Name: analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions
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This work was supported by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Research and Innovation.

1. Voorbereidingen voor Cultuur van de Cel <ol><li> Schaf commercieel beschikbare voorraden van menselijke cellen. LET OP: Dit protocol maakt gebruik van HCT 116 colorectaal carcinoom en primaire menselijke nier proximale tubulaire epitheelcellen (HRPTEC). </li><li> Voeg 500 ml medium voor kweek van HCT116: Dulbecco&#39;s Modified Eagle Medium (DMEM) / hoge glucose-medium aangevuld met 7,5% foetaal runderserum (FBS) en 1% penicilline / streptomycine (P / S). </li><li> Voeg 500 ml medium voor kweek van HRPTEC: E...

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De analyse van cap-bindende eiwitten in menselijke cellen blootgesteld aan fysiologische zuurstofarme omstandigheden kunnen zorgen voor de identificatie van nieuwe zuurstof gereguleerde translatie initiatie factoren. De affiniteit van deze factoren de 5 &#39;cap van mRNA of andere cap geassocieerde eiwitten kunnen worden gemeten door de kracht van hun associatie met m 7 GTP-gekoppelde agarose parels. Een nadeel van deze techniek is dat het meet de dop bindende eiwitten potentieel van post-lysis, maar het word...

Translationele controle is in opkomst als een even belangrijke stap om gentranscriptieregulatie in genexpressie, vooral in perioden van cellulaire stress 1. Een brandpunt van de vertaling controle is op de snelheidsbeperkende stap van initiatie, waar de eerste stappen van de eiwitsynthese betrekking hebben op de binding van de eukaryote initiatie factor 4E (eIF4E) naar de 7-methylguanosine (m 7 GTP) 5 &#39;cap van mRNA 2 . eIF4E maakt deel uit van een trimere complex genaamd eIF4F dat eIF4A, een RNA-helicase en eIF4G, een steiger eiwit nodig is voor de indienstneming van andere vertaling factoren en de 40S ribosoom 3 bevat. Onder normale fysiologische omstandigheden, de meeste mRNA vertaald door een cap-afhankelijk mechanisme, maar onder perioden van cellulaire stress ongeveer 10% van humane mRNAs bevatten 5 &#39;UTR die cap-onafhankelijke translatie waardoor inwijding 1,4. Cap-afhankelijke vertaling is van oudsher synoniemlende met eIF4F echter, stress-specifieke variaties van eIF4F hebben een trending topic 5-8 geworden. Verschillende cellulaire stress veroorzaken eIF4E activiteit die moet worden onderdrukt via de zoogdieren doelwit van rapamycine complex 1 (mTORC1). Dit kinase minder wordt onder stress, wat resulteert in de verhoogde activiteit van een van de doelstellingen, de 4E-bindend eiwit (4E-BP). Niet-gefosforyleerde 4E-BP bindt aan en blokkeert eIF4E zijn vermogen tot interactie met eIF4G waardoor de onderdrukking van cap-afhankelijke translatie 9,10. Interessant is dat een homoloog van genoemd eIF4E eIF4E2 (of 4EHP) een veel lagere affiniteit voor 4E-BP 11, waardoor het mogelijk stress gemedieerde repressie onttrekken. Inderdaad, aanvankelijk gekarakteriseerd als een repressor van translatie vanwege het gebrek aan interactie met eIF4G 12, eIF4E2 initieert de translatie van mRNAs die honderden RNA hypoxieresponselementen elementen in de 3 &#39;UTR bevatten in hypoxische spanning 6,13. Deze activering is bereikt door interactie met eIF4G3, RNA bindend eiwit motief 4, en de hypoxia induceerbare factor (HIF) 2α een hypoxische eIF4F complex of eIF4F H 6,13 vormt. Als een repressor onder normale omstandigheden, eIF4E2 bindt met GIGYF2 en ZNF598 14. Deze complexen werden gedeeltelijk geïdentificeerd door middel van agarose-gekoppelde m 7 GTP affiniteitsharsen. Dit klassieke methode 15 is standaard op het gebied van vertaling en is de beste en meest gebruikte techniek om-cap bindende complexen te isoleren in pull-down en in vitro bindingstesten 16-19. Als de cap-afhankelijke translatie machinerie is in opkomst als flexibel en aanpasbaar met inter-wisselende delen 6-8,13, deze methode is een krachtig instrument om nieuwe-cap-bindende eiwitten die betrokken zijn bij de reactie op stress snel kan identificeren. Bovendien verschillen in eIF4F kan grote gevolgen hebben als een aantal eukaryote modelsystemen blijken een eIF4E2 homoloog voor stressreacties zoals gebruikenals A. thaliana 20, S. Pombe 21, D. melanogaster 22 en 23 C. elegans. Er zijn aanwijzingen dat variaties in eIF4F niet strikt kan beperkt stresssituaties, maar worden in normale fysiologie 24. De zuurstoftoevoer naar weefsels (at capillair uiteinden) of binnen weefsels (gemeten met micro-elektroden) varieert van 2-6% in de hersenen 25, 3-12% in de longen 26, 3,5-6% in de darm 27, 4% in de lever 28, 7-12% in de nier 29, 4% in de spieren 30 en 6-7% in het beenmerg 31. Cellen en mitochondriën bevatten minder dan 1,3% zuurstof 32. Deze waarden zijn veel dichter bij hypoxie dan de omgevingslucht wanneer cellen worden routinematig gekweekt. Dit suggereert dat wat eerder werd gezien als hypoxie-specifieke cellulaire processen betrokken in een fysiologische omgeving zijn. Interessant, eIF4F en eIF4F H </sup&gt; actief deelnemen aan de translatie-initiatie van verschillende zwembaden of klassen van mRNA in verschillende menselijke cellijnen blootgesteld aan fysiologische zuurstof of &quot;physioxia&quot; 24. Lage zuurstof rijdt ook een goede ontwikkeling van de foetus 33 en cellen hebben in het algemeen een hogere proliferatie tarieven, langere levensduur, minder DNA-schade en minder algemene stressreacties in physioxia 34. Daarom eIF4F H is waarschijnlijk een belangrijke factor in de expressie van geselecteerde genen onder fysiologische omstandigheden. Hier bieden we een protocol om kweekcellen in vaste fysiologische zuurstofarme omstandigheden of in een dynamisch fluctuerende bereik dat waarschijnlijk meer representatief is voor het weefsel micro-omgevingen. Een voordeel van deze methode is dat de cellen worden gelyseerd in de hypoxie werkstation. Het is vaak niet duidelijk hoe de overgang van hypoxische celkweek cellysis wordt uitgevoerd in andere protocollen. De cellen worden vaak eerst verwijderd uit een klein hypoxie incubator wordenvoorgrond lysis, maar deze blootstelling aan zuurstof kunnen beïnvloeden biochemische pathways de cellulaire reactie op zuurstof snel (één of twee min) 35. Bepaalde-cap-bindende eiwitten nodig interacties met een tweede base of kan de m 7 GTP, daarom enkele cap interactors kan worden gemist in het zuiveringsproces te hydrolyseren. -Agarose gebonden enzymatisch resistente cap analoga kunnen worden gesubstitueerd in dit protocol. Het verkennen van de activiteit en de samenstelling van eIF4F H en andere variaties van eIF4F door middel van de hier beschreven methode zal licht werpen op de ingewikkelde genexpressie machines dat cellen gebruiken tijdens fysiologische omstandigheden of stressreacties.

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			<td style="width:205px;">Agarose-linked m7GTP</td>
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			<td height="21" style="height:21px;">150 mm culture dish</td>
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			<td height="21" style="height:21px;">AEBSF Hydrochloride</td>
			<td>ACROS Organics</td>
			<td>A0356829</td>
			<td>AEBSF</td>
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			<td height="21" style="height:21px;">1.5 mL Centrifuge Tubes</td>
			<td>FroggaBio</td>
			<td>1210-00S</td>
			<td>Used to centrifuge small volumes</td>
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		<tr height="21">
			<td height="21" style="height:21px;">15 mL Conical Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>1495970C</td>
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			<td height="21" style="height:21px;">Dithiothreital</td>
			<td>Fisher</td>
			<td>BP172-25</td>
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			<td height="21" style="height:21px;">Epithelial cell medium (complete kit)</td>
			<td>ScienCell</td>
			<td>4101</td>
			<td>Includes serum and growth factor supplements)</td>
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			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
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			<td height="21" style="height:21px;">100 mM Guanosine 5&#39;-triphosphate, 1 mL</td>
			<td>Jena Bioscience</td>
			<td>272076-0251M</td>
			<td>GTP</td>
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			<td height="21" style="height:21px;">HCT116 colorectal carcinoma</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>Human cancer cell line</td>
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			<td height="21" style="height:21px;">Human renal proximal tubular epithelial cells</td>
			<td>ATCC</td>
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			<td>GE Life Sciences</td>
			<td>SH30022.01</td>
			<td>Standard media for human cell culture</td>
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			<td height="21" style="height:21px;">Hyclone Penicillin-Streptomycin solution</td>
			<td>GE Life Sciences</td>
			<td>SV30010</td>
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			<td height="21" style="height:21px;">Igepal CA-630</td>
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			<td height="25" style="height:25px;">Potassium chloride</td>
			<td>Fisher</td>
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			<td>Fisher</td>
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			<td>MgCl2</td>
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			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td>NaCl</td>
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			<td height="21" style="height:21px;">Sodium fluoride</td>
			<td>Fisher</td>
			<td>5299-100</td>
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			<td height="25" style="height:25px;">Disodium phosphate</td>
			<td>Fisher</td>
			<td>5369-500</td>
			<td>Na2HPO4</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Premium Grade Fetal Bovine Serum</td>
			<td>Seradigm</td>
			<td>1500-500</td>
			<td>FBS</td>
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			<td height="21" style="height:21px;">Protease Inhibitor Cocktail (100 x)</td>
			<td>Cell Signalling</td>
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analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Analyse van Cap-bindend vermogen in reactie op Oxygen van eIF4E en eIF4E2 in een m 7 GTP Affinity Column Figuren 1 en 2 geven western blots typische m 7 GTP affiniteitszuivering van twee zware cap-bindende eiwitten in reactie op zuurstof fluctuaties in twee menselijke cellijnen: primaire humane renale proximale tubulaire epitheelcellen (HRPTEC) in F igure 1 ...

Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Analyse van Cap-bindende eiwitten in menselijke cellen blootgesteld aan Physiological Oxygen Voorwaarden

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

The overall goal of this experiment is to capture cap-binding proteins in conditions of low oxygen. This method utilizes an agarose linked seven methyl-guanosine cap analog to investigate cap-binding factors and their interacting partners. This method will help answer key questions in the field of protein synthesis such as identifying novel oxygen regulated factors involved in cap dependent translation.The main advantage of this technique is that cells are kept in a hypoxia workstation up until the point of lysis. The implications of this technique extend towards cancer therapy because cancer cells within a tumor are exposed to very low levels of oxygen and translational control plays a key role in tumor progression. We first had the idea for this method when we read a recent publication from Corona's colleagues reporting that tissue oxygenation is actually closer to what we call hypoxia rather than the normaxia that cells are routinely cultured in.Begin this procedure will cell culture as described in the text protocol. After counting the cells using a hemocytometer feed 150 millimeter cultured dishes containing 15 milliliters of complete medium to obtain a cell density of 2500 to 5000 cells per square centimeter. Use two 150 millimeter dishes for each cap-binding assay.Place the seeded culture dishes in the incubator at 37 degrees celsius and a five percent carbon dioxide atmosphere. After the cells have incubated at least 24 hours place the cells into a hypoxia workstation for the desired time. Then, set the workstation to the appropriate physioxia.Pre-warm one X phosphate buffered saline, or PBS, and trypsin in a 37 degree celsius water bath for 30 minutes. Aliquot 980 microliters of lysis buffer into a 1.5 microliter centrifuge tube for each cap-binding assay being performed. 10 microliters of 100X protease inhibitor cocktail and 10 microliters of 4-benzenesulfonyl fluoride hydrocloride to the 980 microliters of lysis buffer on ice.Discard the media from each 150 millimeter dish in a waste container within the hypoxia workstation. Then, wash the cells with three to four milliliters of warm one X PBS and discard the liquid. Add one milliliter of warm 05 percent trypsin EDTA to each dish and let the dishes sit for two minutes or until the cells are no longer adhered to the plate.Using a pipette transfer the cells of one plate to a 1.5 milliliter micro-centrifuge tube. Repeat as needed so that each plate of cells is transferred to a separate 1.5 milliliter micro-centrifuge tube. Centrifuge the micro-centrifuge tubes at 6000 times g for 90 seconds to pellet the cells.Following centrifugation aspirate the trypsin using a pipette without disturbing the pellet. Wash the cells with warm one X PBS by gently pipetting 200 microliters of PBS into the tube just above the pellet. Then aspirate the PBS without disturbing the pellet.Next, pipette 500 microliters from the one milliliter prepared lysis buffer solution onto the first cellular pellet. Re-suspend the pellet entirely by pipetting up and down. Combine the re-suspended cells with the second cell pellet and re-suspend the second pellet by pipetting up and down.Repeat this process until all pellets have been combined and re-suspended for each sample. Next, combine the lysate with the remaining 500 microliters of lysis buffer in a 1.5 liter micro-centrifuge tube. Remove the samples from the hypoxia workstation.Lyse the cells using gentle agitation by rotating the samples at four degrees celsisus for 1.5 to two hours. Following incubation, centrifuge the lysed cells at 1200 times g for 15 minutes at four degrees celsius to remove cellular debris. Transfer the lysate to a new 1.5 milliliter micro-centrifuge tube and discard the pellet.Reserve a portion of the supernatant to be used as whole cell lysate input control for western blot analysis. To prepare for the cap-binding assay use scissors to remove the tip of the pipette to facilitate collection of the bead slurry. For each sample transfer 50 microliters of the blank agarose bead control slurry and 50 microliters of the m7GTP agarose C10-linked beads slurry to separate 1.5 milliliter micro-centrifuge tubes.Then, pellet the beads by centrifuging the slurry at 500 times g for 30 seconds. Remove the supernatant carefully and re-suspend the beads in 500 microliters of TBS. After repeating these steps pellet the beads at 500 times g for 30 seconds and remove the supernatant.Next, transfer the supernatant containing the lysate to the 1.5 milliliter micro-centrifuge tube containing the blank agarose beads. Incubate for 10 minutes at four degrees celsius with gentle agitation. After 10 minutes, pellet the blank agarose beads by centrifuging at 500 times g for 30 seconds.Following centrifugation transfer the lysate that doesn't bind to the beads to the 1.5 milliliter micro-centrifuge tube containing the m7GTP agarose C10-linked beads. Wash the blank agarose beads by re-suspending the beads in 500 microliters of lysis buffer, pelleting the beads by centrifuging at 500 time g for 30 seconds, and discarding the supernatant. Repeat this four times for a total of five washes.Re-suspend the blank agarose beads in one X SDS-PAGE sample buffer. And boil the beads for 90 seconds at 95 degrees celsius. Store the sample at 20 degrees celsius for future western blot analysis to observe weather the protein of interest binds non-specifically to the bead.Incubate the lysate with the m7GTP agarose C10-linked beads with gentle agitation for one hour at four degrees celsuis to capture cap-binding proteins. Following agitation, pellet the m7GTP agarose C10-linked beads by centrifuging at 500 times g for 30 seconds. After discarding the supernatant, wash the m7GTP agarose C10-linked beads as before.Re-suspend the beads in 600 microliters of lysis buffer and add GTP to a final concentration of one millimolar. Incubate the m7GTP agarose C10-linked beads plus one millimolar GTP with gentle agitation for one hour at four degrees celsius. This will dissociate proteins that non-specifically interact with m7GTP.Pellet the m7GTP agarose C10-linked beads by centrifuging at 500 time g for 30 seconds. Transfer the supernatant to a new 1.5 milliliter micro-centrifuge tube, and add 200 microliters of four X SDS-PAGE sample buffer. After washing the beads as before, re-suspend the beads in one X SDS-PAGE sample buffer and boil at 95 degrees celsius for 90 seconds.Shown here are representative western blots of a typical m7GTP affinity purification of two major cap-binding proteins in response to oxygen fluctuations in primary human renal proximal tubule epithelial cells. The cell lysates were exposed to eight percent, five percent, three percent, or one percent oxygen for 24 hours. The lanes include 10 percent of the whole cell lysate as the input, a GTP washed to measure the specificity for m7GTP, and the proteins bound to m7GTP beads after the GTP wash.Data from at least three independent experiments were quantified by Image J and expressed as relative density units relative to 10 percent input of the whole cell lysate. Results indicate that human cells cultured under physiological oxygen utilize two cap-binding proteins to recruit distinct mRNAs for translation. After watching this video you should have a good understanding of how to capture cap-binding proteins in low oxygen conditions.While attempting this procedure it's important to remember to keep cells in the hypoxia workstation up until the point of lysis, as oxygen can rapidly alter the biochemical properties of these translation initiation factors. Following this procedure other methods like polysome fractionation can be performed to answer additional questions such as which cap-binding proteins associate with active translation under conditions of low oxygen.

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