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 ...

The overall goal of this experiment is to spectroscopically follow the unnatural communication between two proteins induced by a synthetic chemical transducer. The will demonstrate the possibility of developing synthetic analogs of signalling proteins that will be able to carry out signal transduction pathways not found in nature. The ability to follow unnatural protein to protein communication by common spectrophotometer could help develop and improve the function of artificial chemical transducers and bring us closer to achieving artificial signal transduction therapy.The chemical transducer is composed of a platelet derived growth factor, or PDGF aptamer. A bivalent ethacrynic amide GST inhibitor, a fluorophore and a quencher. The synthesis of the chemical transducer will not be demonstrated in this video.But is described in the text protocol. The operating principles of the chemical transducer are illustrated here. The chemical transducer inhibits the enzymatic activity of GST, when the two EA groups bind the enzymes active sites.Addition of PDGF leads to the formation of the PDGF chemical transducer complex. Which disrupts the transducer-GST interaction, therefore restoring GST enzymatic activity. The following addition of an unmodified PDGF aptamer releases the chemical transducer and allows it to re-inhibit the enzyme.Prior to starting the experiments for examining control of GST activity by PDGF, set up an experimental procedure in the plate reader for kinetic measurement. Create a new experiment as a standard protocol. Select Procedure to open the Procedure Settings window.In the pop-up list on the upper side of the window, choose the 384 Plate Type according to the plate manufacturer. Select Read on the left menu. Choose Absorbance for the Detection Method.And Endpoint for the Read Type. In the Wavelength window, type 340 nanometers. Select Full Plate, and choose the wells to be measured.Then click OK to close the Read window. On the left menu, choose Start Kinetic. Set the Run Time as 10 minutes.Select the Minimum Intervals option. Then click OK to close the Kinetic window. Drag the Read Line into the Kinetic Measurement.Select the Validate button, followed by the OK button. Save the experiment. Select the Play button.A dialog box will appear, but do not select the OK button until ready to start the kinetic measurement. Once the plate reader has been set up, the next step is to prepare the samples for measuring GST activity in the presence of the chemical transducer and PDGF. In order to test four concentrations of PDGF in triplicate, prepare four samples where each contains 3.25 microliters of the chemical transducer, and 3.25 microliters of GST M-1.Add to each sample, zero, 1.2, 2.4 or 4.9 microliters of PDGF respectively. Then add the appropriate volume of the assay buffer to each sample to bring the total volume to 130 microliters. Incubate the samples at room temperature for 10 minutes.After 10 minutes, transfer 40 microliters of each sample to the wells of a 384 transparent well plate. Transfer each of the four samples to either the odd or the even wells in the same row only. To allow the use of a multi-pipetter for substrate addition.Next, use a 12-channel multi-pipetter to quickly add to each sample 10 microliters of each substrate previously prepared in a 96 well plate. Mix quickly, but gently, to avoid bubbles. Immediately insert the plate into the plate reader, and start the kinetic measurement by selecting the OK button in the dialog box.To examine GST activation inhibition cycles mediated by the chemical transducer, prepare five samples, each containing 84.5 microliters of assay buffer. 3.25 microliters of chemical transducer, and 3.25 microliters of GST M1-1. Incubate at room temperature for three minutes.Add 3.65 microliters of assay buffer to sample one, and 3.65 microliters of PDGF to samples two, three, four and five. Incubate at room temperature for three minutes. Add 3.12 microliters of assay buffer to samples one and two, and 3.12 microliters of PDGF aptamer to samples three, four and five.Incubate at room temperature for three minutes. Add 24.4 microliters of assay buffer to samples one, two and three. And 24.4 microliters of PDGF to samples four and five.Incubate at room temperature for three minutes. Add 7.8 microliters of assay buffer to samples one to four, and 7.8 microliters of PDGF aptamer to sample five. Incubate at room temperature for five minutes.Transfer the samples to a 384 transparent well plate. To perform the experiment in triplicate, and quickly add the substrate to the samples. Insert the plate into the reader, and start the kinetic measurement.To begin this assay, set up an experimental procedure in the plate reader for kinetic measurement as described in the protocol text. Prepare two samples in two wells of a 384 transparent well plate. For each well, mix one microliter of GST M1-1 and one microliter of the chemical transducer in 38 microliters of assay buffer.Leave an empty well between the two wells. Using a 12-channel multi-pipetter, quickly add 10 microliters of substrate to each well. And mix gently and quickly to avoid bubbles.Immediately insert the plate into the reader and start the kinetic measurement. When the plate reader opens up after 3.5 minutes, quickly add 1.125 microliters of PDGF to one of the wells, mix gently, and allow the plate to re-enter the reader for the remaining kinetic measurements. Prior to preparing the samples for this experiment, set up an experimental procedure in the plate reader for kinetic measurement as described in the protocol text.A nitrite/nitrate colorimetric kit will be used for measuring nitric oxide production. In a 96 well plate, dispense 50 microliters of assay buffer per well into the first row. 70 microliters of Griess One Reagent per well into the second row.And 70 microliters of Griess Two Reagent per well into the third row. Prepare four samples, each containing 4.8 microliters of the chemical transducer. To sample one, add 155.2 microliters of assay buffer.To sample two, add 3.2 microliters of GST M1-1, and 152 microliters of assay buffer. To sample three, add 9.6 microliters of PDGF, and 145.6 microliters of assay buffer. And to sample four, add 3.2 microliters of GST M1-1, 9.6 microliters of PDGF, and 142.4 microliters of assay buffer.Incubate the samples at room temperature for 10 minutes. Dispense 50 microliters of sample per well in a 384 transparent well plate to perform the experiment in triplicate. Dispense samples only to the odd or even wells in the same line to allow the use of a multi-pipetter for substrate addition.Add 0.54 microliters of JS-K prodrug to each sample. Quickly add to each sample 10 microliters of glutathione solution and mix gently and quickly to avoid bubbles. Insert the plate into the reader, and start the kinetic measurement.Immediately after the kinetic measurement, transfer 50 microliters of each sample to each well of the first row of the 96 well plate with assay buffer. Then transfer 50 microliters from each well of the second row of the plate into the wells of the first row of the plate. And 50 microliters per well from the third row of the plate into the wells of the first row of the plate.Incubate while protecting from light for 10 minutes at room temperature. Before measuring the absorbance at 550 nanometers. Measuring GST activity without the chemical transducer, and with the chemical transducer showed that the chemical transducer inhibits GST activity.GST is reactivated by the addition of increasing concentrations of PDGF to the GST-chemical transducer complex. Sequential additions of PDGF, and an unmodified PDGF aptamer to the GST-chemical transducer complex revealed that the communication between PDGF and GST was reversible. The addition of PDGF to the GST-transducer complex resulted in a rapid increase in GST activity, 3.5 minutes after adding substrates.While the addition of a PDGF aptamer to a mixture of GST, PDGF and the chemical transducer resulted in a decrease in GST activity. These results indicate that the system responds in real-time to changes in its environment. Subsequently, the ability of this system to control prodrug activation was investigated using JS-K, an anti-cancer prodrug that is activated by GST to release toxic nitric oxide.Measurements of nitric oxide amounts released upon the addition of JS-K to the chemical transducer and different combinations of GST and PDGF confirmed that only the presence of both GST and PDGF activates JS-K. This all shows how a synthetic agent can enable a growth factor to trigger the catalytic activity of Glutathione S-Transferase, which is not its natural enzyme partner. Future biomimetics of this class may therefore be used to alter the response of cells to environmental signals or provide them with new properties.

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

Research

JoVE Journal

Biochemistry

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 several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

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

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

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 the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

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

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

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

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

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 importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

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

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 solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

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

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

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 PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA 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

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

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

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

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 being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

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

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An important first step in any membrane-dependent reaction is binding of protein on the phospholipid membrane. An approach to studying protein interaction with lipid membranes has been developed using fluorescently labeled proteins and flow cytometry. This method allows the study of protein-membrane interactions using live cells and natural or artificial phospholipid vesicles. The advantage of this method is the simplicity and availability of reagents and equipment. In this method, proteins are labeled using fluorescent dyes. However, both self-made and commercially available, fluorescently labeled proteins can be used. After conjugation with a fluorescent dye, the proteins are incubated with a source of the phospholipid membrane (microvesicles or cells), and the samples are analyzed by flow cytometry. The obtained data can be used to calculate the kinetic constants and equilibrium Kd. In addition, it is possible to estimate the approximate number of protein binding sites on the phospholipid membrane using special calibration beads.

Here, we describe a set of methods for characterizing the interaction of proteins with membranes of cells or microvesicles.

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...