Name: identification of small molecule-binding proteins in a native cellular environment by live-cell photoaffinity labeling
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling at JoVE.com

We thank Dr. Ben Nacev for advice on design of the photoaffinity labeling protocol, Dr. Wei Shi for synthesizing the itraconazole photoaffinity probe, Dr. Yongjun Dang for advice on affinity pull-down experiments, and other members of the J.O.L. laboratory for helpful comments and support. This work was supported in part by a PhRMA Foundation Fellowship in Pharmacology/Toxicology (to S.A.H.); National Cancer Institute Grant R01CA184103; the Flight Attendant Medical Research Institute; Prostate Cancer Foundation (J.O.L.); and the Johns Hopkins Institute for Clinical and Translational Research, which is funded in part by Grant UL1 TR 001079 from the National Center for Advancing Translational Sciences (NCATS).

BEMÆRK: Denne protokol blev tilpasset fra MacKinnon og Taunton 10 til brug i levende celler. 1. Fremstilling af dyrkede celler <ol><li> Fremstille sterile 6-cm cellekulturskåle for antallet af prøver ønskede (se nedenfor). BEMÆRK: En skål af celler anvendes per behandling tilstand, men hvis mere protein er påkrævet 2 eller 3 retter kan fremstilles pr tilstand og kombineres efter UV-bestråling. <ol><li> Forbered mindst 3 retter af celler; Negativ kontrol med DMSO ...

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Forskellige tilgange til at identificere målene i små molekyler kan groft inddeles i to kategorier: top-down, hvor den cellulære fænotype af lægemidlet bruges til at indsnævre sine potentielle mål baseret på deres kendte funktioner, eller bottom-up, hvor målet identificeres direkte ved kemisk eller genetisk middel 3. Top-down eller fænotypiske undersøgelser kan identificere visse cellulære processer, der berøres af lægemidlet (fx transkription / translation / DNA-syntese, cellecyklus bl...

Bioaktive små molekyler fundamentalt arbejde ved at interagere med og ændre funktionen af ​​en eller flere &quot;target&quot; molekyler, mest almindeligt proteiner, i cellen. I lægemiddelforskning, når en aktiv forbindelse er opdaget gennem fænotypisk screening, identifikation af det molekylære mål (r) af denne forbindelse er afgørende, ikke kun for at forstå virkningsmekanisme og potentielle bivirkninger af forbindelsen, men også for potentielt at opdage ny biologi ligger til grund for sygdommen model og bane vejen for udvikling af nye mekanistiske klasser af lægemidler 1. Selvom identifikation mål ikke er nødvendig for et lægemiddel, der skal anvendes terapeutisk, i de senere år har der været en stigende erkendelse af, at nye lægemiddelkandidater er mere tilbøjelige til at lykkes i kliniske forsøg, og derfor giver bedre afkast på investeringer, hvis en valideret target er kendt 2. Der har således været en stigende interesse for fremgangsmåder til identifikation af småmolekyle målproteiner. En klassisk target identifikation Eksperimentet afhænger typisk af affinitetsoprensning, hvor den lille molekyle af interesse immobiliseres på en harpiks og inkuberet med helcellelysater, hvorefter ubundne proteiner vaskes bort, og de ​​resterende proteiner elueres og identificeres 3. Mens denne teknik er blevet anvendt til at identificere målene for mange små molekyler 4, den er uegnet som en universel target ID metode af flere grunde. For det første skal målproteinet bevare sin native konformation ved cellelyse, for at bevare sin evne til at binde til den lille molekyle. Dette kan være særligt problematisk for membranproteiner, som ofte undergår konformationelle ændringer efter at være blevet fjernet fra deres native miljø, eller blot aggregat og udfældes fra opløsningen. For det andet skal den lille molekyle modificeres kemisk på en sådan måde, at det kan immobiliseres på harpiksen under opretholdelsedets evne til at binde målproteinet. Dybe bindingslommer kan derfor blive utilgængelige for et lille molekyle, når den er fastgjort til harpiksen. For det tredje skal bindingsaffiniteten være tilstrækkelig høj, at interaktionen opretholdes under vasketrinene, hvilket gør identifikation af lavere affinitet interaktioner udfordrende. Fjerde, miljøforhold såsom pH, ion koncentration, eller tilstedeværelsen af ​​andre endogene molekyler kan variere rumligt inden i cellen, og til tider er forudsætninger for lægemiddel-target interaktioner. Således, at finde de korrekte betingelser til at tillade og vedligeholde bindende ydersiden af ​​cellen kan kræve en betydelig mængde af trial and error. Fotoaffinitetsmærkning omgår disse problemer ved at tillade kovalent binding af et lille molekyle og dets mål inden den native forbindelse med en celle. Snarere end at immobilisere det lille molekyle til en stor voluminøs harpiks, er molekylet i stedet kemisk modificeret til at installere to små funktionelle groups: en fotoaktiverbart del, der tillader kovalent tværbinding til målproteinet ved bestråling med en bestemt bølgelængde af lys, og en reportergruppe, der tillader at målproteinet blive opdaget og efterfølgende isoleret. Levende celler behandles med fotoaffinitets probe, proben binder og kovalent tværbinder til målproteinet, og proben-proteinkomplekset isoleres derefter intakt. Specificiteten af ​​proben binding til målet påvises ved at udføre en konkurrence eksperiment parallelt, hvor et overskud af stamforbindelsen anvendes til at konkurrere væk binding af proben til målproteinet. Den design og syntese af fotoaffinitet sonder varierer meget fra den ene lille molekyle til et andet, og vil ikke blive dækket i denne protokol; har dog flere gode diskussioner om emnet blevet offentliggjort 5-9. Den væsentligste overvejelse er, at sonden bevarer bioaktivitet af stamforbindelsen, derfor presumably binding til det samme target (s). Struktur-aktivitetsforhold (SAR) -undersøgelser skal udføres for at bestemme, hvilke dele af molekylet kan modificeres uden tab af bioaktivitet. En række forskellige kemiske grupper er blevet anvendt som fotoaktiverbare tværbindere, herunder diazirine, benzophenon, og aryl azid, som hver har fordele og ulemper 10. Ligeledes er der flere reporter tags, der er blevet anvendt til at isolere probe-bindende proteiner. Reportergrupper kan være funktionel på egen hånd, såsom den almindeligt anvendte biotin eller fluorescerende mærker, eller kan være forstadier, der kræver yderligere funktionalisering efter fototværbindingen trin, som har den fordel at være mindre og dermed mindre tilbøjelige til at gå på kompromis bioaktivitet 11. I denne protokol, har vi brugt en fotoaffinitet sonde, der indeholder en diazirine fototværbinding gruppe, og en terminal alkyn til fastgørelse af en reporter gruppen gennem en Cu (I) -catalyzed azid-Alkyn Sharpless-Huisgen cycloaddition (eller klik) reaktion 12-15. SAR studier, probe design og syntese, og resultaterne af disse undersøgelser er blevet offentliggjort andre steder 16-18.

<table border="0" cellpadding="0" cellspacing="0" width="1169">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td style="width:188px;">Life Technologies</td>
			<td style="width:117px;">20490</td>
			<td style="width:481px;">Make fresh day of use. Prepare 100 mM stock in water with 4 eq NaOH.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA)&#160;</td>
			<td>AnaSpec</td>
			<td>63360-50&#160;</td>
			<td>Prepare 1.7 mM stock in a 4:1 ratio of t-butanol to DMSO, store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Copper Sulfate (CuSO4-5H2O)</td>
			<td>LabChem, Inc.</td>
			<td>LC13440-1&#160;</td>
			<td>Prepare 50 mM stock in water, store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin-azide</td>
			<td>Click Chemistry Tools</td>
			<td>AZ104-100</td>
			<td>Prepare 10 mM stock in DMSO, store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647-azide&#160;</td>
			<td>Life Technologies</td>
			<td>A10277</td>
			<td>Prepare 1 mM stock in DMSO, store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">365 nm UV lamp</td>
			<td>Spectroline</td>
			<td>FC100</td>
			<td>UV-blocking glasses should be worn while operating.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor tablets, EDTA-free</td>
			<td>Roche Life Science</td>
			<td>11873580001</td>
			<td>Prepare 50x solution in water and store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator</td>
			<td>Branson</td>
			<td>Sonifier 250</td>
			<td>Set to output 1, duty 30%.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescent gel scanner</td>
			<td>GE Healthcare Life Sciences</td>
			<td>FLA 9500</td>
			<td>Use red laser to detect Alexa-fluor 647.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Detergent-compatible Dc protein assay kit</td>
			<td>Bio-Rad</td>
			<td>5000112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High Capacity Streptavidin Agarose beads&#160;</td>
			<td>Life Technologies</td>
			<td>20359</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagles Medium, low glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>11885092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum, qualified</td>
			<td>ThermoFisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptamycin solution</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS Sample Buffer (2X)</td>
			<td>ThermoFisher Scientific</td>
			<td>LC2676</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of small molecule-binding proteins in a native cellular environment by live-cell photoaffinity labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

Resultaterne vist her blev opnået med et foto-affinitet-proben af det antifungale lægemiddel itraconazol, hvis anvendelse tidligere er blevet publiceret 16.. Disse resultater viser anvendelsen af ​​den levende celle teknik fotoaffinitetsmærkning til at identificere en større itraconazol-bindende protein som den 35 kDa membranprotein spændingsafhængige Anion kanal 1 (VDAC1). Den ovennævnte protokol blev ud...

Watch this Scientific Journal Video about Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling at JoVE.com

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifikation af små molekyler-bindende proteiner i en Native Cellular Environment af live-cell fotoaffinitetsmærkning

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

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Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

Opsamling og identifikation af RNA-bindende proteiner ved hjælp af klik kemi-assisteret RNA-interactome Capture (CARIC) strategi

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Analyse af proteinfoldning, Transport og nedbrydning i levende celler af radioaktivt puls Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Hybrid Clear/blå Native elektroforese til separation og analyse af Mitokondrial respirations kæde Superkomplekser

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Identifikation af inositol fosfat eller Phosphoinositide interagerende proteiner ved affinitets kromatografi koblet til Western blot eller massespektrometri

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

Måling af interaktioner mellem fluorescerende sonder og lignin i plante sektioner af sFLIM baseret på naturlig Auto Luorescens

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Karakteriserecellulære proteiner med in-cell hurtig fotokemisk oxidation af proteiner

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...