JoVE Journal > Genetics
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Genetics

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

Published: December 28, 2016
doi:
10.3791/55112
, , , , ,
1Department of Molecular and Cellular Biology,University of Guelph

Summary

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.

Abstract

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.

Introduction

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 'cap af mRNA'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 'UTR'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'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> deltage aktivt i oversættelsen initiering af distinkte puljer eller klasser af mRNA i flere forskellige humane cellelinjer udsat for fysiologisk ilt eller "physioxia" 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.

Protocol

1. Forberedelse til Cell Culture Køb kommercielt tilgængelige lagre af humane celler. BEMÆRK: Denne protokol udnytter HCT116 kolorektal carcinom og primære humane renale proximale tubulære epitelceller (HRPTEC). 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). Gør 500 ml medium til dyrkning af HRPTEC: epitelcelle medium suppleret med 5% …

Representative Results

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…

Discussion

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'-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, …

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

Materials

γ-aminophenyl-m7GTP agarose C10-linked beads Jena Bioscience AC-1555 Agarose-linked m7GTP
100 mm culture dish Corning 877222 10-cm culture dish
150 mm culture dish Thermofisher 130183 15-cm culture dish
AEBSF Hydrochloride ACROS Organics A0356829 AEBSF
Agarose Beads Jena Bioscience  AC-0015 Agarose bead control
Bromophenol Blue Fisher BP112-25 Component of SDS-PAGE loading buffer
1.5 mL Centrifuge Tubes FroggaBio 1210-00S Used to centrifuge small volumes
15 mL Conical Centrifuge Tubes Fisher 1495970C Used in culturing primary cells
Defined trypsin inhibitor Fisher R007100 DTI
Dithiothreital Fisher BP172-25 DTT
Epithelial cell medium (complete kit) ScienCell 4101 Includes serum and growth factor supplements)
Glycerol Fisher BP229-1 Component of SDS-PAGE loading buffer
100 mM Guanosine 5'-triphosphate, 1 mL Jena Bioscience 272076-0251M GTP
HCT116 colorectal carcinoma ATCC CCL-247 Human cancer cell line
Human renal proximal tubular epithelial cells ATCC PCS-400-010 HRPTEC
Hyclone DMEM/High Glucose GE Life Sciences SH30022.01 Standard media for human cell culture
Hyclone Penicillin-Streptomycin solution GE Life Sciences SV30010 Antibiotic component of DMEM
H35 HypOxystation Hypoxygen N/A Hypoxia workstation
Igepal CA-630 MP Biomedicals 2198596 Detergent component of lysis buffer
Monopotassium phosphate Fisher P288-500 KH2PO4
Potassium chloride Fisher P217-500 KCl
Magnesium chloride Fisher M33-500 MgCl2
Sodium chloride Fisher BP358-10 NaCl
Sodium fluoride Fisher 5299-100 NaF (phosphatase inhibitor component of lysis buffer)
Disodium phosphate Fisher 5369-500 Na2HPO4
Premium Grade Fetal Bovine Serum Seradigm 1500-500 FBS
Protease Inhibitor Cocktail (100 x) Cell Signalling 58715 Component of lysis buffer
Sodium Dodecyl Sulfate Fisher BP166-100 SDS
Sodium Orthovanadate Sigma 56508 Na3VO4
Tris Base Fisher BP152-5 Component of buffers
0.05% Trypsin-EDTA (1x) Life Technologies 2500-067 Trypsin used to detach adherent cells
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Tags

Cap-binding ProteinsPhysiological Oxygen ConditionsAgarose Linked Seven Methyl-guanosine Cap AnalogCap-binding FactorsProtein SynthesisOxygen Regulated FactorsCap Dependent TranslationHypoxiaCancer TherapyTumor ProgressionNormaxiaCell CultureLysis BufferProtease Inhibitor Cocktail4-benzenesulfonyl Fluoride HydroclorideTrypsin EDTA

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Timpano, S., Melanson, G., Evagelou, S. L., Guild, B. D., Specker, E. J., Uniacke, J. Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions. J. Vis. Exp. (118), e55112, doi:10.3791/55112 (2016).
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