Name: analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions
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Description: Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

This work was supported by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Research and Innovation.

1. Forberedelse til Cell Culture <ol><li> Køb kommercielt tilgængelige lagre af humane celler. BEMÆRK: Denne protokol udnytter HCT116 kolorektal carcinom og primære humane renale proximale tubulære epitelceller (HRPTEC). </li><li> Gør 500 ml medium til dyrkning af HCT116: Dulbeccos modificerede Eagle-medium (DMEM) / High glucose medium suppleret med 7,5% kalvefosterserum (FBS) og 1% penicillin / streptomycin (P / S). </li><li> Gør 500 ml medium til dyrkning af HRPTEC: epitelcelle medium suppleret med 5% ...

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Analysen af ​​cap-bindende proteiner i humane celler eksponeret for fysiologiske iltindhold kan åbne mulighed for identifikation af nye oxygen-regulerede translationsinitierings- faktorer. Affiniteten af disse faktorer for 5&#39;-cap af mRNA eller andre cap-associerede proteiner kan måles ved styrken af deres tilknytning til m 7 GTP-bundet agaroseperler. En advarsel ved denne teknik er, at den måler cap-bindende potentiale af proteiner efter lyse, men den udføres under ikke-denaturerende betingelser, ...

Translationel kontrol fremstår som en lige så vigtig skridt til transkriptionel regulering i genekspression, især i perioder med cellulært stress 1. En omdrejningspunktet for oversættelse kontrol er det hastighedsbegrænsende trin i indledningen, hvor de første skridt i proteinsyntese involverer binding af den eukaryote indledning faktor 4E (eIF4E) til 7-methylguanosin (m 7 GTP) 5 &#39;cap af mRNA&#39;er 2 . eIF4E er en del af en trimert kompleks opkaldt eIF4F der omfatter eIF4A, en RNA helicase, og eIF4G, et stillads protein kræves for rekruttering af andre oversættelse faktorer og 40S ribosom 3. Under normale fysiologiske betingelser, er det store flertal af mRNA oversættes via en cap-afhængig mekanisme, men under perioder med cellulære stress ca. 10% af de menneskelige mRNA indeholder 5 &#39;UTR&#39;er der kan tillade cap-uafhængig oversættelse intiation 1,4. Cap-afhængige oversættelse har været historisk synonymskellige med eIF4F dog stress-specifikke variationer af eIF4F er blevet et trending emne 5-8. Forskellige cellulære belastninger forårsager eIF4E aktivitet, der skal undertrykkes via pattedyr mål for rapamycin kompleks en (mTORC1). Denne kinase bliver forringet under stress, hvilket resulterer i forøget aktivitet af et af sine mål, den 4E-bindende protein (4E-BP). Ikke-phosphoryleret 4E-BP binder sig til eIF4E og blokerer dens evne til at interagere med eIF4G forårsager undertrykkelse af cap-afhængige oversættelse 9,10. Interessant, en homolog af eIF4E opkaldt eIF4E2 (eller 4EHP) har en meget lavere affinitet for 4E-BP 11, måske gør det muligt at omgå stress-medieret repression. Faktisk oprindeligt karakteriseret som en repressor af oversættelse på grund af sin mangel på interaktion med eIF4G 12, eIF4E2 indleder oversættelse af hundredvis af mRNA, der indeholder RNA hypoxi respons elementer i deres 3&#39;UTR under hypoxisk stress 6,13. Denne aktivering iS opnåede gennem interaktioner med eIF4G3, RNA-bindende protein-motiv 4, og hypoxi inducerbar faktor (HIF) 2α at udgøre en hypoxisk eIF4F kompleks eller eIF4F H 6,13. Som en repressor under normale forhold, eIF4E2 binder med GIGYF2 og ZNF598 14. Disse komplekser blev delvist identificeret ved agarose-bundne m 7 GTP affinitetsresiner. Denne klassiske metode 15 er standard inden for oversættelse og er de bedste og mest anvendte teknik til at isolere cap-bindende komplekser i pull ned og in vitro bindingsassays 16-19. Da hætten-afhængige oversættelse maskiner er på vej som fleksibel og tilpasningsdygtig med indbyrdes skiftende dele 6-8,13, denne metode er et stærkt værktøj til hurtigt at identificere nye cap-bindende proteiner involveret i stress respons. Endvidere kunne variationer i eIF4F har brede implikationer, da flere eukaryote modelsystemer synes at bruge en eIF4E2 homolog til stressreaktioner sådannesom A. thaliana 20, S. pombe 21, D. melanogaster 22, og C. elegans 23. Meget tyder på, at variationer i eIF4F ikke kan begrænses til stress betingelser, men være involveret i normal fysiologi 24. Iltforsyning til vævene (ved kapillære ender) eller inden væv (målt via mikroelektroder) varierer fra 2-6% i hjernen 25, 3-12% i lungerne 26, 3,5-6% i tarmen 27, 4% i leveren 28, 7-12% i nyrerne 29, 4% i muskel 30, og 6-7% i knoglemarven 31. Celler og mitokondrier indeholder mindre end 1,3% oxygen 32. Disse værdier er meget tættere på hypoxi end den omgivende luft, hvor celler dyrkes rutinemæssigt. Dette antyder, at hvad der tidligere blev opfattet som hypoxi-specifikke cellulære processer kan være relevant i en fysiologisk indstilling. Interessant eIF4F og eIF4F H </sup&gt; deltage aktivt i oversættelsen initiering af distinkte puljer eller klasser af mRNA i flere forskellige humane cellelinjer udsat for fysiologisk ilt eller &quot;physioxia&quot; 24. Lav ilt driver også korrekt fosterudvikling 33 og celler generelt har højere spredning satser, længere livslængde, mindre DNA skader og færre generelle stressreaktioner i physioxia 34. Derfor eIF4F H sandsynligvis en nøglefaktor i ekspression af udvalgte gener under fysiologiske betingelser. Her giver vi en protokol til kultur celler i faste fysiologiske iltforhold eller i en dynamisk svingende område, der er sandsynligvis mere repræsentativ for væv mikromiljøer. En fordel ved denne fremgangsmåde er, at cellerne lyseres i hypoxi arbejdsstation. Det er ikke ofte klart, hvordan overgangen fra hypoksisk cellekultur til cellelyse udføres i andre protokoller. Celler ofte først fjernes fra en lille hypoxi inkubator væreforgrunden lysis, men denne eksponering til oxygen kan påvirke biokemiske veje som det cellulære respons på oxygen er hurtig (en eller to min) 35. Visse cap-bindende proteiner kræver interaktioner med en anden base eller kan hydrolysere m 7 GTP, kan derfor glip af nogle cap interaktionskandidater i rensningsprocessen. Agarose-forbundet til enzymatisk resistente cap-analoger kan erstattes i denne protokol. Udforskning af aktivitet og sammensætningen af eIF4F H og andre variationer af eIF4F gennem den her beskrevne metode vil kaste lys over de indviklede genekspression machineries at celler udnytter under fysiologiske betingelser eller stressreaktioner.

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			<td height="26" style="height:26px;width:380px;">&#947;-aminophenyl-m7GTP agarose C10-linked beads</td>
			<td style="width:151px;">Jena Bioscience</td>
			<td style="width:165px;">AC-1555</td>
			<td style="width:205px;">Agarose-linked m7GTP</td>
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		<tr height="21">
			<td height="21" style="height:21px;">100 mm culture dish</td>
			<td>Corning</td>
			<td>877222</td>
			<td>10-cm culture dish</td>
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		<tr height="21">
			<td height="21" style="height:21px;">150 mm culture dish</td>
			<td>Thermofisher</td>
			<td>130183</td>
			<td>15-cm 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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		<tr height="21">
			<td height="21" style="height:21px;">Agarose Beads</td>
			<td>Jena Bioscience&#160;</td>
			<td>AC-0015</td>
			<td>Agarose bead control</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol Blue</td>
			<td>Fisher</td>
			<td>BP112-25</td>
			<td>Component of SDS-PAGE loading buffer</td>
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		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Conical Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>1495970C</td>
			<td>Used in culturing primary cells</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Defined trypsin inhibitor</td>
			<td>Fisher</td>
			<td>R007100</td>
			<td>DTI</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreital</td>
			<td>Fisher</td>
			<td>BP172-25</td>
			<td>DTT</td>
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		<tr height="21">
			<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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		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td>Component of SDS-PAGE loading buffer</td>
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		<tr height="21">
			<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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		<tr height="21">
			<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>
			<td>PCS-400-010</td>
			<td>HRPTEC</td>
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			<td height="21" style="height:21px;">Hyclone DMEM/High Glucose</td>
			<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>
			<td>Antibiotic component of DMEM</td>
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			<td height="21" style="height:21px;">H35 HypOxystation</td>
			<td>Hypoxygen</td>
			<td>N/A</td>
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			<td height="21" style="height:21px;">Igepal CA-630</td>
			<td>MP Biomedicals</td>
			<td>2198596</td>
			<td>Detergent component of lysis buffer</td>
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			<td height="24" style="height:24px;">Monopotassium phosphate</td>
			<td>Fisher</td>
			<td>P288-500</td>
			<td>KH2PO4</td>
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		<tr height="25">
			<td height="25" style="height:25px;">Potassium chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td>KCl</td>
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		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride</td>
			<td>Fisher</td>
			<td>M33-500</td>
			<td>MgCl2</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td>NaCl</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sodium fluoride</td>
			<td>Fisher</td>
			<td>5299-100</td>
			<td>NaF (phosphatase inhibitor component of lysis buffer)</td>
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		<tr height="25">
			<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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		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail (100 x)</td>
			<td>Cell Signalling</td>
			<td>58715</td>
			<td>Component of lysis buffer</td>
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			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate</td>
			<td>Fisher</td>
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			<td>56508</td>
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			<td height="21" style="height:21px;">Tris Base</td>
			<td>Fisher</td>
			<td>BP152-5</td>
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			<td>Life Technologies</td>
			<td>2500-067</td>
			<td>Trypsin used to detach adherent cells</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 af Cap-bindende evne i Reaktion på Oxygen af eIF4E og eIF4E2 i en m 7 GTP Affinity Column Figur 1 og 2 repræsenterer western blots af typiske m 7 GTP affinitetsoprensning af to store cap-bindende proteiner som respons på oxygen udsving i to humane cellelinier: primære humane renale proximale tubulære epitelceller (HRPTEC) i F igur 1 og colorecta...

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 af Cap-bindende proteiner i Human celler udsat for Fysiologiske iltforhold

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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