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

Denne forskningen ble støttet av Minerva Foundation, HFSP Organization, og en European Research Council Grant (Starting Grant 338265).

1. Syntese av &quot;Chemical Svinger &#39; <ol><li> foreløpige Forberedelser <ol><li> Fremstille 2 M trietylammoniumacetat (TEAA) buffer ved å blande 278 ml trietylamin med 114 ml eddiksyre og 400 ml ultrarent vann. Juster pH-verdien til 7 og tilsett vann til et endelig volum på 1 L. holde den i en mørk flaske. Merk: Denne løsningen er stabil i år. </li><li> Fremstille en 5 mM askorbinsyre oppløsning ved å oppløse 18 mg askorbinsyre i 20 ml ultrarent vann. Bruk en ny løsning; oppløsningen er stabil i...

<ol>
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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...

Signaltransduksjonsveier spille en betydelig rolle i praktisk talt alle cellulære prosessen og tillate cellen å reagere raskt på miljøsignaler. 1. Disse banene ofte utløses ved binding av et signalmolekyl til en ekstracellulær reseptor, noe som resulterer i aktivering av intracellulære enzymer. Amplifisering og forplantning av dette signal i cellen er formidlet ved funksjon av signaleringsproteiner som danner et nettverk av protein-protein interaksjoner hvori enzymene er reversibelt aktivert med høy spesifisitet. På grunn dysregulering av disse nettverkene ofte fører til kreftutvikling, har det vært stor interesse for å etablere &#39;signaltransduksjon terapi av kreft&#39;, 2 hvorved medikamenter er utviklet for å forstyrre maligne signalveier. Vi har nylig foreslått en alternativ tilnærming til signaltransduksjon terapi som er avhengig av evnen til medikamenter for å generere unaturlige signaltransduksjonsveier. <sup&gt; 3 I særdeleshet, tror vi at ved å utforme syntetiske stoffer som etterligner funksjon av signaliseringsproteiner, ville det være mulig å modulere cellens funksjon indirekte. For eksempel kan disse kunstige nettverk gjør det mulig protein biomarkører for å aktivere enzymer som spalter forløpere. Alternativt kan disse signal protein-mimetika være i stand til å aktivere unaturlige cellesignalbaner, noe som resulterer i terapeutiske effekter. For å demonstrere gjennomførbarheten av denne tilnærmingen, har vi nylig opprettet et syntetisk &quot;kjemiske transduser &#39;4 som gjør det mulig blodplateavledet vekstfaktor (PDGF) for å utløse den spaltning av et anticancer prodrug ved aktivering av glutation-s-transferase (GST), som er ikke sin naturlige bindende partner. Strukturen av denne &quot;transduser&quot; består av et anti-PDGF-DNA aptamer som er modifisert med en bivalent inhibitor for GST. Således tilhører denne syntesemiddel til en familie av molekyler med bindingsseter for åforskjellige proteiner, 5-7 eksempel kjemiske indusere av dimerization (KBS) 8-10 og også til gruppen av protein-bindemidler basert på oligonukleotid-syntetisk molekyl konjugater. 11-21 De generelle prinsipper som ligger til grunn for utformingen av slike systemer er beskrevet her, og detaljerte protokoller for syntetisering og teste funksjonen av denne &quot;transduser&quot; med konvensjonelle enzymatiske analysene er gitt. Dette arbeidet er ment å forenkle utviklingen av flere transdusere &#39;&#39; av denne klasse som kan brukes til å formidle intracellulært protein-protein-kommunikasjon og følgelig, for å indusere kunstig celle signalveier. Figur 1 beskriver skjematisk drifts prinsippene for syntetisk &#39;kjemiske transdusere &quot;som kan megle unaturlig protein-protein kommunikasjon. I denne illustrasjonen et &quot;kjemisk transduser &#39;, som integrerer syntetiske bindemidler for proteins I og II (bindemidler I og II), gjør det mulig for protein II for å utløse den katalytiske aktivitet av protein I, som ikke er dens naturlig bindingspartner. I fravær av protein II, binder transduceren det katalytiske sete i enzymet (protein I) og inhiberer dets aktivitet (figur 1, tilstand ii). Bindingen av den &quot;transduser&quot; til protein II, men fremmer interaksjon mellom bindemiddel I og overflaten av proteinet II (figur 1, tilstand iii), noe som reduserer dens affinitet overfor protein I. Som et resultat av den effektive konsentrasjonen av &#39; fri &#39;transduser i oppløsningen er redusert, noe som fører til dissosiasjon av transduseren-protein i-komplekset og til reaktivering av protein i (Figur 1, tilstand iv). Samlet utgjør disse trinnene frem tre grunnleggende prinsipper for utforming av effektive &#39;transdusere&#39;: (1) en &quot;svinger&quot; skal ha en bestemt bindemiddel for hver av protein mål, (2) samspillet betweno bindemiddel II og protein II skal være sterkere enn vekselvirkningen mellom bindemiddel I og protein I, og (3) bindemiddel I må være i stand til å samhandle med overflaten av proteinet II. Dette siste prinsippet ikke nødvendigvis krever at bindemidlet jeg alene ville ha en høy affinitet og selektivitet overfor protein II. I stedet er det basert på vår nylige studier som viste at bringe et syntetisk molekyl i tilknytning til et protein som er egnet til å fremme interaksjon mellom dette molekylet og overflaten av proteinet. 19,22,23 <img alt="Figur 1" src="/files/ftp_upload/54396/54396fig1.jpg" /> Fig. 1: Drifts prinsipper &#39;kjemiske transdusere&#39; Når den &quot;kjemiske transduseren &#39;tilsettes til et aktivt protein I (tilstand i), bindes det til dets aktive sete gjennom bindemiddel I og inhiberer dets aktivitet (tilstand II). I nærvær av protein II, men det ubundne &#39;kjemisk transducer &#39;samvirker med protein II gjennom bindemiddel II, som fremmer interaksjon mellom bindemiddel I og overflaten av proteinet II. Dette indusert bindemiddel I-protein II interaksjon reduserer den effektive konsentrasjonen av bindemiddel jeg, noe som fører til dissosiasjon av &quot;transducer&#39;-protein jeg kompleks og protein jeg reaktive (stat iv). <a href="https://www.jove.com/files/ftp_upload/54396/54396fig1large.jpg" target="_blank">Klikk her for å se en større versjon av dette tallet .</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.

Utformingen, syntese, og virkningsmekanismen til en &quot;kjemiske transduser&quot; som kan indusere kunstig kommunikasjon mellom PDGF og GST er presentert i figur 2. Strukturen av &quot;transduser&quot; omfatter en PDGF DNA aptamer og en bis-ethacrynic amid (Bea ), som er en kjent GST-inhibitor (figur 2a). 19 Disse bindemidler gjør det mulig for &#39;svingeren &quot;for å binde både PDGF og GST med forskjellige affiniteter, nemlig med diss...

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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.

Ligne Funksjon Melde Proteiner: Mot Kunstig signaltransduksjon Therapy

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

Måling av G-proteinkoblet reseptor-signalering via radiomerket 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

Rensing av biotinylerte celleoverflateproteiner fra<em&gt; Rhipicephalus microplus</em&gt; Epiteliale gutceller

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

Optimalisert protokoll for ekstraksjon av proteiner fra 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

Kombinere Røntgenkrystallografi med liten vinkel X-Ray spredning modell ustrukturert regioner i Nsa1 fra 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

Manipulere levende celler å konstruere stabil 3D Cellular samling uten kunstige stillas

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

Lokalisering av SUMO-modifisert proteiner bruke fluorescerende Sumo-fangst proteiner

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

Karakterisering av proteiner etter størrelses-ekskludering kromatografi koblet til multi-vinkel lysspredning (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

Ikke-radioaktiv analyse for å måle polynukleotidfosforylering av små nukleotids underlag

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

Tverrgående seksjonering av eldre ris (Oryza sativa L.) Kjerner for skanning av elektronmikroskopiavbildning ved hjelp av pipettespisser som immobiliseringsstøtte

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

Polysomprofilering uten gradientopprettere eller fraksjoneringssystemer

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