Name: mimicking the function of signaling proteins: toward artificial signal transduction therapy
Uploaded: 2016-09-29T14:00:00
Description: Watch this Scientific Journal Video about Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy at JoVE.com

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 ...

<ol>
	<li>Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling—2000+and+Beyond.">Signaling—2000 and Beyond.</a> Cell. 100, 113-127 (2000).</li><li>Levitzki, A., Klein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+therapy+of+cancer.">Signal transduction therapy of cancer.</a> Mol Aspects Med. 31, 287-329 (2010).</li><li>Peri-Naor, R., Motiei, L., Margulies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+signal+transduction+therapy:+a+futuristic+approach+to+disease+treatment.">Artificial signal transduction therapy: a futuristic approach to disease treatment.</a> Future Med. Chem. 7, 2091-2093 (2015).</li><li>Peri-Naor, R., Ilani, T., Motiei, L., Margulies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Protein+Communication+and+Enzyme+Activation+Mediated+by+a+Synthetic+Chemical+Transducer.">Protein-Protein Communication and Enzyme Activation Mediated by a Synthetic Chemical Transducer.</a> J. Am. Chem. Soc. 137, 9507-9510 (2015).</li><li>Corson, T. W., Aberle, N., Crews, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Applications+of+Bifunctional+Small+Molecules:+Why+Two+Heads+Are+Better+Than+One.">Design and Applications of Bifunctional Small Molecules: Why Two Heads Are Better Than One.</a> ACS Chem. Biol. 3, 677-692 (2008).</li><li>Rutkowska, A., Schultz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Tango:+The+Toolbox+to+Capture+Interacting+Partners.">Protein Tango: The Toolbox to Capture Interacting Partners.</a> Angew. Chem. Int. Ed. 51, 8166-8176 (2012).</li><li>Meyer, C., K&#246;hn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Molecular+T&#234;te-&#224;-T&#234;te+Arranged+by+a+Designed+Adaptor+Protein.">A Molecular T&#234;te-&#224;-T&#234;te Arranged by a Designed Adaptor Protein.</a> Angew. Chem. Int. Ed. 51, 8160-8162 (2012).</li><li>Klemm, J. D., Schreiber, S. L., Crabtree, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerization+as+a+Regulatory+Mechanism+in+Signal+Transduction.">Dimerization as a Regulatory Mechanism in Signal Transduction.</a> Annu. Rev. Immunol. 16, 569-592 (1998).</li><li>DeRose, R., Miyamoto, T., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+signaling+at+will:+chemically-inducible+dimerization+(CID)+techniques+resolve+problems+in+cell+biology.">Manipulating signaling at will: chemically-inducible dimerization (CID) techniques resolve problems in cell biology.</a> Pflugers Arch. 465, 409-417 (2013).</li><li>Gestwicki, J. E., Marinec, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+control+over+protein-protein+interactions:+beyond+inhibitors.">Chemical control over protein-protein interactions: beyond inhibitors.</a> Comb. Chem. High Throughput. Screen. 10, 667-675 (2007).</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. 25, 848-862 (2013).</li><li>Diezmann, F., Seitz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-guided+display+of+proteins+and+protein+ligands+for+the+interrogation+of+biology.">DNA-guided display of proteins and protein ligands for the interrogation of biology.</a> Chem. Soc. Rev. 40, 5789-5801 (2011).</li><li>R&#246;glin, L., Ahmadian, M. R., Seitz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-Controlled+Reversible+Switching+of+Peptide+Conformation+and+Bioactivity.">DNA-Controlled Reversible Switching of Peptide Conformation and Bioactivity.</a> Angew. Chem. Int. Ed. 46, 2704-2707 (2007).</li><li>R&#246;glin, L., Altenbrunn, F., Seitz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+and+RNA-Controlled+Switching+of+Protein+Kinase+Activity.">DNA and RNA-Controlled Switching of Protein Kinase Activity.</a> ChemBioChem. 10, 758-765 (2009).</li><li>Harris, D. 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=DNA-Small+Molecule+Chimera+with+Responsive+Protein-Binding+Ability.">DNA-Small Molecule Chimera with Responsive Protein-Binding Ability.</a> J. Am. Chem. Soc. 130, 14950-14951 (2008).</li><li>Harris, D. C., Saks, B. R., 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=Protein-Binding+Molecular+Switches+via+Host-Guest+Stabilized+DNA+Hairpins.">Protein-Binding Molecular Switches via Host-Guest Stabilized DNA Hairpins.</a> J. Am. Chem. Soc. 133, 7676-7679 (2011).</li><li>Kim, Y., Cao, Z., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+assembly+for+high-performance+bivalent+nucleic+acid+inhibitor.">Molecular assembly for high-performance bivalent nucleic acid inhibitor.</a> Proc. Nat. Acad. Sci. U.S.A. 105, 5664-5669 (2008).</li><li>Han, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Logical+Molecular+Circuit+for+Programmable+and+Autonomous+Regulation+of+Protein+Activity+Using+DNA+Aptamer-Protein+Interactions.">A Logical Molecular Circuit for Programmable and Autonomous Regulation of Protein Activity Using DNA Aptamer-Protein Interactions.</a> J. Am. Chem. Soc. 134, 20797-20804 (2012).</li><li>Motiei, L., Pode, Z., Koganitsky, A., Margulies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Protein+Surface+Sensors+as+a+Tool+for+Analyzing+Small+Populations+of+Proteins+in+Biological+Mixtures.">Targeted Protein Surface Sensors as a Tool for Analyzing Small Populations of Proteins in Biological Mixtures.</a> Angew. Chem. Int. Ed. 53, 9289-9293 (2014).</li><li>Ranallo, S., Rossetti, M., Plaxco, K. W., Vall&#233;e-B&#233;lisle, A., Ricci, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Modular,+DNA-Based+Beacon+for+Single-Step+Fluorescence+Detection+of+Antibodies+and+Other+Proteins.">A Modular, DNA-Based Beacon for Single-Step Fluorescence Detection of Antibodies and Other Proteins.</a> Angew. Chem. Int. Ed. 54, 13214-13218 (2015).</li><li>Franzini, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Structure-Activity+Relationships+from+Screening+a+Structurally+Compact+DNA-Encoded+Chemical+Library.">Identification of Structure-Activity Relationships from Screening a Structurally Compact DNA-Encoded Chemical Library.</a> Angew. Chem. Int. Ed. 54, 3927-3931 (2015).</li><li>Unger-Angel, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+recognition+by+bivalent,+'turn-on'+fluorescent+molecular+probes.">Protein recognition by bivalent, 'turn-on' fluorescent molecular probes.</a> Chem. Sci. 6, 5419-5425 (2015).</li><li>Nissinkorn, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+Protein+Surfaces+with+Targeted+Fluorescent+Receptors.">Sensing Protein Surfaces with Targeted Fluorescent Receptors.</a> Chem. Eur. J. 21, 15981-15987 (2015).</li><li>Huber, C. G., Oefner, P. J., Bonn, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Liquid+Chromatography+of+Oligonucleotides+on+Nonporous+Alkylated+Styrene-Divinylbenzene+Copolymers.">High-Resolution Liquid Chromatography of Oligonucleotides on Nonporous Alkylated Styrene-Divinylbenzene Copolymers.</a> Anal. Biochem. 212, 351-358 (1993).</li><li>Lyon, R. P., Hill, J. J., Atkins, W. 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>

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/files/ftp_upload/54396/54396fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1144">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">1-chloro-2,4-dinitrobenzene</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">237329</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Acetic acid</td>
			<td style="width:344px;">Bio Lab</td>
			<td style="width:397px;">01070521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Acetnitrile</td>
			<td style="width:344px;">J.T.Baker</td>
			<td style="width:397px;">9017-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Ascorbic acid</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">A4544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Copper(II) Sulfate pentahydrate</td>
			<td style="width:344px;">Merck-Millipore</td>
			<td style="width:397px;">102790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Dimethyl sulfoxide</td>
			<td style="width:344px;">Merck-Millipore</td>
			<td style="width:397px;">802912</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:236px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:344px;">Biological Industries</td>
			<td style="width:397px;">02-023-5A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Ethacrynic acid</td>
			<td style="width:344px;">Tokyo Chemical Industry Co. Ltd</td>
			<td style="width:397px;">E0526</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:236px;">Glutathione-s-transferase M1-1</td>
			<td style="width:344px;">Israel Structural Proteomics Center (Weizmann Institute of Science, Rehovot, Israel)</td>
			<td style="width:397px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">JS-K</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">J4137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">L-glutathione reduced</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">G4251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Magnesium Chloride</td>
			<td style="width:344px;">J.T.Baker</td>
			<td style="width:397px;">0162</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:236px;">nitrate/nitrite colorimetric assay kit</td>
			<td style="width:344px;">Cayman Chemical</td>
			<td style="width:397px;">780001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:236px;">Oligonucleotides</td>
			<td style="width:344px;">W. M. Keck Foundation Biotechnology at Yale University</td>
			<td style="width:397px;"></td>
			<td>custom order</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:236px;">PDGF-BB</td>
			<td style="width:344px;">Israel Structural Proteomics Center (Weizmann Institute of Science, Rehovot, Israel)</td>
			<td style="width:397px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">TBTA</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">678937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Triethylamine</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">T0886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Desalting column</td>
			<td style="width:344px;">GE Healthcare</td>
			<td style="width:397px;">illustra MicroSpin G-25 Columns</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">HPLC</td>
			<td style="width:344px;">Waters&#160;</td>
			<td>2695 separation module</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">HPLC column</td>
			<td style="width:344px;">Waters</td>
			<td style="width:397px;">XBridgeTM OST C18 column (2.5 &#956;M, 4.6 mm &#215; 50 mm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">HPLC column</td>
			<td style="width:344px;">Waters&#160;</td>
			<td style="width:397px;">XBridgeTM OST C18 column (2.5&#956;M, 10 mm &#215; 50 mm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Plate reader</td>
			<td style="width:344px;">BioTek</td>
			<td style="width:397px;">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 ...

Research

JoVE Journal

Biochemistry

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...