6 &#181;M of the dimer.</li>
			<li>Dilute the &#39;chemical transducer&#39; to a 30 &#181;M stock solution.</li>
			<li>Dilute PDGF to a final concentration of 40 &#181;M.</li>
			<li>Dilute PDGF aptamer to a final concentration of 250 &#181;M.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Measure the GST Activity in the Presence of the &#39;chemical transducer&#39; and PDGF.
	<ol>
		<li>Set up an experimental procedure in the plate reader for kinetic measurement.
		<ol>
			<li>Create a new experiment as a &#39;standard protocol&#39;.</li>
			<li>Press on &#39;procedure&#39; to open up the procedure settings window.</li>
			<li>In the pop up list on the upper side of the window, choose &#39;384 plate&#39; type according to the plate manufacturer.</li>
			<li>Press &#39;Read&#39; on the left menu.</li>
			<li>Regarding the detection method choose &#39;Absorbance&#39;.</li>
			<li>Regarding the read type choose &#39;End point&#39;.</li>
			<li>Write 340 nm on the wavelength window.</li>
			<li>Press on the &#39;Full plate&#39; bottom on the upper-right side and choose the well to be measured.</li>
			<li>Press &#39;ok&#39; to close the &#39;Read&#39; window.</li>
			<li>Choose &#39;start kinetic&#39; on the left menu.</li>
			<li>Make the run time 10 min.</li>
			<li>Select the minimum intervals option.</li>
			<li>Press &#39;ok&#39; to close the kinetic window.</li>
			<li>Drag the &#39;Read&#39; line into the kinetic measurement.</li>
			<li>Press the &#39;validate&#39; button and then the &#39;ok&#39; button.</li>
			<li>Save the experiment.</li>
			<li>Press the &#39;play&#39; button. A dialog box will appear &#8212; press the &#39;ok&#39; button only when the measurement should be started.</li>
		</ol>
		</li>
		<li>In order to perform the triplicates experiment, prepare four samples each containing 3.25 &#181;l of the &#39;chemical transducer&#39; and 3.25 &#181;l of GST M1-1. Add to each sample 0, 1.2, 2.4, or 4.9 &#181;l of PDGF and 123.5, 122.3, 121.1, or 118.6 &#181;l of the Assay buffer, respectively.</li>
		<li>Incubate the solution at room temperature for 10 min.</li>
		<li>In a 384-transparent well plate, insert 40 &#181;l of sample to each well. Insert the samples only in the odd wells or only in the even wells in the same line to allow the use of a multi-pipettor for substrate addition.</li>
		<li>Using a 12-channel multi-pipettor, quickly add 10 &#181;l from each of the substrates that were pre-prepared in the 96-well plate (section 2.1.4). Mix gently and quickly to avoid bubbles. Insert the plate into the reader and start the kinetic measurement. Since GST kinetics is quite fast, try to minimize the time between substrate addition and the beginning of the kinetic measurement.</li>
	</ol>
	</li>
	<li>GST Activation/Inhibition Cycles Mediated by the &#39;chemical transducer&#39;.
	<ol>
		<li>In order to perform the triplicates experiment, prepare 5 samples each containing 84.5 &#181;l of Assay buffer, 3.25 &#181;l of &#39;chemical transducer&#39;, and 3.25 &#181;l of GST M1-1. Incubate at room temperature for 3 min.</li>
		<li>Add 3.65 &#181;l of Assay buffer to sample 1 and 3.65 &#181;l of PDGF to samples 2-5. Incubate at room temperature for 3 min.</li>
		<li>Add 3.12 &#181;l of Assay buffer to samples 1-2 and 3.12 &#181;l of PDGF aptamer to samples 3-5. Incubate at room temperature for 3 min.</li>
		<li>Add 24.4 &#181;l of Assay buffer to samples 1-3 and 24.4 &#181;l of PDGF to samples 4-5. Incubate at room temperature for 3 min.</li>
		<li>Add 7.8 &#181;l of Assay buffer to samples 1-4 and 7.8 &#181;l of PDGF aptamer to sample 5. Incubate at room temperature for 5 min.</li>
		<li>In a 384-transparent well plate, insert 40 &#181;l of sample into each well. Insert samples only into the odd or only into the even wells in the same line.</li>
		<li>Using a 12-channel multi-pipettor, quickly add 10 &#181;l from each substrate (pre-prepared in the 96-well plate). Mix gently and quickly to avoid bubbles. Insert the plate into the reader and start the kinetic measurement.</li>
		<li>Calculate the V0 [mOD/min] under each condition by subtracting the OD measured at 340 nm at t = 0.5 min from the OD measured at 340 nm at t = 1.5 min to assess the activation/inhibition recyclability.25</li>
	</ol>
	</li>
	<li>Evaluate the Real-time Response of the &#39;chemical transducer&#39; to Changes in the Environment.
	<ol>
		<li>Real-time effect of PDGF addition
		<ol>
			<li>Set up an experimental procedure in the plate reader for kinetic measurement.</li>
			<li>Repeat steps 2.2.1.1-2.2.1.10.</li>
			<li>Make the run time 3.5 min.</li>
			<li>Select the minimum intervals option.</li>
			<li>Press &#39;ok&#39; to close the kinetic window.</li>
			<li>Drag the &#39;Read&#39; line into the kinetic measurement.</li>
			<li>Choose Plate Out/In on the left menu.</li>
			<li>Choose the option &#39;plate out (no dialog)&#39;.</li>
			<li>Choose the &#39;Delay&#39; option on the left menu and enter 30 sec.</li>
			<li>Choose Plate Out/In on the left menu.</li>
			<li>Choose the option &#39;plate in (no dialog)&#39;.</li>
			<li>Create a second kinetic measurement by repeating steps 2.2.1.4 - 2.2.1.14 but in section 2.2.1.11 set the kinetic to be 25 min instead of 10 min.</li>
			<li>Press the &#39;validate&#39; button and then the &#39;ok&#39; button.</li>
			<li>Save the experiment.</li>
			<li>Press the &#39;play&#39; button. A dialog box will appear&#160;&#8211; press the &#39;ok&#39; button only when the measurement should be started.</li>
			<li>Prepare two samples by mixing 1 &#181;l of GST M1-1 and 1 &#181;l of the &#39;chemical transducer&#39; in 38 &#181;l of Assay buffer. Insert the samples into two wells of a 384-transparent well plate, leaving an empty well between these two wells.</li>
			<li>Using a 12-channel multi-pipettor, quickly add 10 &#181;l from each substrate, mix gently and quickly to avoid bubbles, insert the plate into the reader and start the kinetic measurement.</li>
			<li>When the plate opens up (after 3.5 min), quickly add 1.125 &#181;l of PDGF to one of the wells, mix gently, and allow the plate to close up for the remaining kinetic measurements.</li>
		</ol>
		</li>
		<li>Real-time effect of adding the PDGF aptamer
		<ol>
			<li>Repeat steps 2.4.1.1-2.4.1.15.</li>
			<li>Prepare two samples by mixing 1 &#181;l of GST M1-1, 1 &#181;l of the &#39;chemical transducer&#39;, and 1.125 &#181;l of PDGF in 36.9 &#181;l of Assay buffer. Insert the samples into two wells of a 384-transparent well plate, leaving an empty well between these two wells.</li>
			<li>Using a 12-channel multi-pipettor, quickly add 10 &#181;l from each substrate, mix gently and quickly to avoid bubbles, insert the plate into the reader, and start the kinetic measurement.</li>
			<li>When the plate opens up (after 1.5 min), quickly add 1.2 &#181;l of PDGF aptamer to one of the wells, mix gently and allow the plate to close up for the remaining kinetic measurements.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Measure JS-K Prodrug Activation by GST in the Presence of the &#39;chemical transducer&#39; and PDGF.
	<ol>
		<li>Set up an experimental procedure in the plate reader for kinetic measurement.
		<ol>
			<li>Create a new experiment as a &#39;standard protocol&#39;.</li>
			<li>Press on &#39;procedure&#39; to open up the procedure settings window.</li>
			<li>In the pop up list on the upper side of the window choose &#39;384 plate&#39; type according to the plate manufacturer.</li>
			<li>Press &#39;Read&#39; on the left menu.</li>
			<li>Regarding the detection method chose &#39;Absorbance&#39;.</li>
			<li>Regarding the read type choose &#39;End point&#39;.</li>
			<li>Write 305 nm on the wavelength window.</li>
			<li>Press on the &#39;Full plate&#39; button on the upper-right side and choose the well to be measured.</li>
			<li>Press &#39;ok&#39; to close the &#39;Read&#39; window.</li>
			<li>Choose &#39;start kinetic&#39; on the left menu.</li>
			<li>Make the run time 10 min.</li>
			<li>Select the minimum intervals option.</li>
			<li>Press &#39;ok&#39; to close the kinetic window.</li>
			<li>Drag the &#39;Read&#39; line into the kinetic measurement.</li>
			<li>Press the &#39;validate&#39; button and then the &#39;ok&#39; button.</li>
			<li>Save the experiment.</li>
			<li>Press the &#39;play&#39; button. A dialog box will appear - press the &#39;ok&#39; button only when the measurement should be started.</li>
		</ol>
		</li>
		<li>For NO production measurements use a nitrite/nitrate calorimetric kit. In a 96-well plate insert 50 &#181;l of assay buffer into one row, 70 &#181;l of Griess I reagent into a second row, and 70 &#181;l of Griess II reagent into a third row.</li>
		<li>In order to perform the triplicates experiment, prepare four samples each containing 4.8 &#181;l of the &#39;chemical transducer&#39;. To sample 1, add 155.2 &#181;l of Assay buffer; to sample 2, add 3.2 &#181;l of GST-M1-1 and 152 &#181;l of Assay buffer; to sample 3, add 9.6 &#181;l of PDGF and 145.6 &#181;l of Assay buffer; and to sample 4, add 3.2 &#181;l of GST-M1-1, 9.6 &#181;l of PDGF, and 142.4 &#181;l of Assay buffer.</li>
		<li>Incubate the solution at room temperature for 10 min.</li>
		<li>In a 384-transparent well plate, insert 50 &#181;l of sample into each well. Insert samples only in the odd or only in the even wells in the same line to allow the use of a multi-pipettor for substrate addition.</li>
		<li>Add 0.54 &#181;l of JS-K (5 mM in DMSO) to each well.</li>
		<li>Using a 12-channel multi-pipettor, quickly add 10 &#181;l from the GSH solution (pre-prepared in the 96-well plate), mix gently and quickly to avoid bubbles. Insert the plate into the reader and start the kinetic measurement.</li>
		<li>Immediately after the kinetic measurement, using a 12-channel multi-pipettor, take 50 &#181;l from each sample into the assay buffer row in the pre-prepared 96-well plate and quickly add to it 50 &#181;l of Griess I reagent and 50 &#181;l of Griess II reagent. Incubate while protecting from light for 10 min at RT and measure the absorbance at 550 nm. 
		Note: The assay buffer and reagents volumes are dependent on the kits&#39; protocol.</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+class+of+bivalent+glutathione+S-transferase+inhibitors.">Novel class of bivalent glutathione S-transferase inhibitors.</a> Biochemistry. 42, 10418-10428 (2003).</li><li>Battle, C., Chu, X., Jayawickramarajah, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligonucleotide-based+systems+for+input-controlled+and+non-covalently+regulated+protein+binding.">Oligonucleotide-based systems for input-controlled and non-covalently regulated protein binding.</a> Supramol. Chem. , 1-16 (2013).</li></ol>

The authors have nothing to disclose.

We presented a method for designing and testing of a 'chemical transducer' that can induce artificial communication between two naturally unrelated proteins, GST and PDGF, without modifying the native proteins. The unnatural GST-PDGF communications could be detected in real time by using enzymatic assays that follow the changes in the activity of GST in the presence of the 'chemical transducer' and increasing the concentrations of PDGF. In addition to detecting the activation of GST by PDGF, these assays were used to fol...

Signal transduction pathways play a significant role in virtually every cellular process and allow the cell to rapidly respond to environmental signals.1 These pathways are often triggered by the binding of a signaling molecule to an extracellular receptor, which results in activation of intracellular enzymes. Amplification and propagation of this signal within the cell is mediated by the function of signaling proteins that form a network of protein-protein interactions in which enzymes are reversibly activated with high specificity. Because dysregulation of these networks frequently leads to cancer development, there has been much interest in establishing &#39;signal transduction therapy of cancer&#39;,2 whereby drugs are designed to disrupt malignant signaling pathways. We have recently proposed an alternative approach to signal transduction therapy that relies on the ability of drugs to generate unnatural signal transduction pathways.3 In particular, we believe that by designing synthetic agents that mimic the function of signaling proteins, it would be possible to modulate the cell&#39;s function indirectly. For example, these artificial networks may enable protein biomarkers to activate enzymes that cleave prodrugs. Alternatively, these signaling protein mimetics might be able to activate unnatural cell signaling pathways, resulting in therapeutic effects.
To demonstrate the feasibility of this approach, we have recently created a synthetic &#39;chemical transducer&#39;4 that enables platelet-derived growth factor (PDGF) to trigger the cleavage of an anticancer prodrug by activating glutathione-s-transferase (GST), which is not its natural binding partner. The structure of this &#39;transducer&#39; consists of an anti-PDGF DNA aptamer that is modified with a bivalent inhibitor for GST. Hence, this synthetic agent belongs to a family of molecules with binding sites to different proteins,5-7 such as chemical inducers of dimerization (CIDs)8-10 and also to the group of protein-binders based on oligonucleotide-synthetic molecule conjugates.11-21
The general principles underlying the design of such systems is described herein and detailed protocols for synthesizing and testing the function of this &#39;transducer&#39; with conventional enzymatic assays are provided. This work is intended to facilitate developing additional &#39;transducers&#39; of this class, which may be used to mediate intracellular protein-protein communication and consequently, to induce artificial cell signaling pathways.
Figure 1 schematically describes the operating principles of synthetic &#39;chemical transducers&#39; that can mediate unnatural protein-protein communications. In this illustration, a &#39;chemical transducer&#39;, which integrates synthetic binders for proteins I and II (binders I and II), enables protein II to trigger the catalytic activity of protein I, which is not its natural binding partner. In the absence of protein II, the transducer binds the catalytic site of the enzyme (protein I) and inhibits its activity (Figure 1, state ii). The binding of the &#39;transducer&#39; to protein II, however, promotes interactions between binder I and the surface of protein II (Figure 1, state iii), which reduces its affinity toward protein I. As a result, the effective concentration of the &#39;free&#39; transducer in the solution is reduced, which leads to dissociation of the transducer-protein I complex and to reactivation of protein I (Figure 1, state iv). Taken together, these steps highlight three fundamental principles underlying the design of efficient &#39;transducers&#39;: (1) a &#39;transducer&#39; should have a specific binder for each of the protein targets, (2) the interaction between binder II and protein II should be stronger than the interaction between binder I and protein I, and (3) binder I must be able to interact with the surface of protein II. This last principle does not necessarily require that binder I alone would have a high affinity and selectivity toward protein II. Instead, it is based on our recent studies which showed that bringing a synthetic molecule in proximity to a protein is likely to promote interactions between this molecule and the surface of the protein.19,22,23
<img alt="Figure 1" src="/files/ftp_upload/54396/54396fig1.jpg" />
Figure 1: Operating principles of &#39;chemical transducers&#39;. When the &#39;chemical transducer&#39; is added to an active protein I (state i), it binds to its active site through binder I and inhibits its activity (state ii). In the presence of protein II, however, the unbound &#39;chemical transducer&#39; interacts with protein II through binder II, which promotes interactions between binder I and the surface of protein II. This induced binder I-protein II interaction reduces the effective concentration of binder I, which leads to dissociation of the &#39;transducer&#39;-protein I complex and to protein I reactivation (state iv). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54396/54396fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>1-chloro-2,4-dinitrobenzene</td><td>Sigma-Aldrich</td><td>237329</td><td></td></tr><tr><td>Acetic acid</td><td>Bio Lab</td><td>01070521</td><td></td></tr><tr><td>Acetnitrile</td><td>J.T.Baker</td><td>9017-03</td><td></td></tr><tr><td>Ascorbic acid</td><td>Sigma-Aldrich</td><td>A4544</td><td></td></tr><tr><td>Copper(II) Sulfate pentahydrate</td><td>Merck-Millipore</td><td>102790</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Merck-Millipore</td><td>802912</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Biological Industries</td><td>02-023-5A</td><td></td></tr><tr><td>Ethacrynic acid</td><td>Tokyo Chemical Industry Co. Ltd</td><td>E0526</td><td></td></tr><tr><td>Glutathione-s-transferase M1-1</td><td>Israel Structural Proteomics Center (Weizmann Institute of Science, Rehovot, Israel)</td><td></td><td></td></tr><tr><td>JS-K</td><td>Sigma-Aldrich</td><td>J4137</td><td></td></tr><tr><td>L-glutathione reduced</td><td>Sigma-Aldrich</td><td>G4251</td><td></td></tr><tr><td>Magnesium Chloride</td><td>J.T.Baker</td><td>0162</td><td></td></tr><tr><td>nitrate/nitrite colorimetric assay kit</td><td>Cayman Chemical</td><td>780001</td><td></td></tr><tr><td>Oligonucleotides</td><td>W. M. Keck Foundation Biotechnology at Yale University</td><td></td><td>custom order</td></tr><tr><td>PDGF-BB</td><td>Israel Structural Proteomics Center (Weizmann Institute of Science, Rehovot, Israel)</td><td></td><td></td></tr><tr><td>TBTA</td><td>Sigma-Aldrich</td><td>678937</td><td></td></tr><tr><td>Triethylamine</td><td>Sigma-Aldrich</td><td>T0886</td><td></td></tr><tr><td>Desalting column</td><td>GE Healthcare</td><td>illustra MicroSpin G-25 Columns</td><td></td></tr><tr><td>HPLC</td><td>Waters&amp;nbsp;</td><td>2695 separation module</td><td></td></tr><tr><td>HPLC column</td><td>Waters</td><td>XBridgeTM OST C18 column (2.5 &amp;mu;M, 4.6 mm &amp;times; 50 mm)</td><td></td></tr><tr><td>HPLC column</td><td>Waters</td><td>XBridgeTM OST C18 column (2.5&amp;nbsp;&amp;mu;M, 10 mm &amp;times; 50 mm)</td><td></td></tr><tr><td>Plate reader</td><td>BioTek</td><td>synergy H4 hybrid</td><td></td></tr></tbody></table>

mimicking the function of signaling proteins: toward artificial signal transduction therapy

Signal transduction pathways, which control the response of cells to various environmental signals, are mediated by the function of signaling proteins that interact with each other and activate one other with high specificity. Synthetic agents that mimic the function of these proteins might therefore be used to generate unnatural signal transduction steps and consequently, alter the cell's function. We present guidelines for designing 'chemical transducers' that can induce artificial communication between native proteins. In addition, we present detailed protocols for synthesizing and testing a specific 'transducer', which can induce communication between two unrelated proteins: platelet-derived growth-factor (PDGF) and glutathione-S-transferase (GST). The way by which this unnatural PDGF-GST communication could be used to control the cleavage of an anticancer prodrug is also presented, indicating the potential for using such systems in 'artificial signal transduction therapy'. This work is intended to facilitate developing additional 'transducers' of this class, which may be used to mediate intracellular protein-protein communication and consequently, to induce artificial cell signaling pathways.

The design, synthesis, and mechanism of action of a &#39;chemical transducer&#39; that can induce artificial communication between PDGF and GST are presented in Figure 2. The structure of the &#39;transducer&#39; integrates a PDGF DNA aptamer and a bis-ethacrynic amide (bEA), which is a known GST inhibitor (Figure 2a).19 These binders enable the &#39;transducer&#39; to bind both PDGF and GST with different affinities, namely, with dissociation ...

Watch this Scientific Journal Video about Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy at JoVE.com

Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy

We present guidelines for developing synthetic 'chemical transducers' that can induce communication between naturally unrelated proteins. In addition, detailed protocols are presented for synthesizing and testing a specific 'transducer' that enables a growth factor to activate a detoxifying enzyme and consequently, to regulate the cleavage of an anticancer prodrug.

Signal transduction pathways, which control the response of cells to various environmental signals, are mediated by the function of signaling proteins ...

mimicking-function-signaling-proteins-toward-artificial-signal

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

6.9K Views.  Weizmann Institute of Science. We present guidelines for developing synthetic 'chemical transducers' that can induce communication between naturally unrelated proteins. In addition, detailed protocols are presented for synthesizing and testing a specific 'transducer' that enables a growth factor to activate a detoxifying enzyme and consequently, to regulate the cleavage of an anticancer prodrug. 

Video: Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Developmental Biology

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.

Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

Genetics

Engineering Intracellular Protein Sensors in Mammalian Cells

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells to sense and respond to these biomarkers may help the understanding of molecular mechanisms underlying pathologies, as well as to develop new cell-based therapies. While several systems that detect extracellular proteins have been developed, a modular framework that can be easily re-engineered to sense different intracellular proteins was missing.
Here, we describe a protocol to implement a modular genetic platform that senses intracellular proteins and activates a specific cellular response. The device operates on intracellular antibodies or small peptides to sense with high specificity the protein of interest, triggering the transcriptional activation of output genes, through a TEV protease (TEVp)-based actuation module. TEVp is a viral protease that selectively cleaves short cognate peptides and is widely used in biotechnology and synthetic biology for its high orthogonality to the cleavage site. Specifically, we engineered devices that recognize and respond to protein-biomarkers of viral infections and genetic diseases, including mutated huntingtin, NS3 serine-protease, Tat and Nef proteins to detect Huntington&#8217;s disease, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections, respectively. Importantly, the system can be hand tailored for the desired input-output functional outcome, such as fluorescent readouts for biosensors, stimulation of antigen presentation for immune response, or initiation of apoptosis to eliminate unhealthy cells.

Here, we present a protocol for engineering genetically-encoded intracellular protein sensor-actuator(s). The device specifically detects target proteins through intracellular antibodies (intrabodies) and responds by switching on gene transcriptional output. A general framework is built to rapidly replace intrabodies, enabling rapid detection of any desired protein, without altering the general architecture.

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Immunology and Infection

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of sphingolipids in the cell membrane, is one of the best-characterized LPLs that regulates a variety of cellular physiological responses via signaling pathways mediated by sphingosine 1-phosphate receptors (S1PRs). This implicated that the S1P-S1PRs signaling system is a remarkable potential therapeutic target for disorders, including multiple sclerosis (MS), autoimmune disorders, cancer, inflammation, and even COVID-19. S1PRs, a small subset of the class A G-protein coupled receptor (GPCR) family, are composed of five subtypes: S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. The lack of detailed structural information, however, impedes the drug discovery targeting S1PRs. Here, we applied the cryo-electron microscopy method to solve the structure of the S1P-S1PRs complex, and elucidated the mechanism of activation, selective drug recognition, and G-protein coupling by using cell-based functional assays. Other lysophospholipid receptors (LPLRs) and GPCRs can also be studied using this strategy.

S1P exerts its diverse physiological effects through the S1P receptors (S1PRs) subfamily. Here, a pipeline is described to expound on the structures and function of S1PRs.

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of ...

Investigating Development with Optogenetic Signaling Manipulation in Zebrafish

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

Author Spotlight: Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Biology

Signal Transduction: Overview

Cells respond to many types of information, often through receptor proteins positioned on the membrane. They respond to chemical signals, such as hormones, neurotransmitters, and other signaling molecules, initiating a series of molecular reactions to produce an appropriate response. This is called signal transduction. Cells also coordinate different responses elicited by the same signaling molecule via mediators, allowing molecular cross-talk.
Typically, signal transduction involves three steps: (1) reception, (2) transduction, and (3)&#160; response. In most signal reception, a membrane-impermeable molecule, or ligand, causes a change in a membrane receptor; however, some signaling molecules, such as hormones, can cross the membrane to reach their internal receptors. The membrane receptor then sends this signal to other cellular molecules, typically intracellular messengers, which convert the message into a cellular response. Commonly known receptors on the cell surface or inside the cell include G protein&#8211;coupled receptors (GPCRs), ligand-gated ion channels, enzyme-linked receptors, and nuclear receptors. GPCRs are membrane-spanning proteins with an extracellular binding ligand binding site. Most drugs target GPCR and activate or block its activity. Once activated, these receptors couple with heterotrimeric G proteins and activate them. The activated heterotrimeric G proteins then move across the membrane to stimulate various downstream effectors to generate second messengers. The second messengers, such as cyclic AMP, carry the signal to target proteins and elicit a cellular response.
Another group of receptors, ligand-gated ion channels, are involved in fast synaptic neurotransmission by rapidly changing the ion flow across the cell. Various drugs bind at the ligand binding site, allosteric site, or channel pore to regulate the opening and closing of the ion channel. This leads to membrane potential changes affecting cellular processes such as neurotransmission, muscle contraction, and muscle relaxation.
On the other hand, the enzyme-linked receptors are unique transmembrane receptors that either have an intracellular enzymatic domain or are attached to an enzyme to start a phosphorylation cascade, phosphorylating downstream protein substrates and activating them.. These receptors are involved in cell proliferation, growth, differentiation, repair, and immune responses.
Lastly, various lipophilic ligands and drugs diffuse through the membrane to bind and activate nuclear receptors in the cytosol or directly bind to DNA inside the nucleus. They are ligand-activated transcription factors that regulate the transcription of genes involved in reproduction and metabolic pathways.
The intracellular response generated through these different types of receptors includes changes at both gene and protein levels. The receptors also bind to a variety of ligands, ranging from neurotransmitters, hormones, vitamins, lipids, or lipoproteins. Pharmaceutical companies exploit this to design drugs of different structural and chemical properties, targeting specific signaling pathways involved in a particular disease.

Cells respond to many types of information, often through receptor proteins positioned on the membrane. They respond to chemical signals, such as ...

Education

JoVE Core

Pharmacology

Unit 1

Pharmacodynamics

KEY TERMS AND CONCEPTS

Interactions Between Signaling Pathways

Signaling cascades usually lack linearity. Multiple pathways interact and regulate one another, allowing cells to integrate and respond to diverse environmental stimuli.
Convergence and divergence, and cross-talk between signaling pathways
Two distinct signaling pathways can converge on a single functional unit, which may either be a single protein or a complex of proteins. The response is either functionally distinct or synergistic between the two pathways but different from the response observed when either pathway is individually activated.
Similarly, pathways diverge when an upstream signaling component interacts with multiple downstream components. For example, the action of a neurotransmitter on a target cell can cause distinct effects to appear together. Together, convergence and divergence create networks of cross-interaction where relatively simple signals elicit complex responses, provide functional flexibility, and preserve signal specificity.
The growth inhibitory effect of the tumor suppressor protein, p53, is due to its interaction with multiple signaling pathways. Depending on the stimulus, it is activated when phosphorylated by one of the several protein kinases. The activated p53 can act as a transcription factor to induce or suppress genes from different signaling pathways.
MAP kinases phosphorylate and activate p53 in response to the signal, which causes cell cycle arrest and apoptosis as well as other p53-mediated cellular responses. p53 can also negatively regulate MAP kinase signaling via the transcriptional activation of phosphatases that inhibit MAP kinases. It is interesting to note that alteration of both p53 and MAPK signaling pathways is observed in a majority of human cancers causing dysregulated cell proliferation.

Signaling cascades usually lack linearity. Multiple pathways interact and regulate one another, allowing cells to integrate and respond to diverse ...

Cell Biology

Principles of Cell Signaling

Amplifying Signals via Second Messengers

Many receptor binding ligands are hydrophilic; they do not cross the cell membrane but bind to cell-surface receptors. Thus, their message must be relayed by second messengers present in the cell cytoplasm. There are several second messenger pathways, each with its own way of relaying information. For example, the G protein-coupled receptors can activate both phosphoinositol and cyclic AMP (cAMP) second messenger pathways. The phosphoinositol pathway is active when the receptor induces phospholipase C to hydrolyze the phospholipid, phosphatidylinositol biphosphate (PIP2), into two second messengers: diacylglycerol (DAG) and inositol triphosphate (IP3). DAG remains near the cell membrane and activates protein kinase C (PKC). IP3 translocates to the endoplasmic reticulum (ER) and becomes the opening ligand for calcium ion channels on the ER membrane, releasing calcium into the cytoplasm.
In the cAMP pathway, the activated receptor induces adenylate cyclase to produce multiple copies of cAMP from nearby adenosine triphosphate (ATP) molecules. cAMP can stimulate protein kinase A (PKA), open calcium ion channels, and activate the enzyme&#8211;&#160; Exchange-protein activated by cAMP (Epac).
Cyclic guanosine monophosphate (cGMP) is similar to cAMP. cGMP is synthesized from guanosine triphosphate (GTP) molecules when guanylyl cyclase is activated. As a second messenger, cGMP induces protein kinase G (PKG), which has many overlapping functions with PKA. However, PKG expression is restricted to vascular tissues, lungs, and the brain.
Phosphatidylinositol triphosphate (PIP3) is a second messenger derived from the phosphorylation of PIP2. This event is triggered when growth factors bind the receptor tyrosine kinase. PIP3 recruits Akt (aka. protein kinase B) to the membrane. This kinase is intimately involved in regulating cell survival pathways&#8211; including proliferation, apoptosis, and migration.

Many receptor binding ligands are hydrophilic; they do not cross the cell membrane but bind to cell-surface receptors. Thus, their message must be relayed ...

Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy

Summary

Explore More Videos

Chapters in this video

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

Engineering Intracellular Protein Sensors in Mammalian Cells

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors

Optogenetic Signaling Activation in Zebrafish Embryos

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Signal Transduction: Overview

Interactions Between Signaling Pathways

Amplifying Signals via Second Messengers