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

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 (eIF4F^H), 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

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 '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.....

Protocol

1. Voorbereidingen voor Cultuur van de Cel

1. 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).
2. Voeg 500 ml medium voor kweek van HCT116: Dulbecco's Modified Eagle Medium (DMEM) / hoge glucose-medium aangevuld met 7,5% foetaal runderserum (FBS) en 1% penicilline / streptomycine (P / S).
3. Voeg 500 ml medium voor kweek van HRPTEC: Epitheliale celmedium aangevuld met 5% FBS, 1% epitheelcel groei supplement, en 1% P / S.
4. Bereid 500 ml 1x fosfaat-gebufferde zoutoplossing (....

Results

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 .......

Discussion

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 '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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Dithiothreitol	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 (100x)	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