Analyse av Cap-bindende proteiner i humane celler eksponert for Fysiologiske oksygenforholdene
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
Translasjonell kontroll fremstår som en like viktig skritt for å transcriptional regulering i genuttrykk, særlig i perioder med cellulært stress en. Et samlingspunkt oversettelse kontroll er på hastighetsbegrensende trinn i initiering hvor de første trinnene av proteinsyntese involverer binding av eukaryote initiering faktor 4E (eIF4E) til 7-methylguanosine (m 7 GTP) 5 'cap av mRNA 2 . eIF4E er en del av en trimere kompleks kalt eIF4F som inkluderer eIF4A, en RNA helicase, og eIF4G, et stillas protein nødvendig for rekruttering av andre oversettelses faktorer og 40S ribosom tre. Under normale fysiologiske forhold, er det store flertallet av mRNA oversettes ved hjelp av en hette-avhengig mekanisme, men under perioder av cellulært stress omtrent 10% av humane mRNA inneholde 5 'UTR som kan tillate cap-uavhengig oversettelses intiation 1,4. Cap-avhengige oversettelsen har vært historisk synonymOUS med eIF4F, men stress-spesifikke variasjoner av eIF4F har blitt en trending topic 5-8.
Ulike cellulære påkjenninger føre eIF4E aktivitet for å bli undertrykt via mammalian target of rapamycin kompleks 1 (mTORC1). Denne kinase blir svekket under stress, noe som resulterer i økt aktivitet av ett av sine mål, 4E-bindende protein (4E-BP). Non-fosforylert 4E-BP binder seg til eIF4E og blokkerer dens evne til å samhandle med eIF4G forårsaker undertrykkelse av cap-avhengige oversettelse 9,10. Interessant, en homolog av eIF4E heter eIF4E2 (eller 4EHP) har en mye lavere affinitet for 4E-BP 11, kanskje slik at det å unngå belastning-mediert represjon. Faktisk, først karakterisert som en repressor av oversettelse på grunn av sin mangel på interaksjon med eIF4G 12, initierer eIF4E2 oversettelsen av hundrevis av mRNA som inneholder RNA hypoksi responselementer i sin 3 'UTR under hypoksiske stresset 6,13. Denne aktiveringen is oppnås gjennom interaksjoner med eIF4G3, RNA-bindende protein motiv 4, og den hypoksi induserbar faktor (HIF) 2α for å utgjøre en hypoksisk eIF4F kompleks, eller eIF4F H 6,13. Som en repressor under normale forhold, binder eIF4E2 med GIGYF2 og ZNF598 14. Disse kompleksene ble delvis identifisert ved agarose-koblede m 7 GTP affinitet harpikser. Denne klassiske metoden 15 er standard innen oversettelse og er de beste og mest brukte teknikk for å isolere cap-bindende komplekser i trekke ned og in vitro bindingsanalyser 16-19. Etter hvert som hetten avhengige oversettelse maskineri fremstår som fleksibel og tilpasningsdyktig med innbyrdes skiftende deler 6-8,13, er denne metoden et kraftig verktøy for raskt å identifisere nye cap-bindende proteiner som er involvert i den stressrespons. Videre variasjoner i eIF4F kunne ha bred betydning som flere eukaryote modellsystemer synes å bruke en eIF4E2 homolog for stressresponser slikesom A. thaliana 20, S. pombe 21, D. melanogaster 22, og C. elegans 23.
Tyder på at variasjoner i eIF4F ikke kan være strengt begrenset til stress forhold, men være involvert i normal fysiologi 24. Oksygentilførselen til vevene (ved kapillær ender) eller i vev (målt via mikroelektroder) varierer fra 2-6% i hjernen 25, 3-12% i lungene 26, 3,5-6% i tarmen 27, 4% i leveren 28, 7-12% i nyrene 29, 4% i muskel 30, og 6-7% i benmargen 31. Celler og mitokondrier inneholder mindre enn 1,3% oksygen 32. Disse verdiene er mye nærmere hypoksi enn den omgivende luft, hvor cellene rutinemessig dyrket. Dette tyder på at det tidligere var tenkt som hypoksi-spesifikke cellulære prosesser kan være relevant i en fysiologisk innstilling. Interessant, eIF4F og eIF4F H </sup> delta aktivt i oversettelsen initiering av ulike bassenger eller klasser av mRNA i flere forskjellige humane cellelinjer som er utsatt for fysiologisk oksygen eller "physioxia" 24. Lavt oksygen driver også riktig fosterutvikling 33 og celler generelt har høyere spredning priser, lengre levetid, mindre DNA skader og mindre generell stressresponser i physioxia 34. Derfor er eIF4F H sannsynligvis en viktig faktor i ekspresjon av utvalgte gener under fysiologiske betingelser.
Her gir vi en protokoll for å dyrke celler i anleggs fysiologiske oksygenforholdene eller i et dynamisk varierende utvalg som sannsynligvis mer representativt for vev microenvironments. En fordel med denne metoden er at cellene blir lysert i hypoksi arbeidsstasjonen. Det er ikke ofte klart hvordan overgangen fra hypoksisk cellekultur til cellelysering blir utført i andre protokoller. Celler blir ofte først fjernes fra en liten hypoksi inkubator bliforgrunnen lyse, men dette eksponering for oksygen kan påvirke biokjemiske reaksjonsveier som den cellulære responsen til oksygen er rask (ett eller to min) 35. Visse cap-bindende proteiner krever samhandling med andre base eller kan hydrolysere m 7 GTP, derfor noen cap interactors kan være savnet i renseprosessen. Agarose-bundet til enzymatisk resistente cap analoger kan være substituert i denne protokollen. Utforsking av aktivitet og sammensetning av eIF4F H og andre varianter av eIF4F gjennom metoden beskrevet her vil belyse intrikate genuttrykk machineries at celler benytter under fysiologiske forhold eller stressresponser.
Protocol
Representative Results
Discussion
Analysen av cap-bindende proteiner i humane celler utsettes for fysiologiske oksygenbetingelser kan gi rom for identifisering av nye oksygen regulert translasjonsinitiering faktorer. Affiniteten av disse faktorene for den 5 'cap av mRNA eller andre cap-assosierte proteiner kan måles ved styrken av deres tilknytning til 7 m GTP bundne agarosekuler. En påminnelse med denne teknikken er at den måler hetten bindende potensial av proteiner post-lysis, men den utføres under ikke-denaturerende betingelser so…
Declarações
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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