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Biochimica

Nuclear Magnetic Resonance Spectroscopy for Identifikation af flere fosforyleringer af Intrinsically Disordered Proteiner

Published: December 27, 2016
doi:
10.3791/55001
, , , , , , , , , , , ,
1UMR8576, CNRS,Lille University, 2UMR-S1172, INSERM CNRS,Lille University

Summary

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Abstract

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer’s disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau’s heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

Introduction

En af de vigtigste udfordringer i sundhedsvæsenet i det 21. århundrede er neurodegenerative sygdomme såsom Alzheimers sygdom (AD). Tau er et microtubulus-associeret protein, der stimulerer mikrotubulus (MT) formation. Tau er lige så involveret i flere neurodegenerative sygdomme, såkaldte tauopatier, hvoraf de mest kendte er AD. I disse lidelser, er Tau selvstændige aggregater i parrede spiralformede filamenter (PHF'er) og fundet ændret mange rester af posttranslationelle modifikationer (PTMS) såsom fosforylering 1. Phosphorylering af Tau-protein er impliceret i både regulering af dets fysiologiske funktion af MT stabilisering og patologisk tab af funktion, der kendetegner AD neuroner.

Endvidere Tau-protein, når de integreres i PHF'er i syge neuroner, er uvægerligt hyperphosphoryleret 2. I modsætning normal tau, der indeholder 2-3 phosphatgrupper, den hyperphosphoryleret Tau i PHF'er indeholder 5 til 9 Phosphate gruppe 3. Hyperphosphorylering af Tau svarer både til en stigning på støkiometri på nogle steder og til fosforylering af yderligere lokaliteter, der kaldes patologiske steder af phosphorylering. Dog eksisterer overlapning mellem AD og normale voksne mønstre af fosforylering trods kvantitative forskelle i niveau 4. Hvor specifikke phosphoryleringsbegivenheder indflydelse funktion og dysfunktion af Tau stort set ukendt. Vi tilstræber at dechifrere Tau regulering af PTMS på det molekylære niveau.

For at uddybe forståelsen af ​​de molekylære aspekter af Tau, vi skulle tage fat tekniske udfordringer. For det første, Tau er i sig selv uordnet protein (IDP) når de er isoleret i opløsning. Sådanne proteiner mangler veldefineret tredimensionel struktur under fysiologiske betingelser og kræver særlige biofysiske metoder til at studere deres funktion (er) og strukturelle egenskaber. Tau er et paradigme for den voksende klasse af internt fordrevne, ofte forbundet medpatologier såsom neurodegenerative sygdomme, dermed øge interessen for at forstå de molekylære parametre der indgår deres funktioner. For det andet karakterisering af Tau-phosphorylering er en analytisk udfordring, med 80 potentielle phosphoryleringssteder langs sekvensen af ​​den længste 441 aminosyre Tau isoform. Et antal antistoffer er blevet udviklet mod phosphorylerede epitoper Tau og anvendes til påvisning af patologisk Tau i neuroner eller hjernevæv. Phosphoryleringsbegivenheder kan finde sted på mindst 20 lokaliteter er omfattet af prolin-rettet kinaser, de fleste af dem i tæt nærhed i Proline-rige region. Den kvalitative (hvilke websteder?) Og kvantitative (hvad støkiometri?) Karakterisering er svært selv af de seneste MS-teknikker 5.

NMR-spektroskopi kan anvendes til at undersøge uordnede proteiner, som er meget dynamiske systemer udgøres af ensembler af konformerer. Høj opløsning NMR spektroskopi var ansøged at undersøge både strukturen og funktionen af ​​tau-proteinet. Desuden kompleksiteten af Tau s phosphorylering profil førte til udviklingen af molekylære værktøjer og nye analysemetoder anvendelse af NMR til identifikation af phosphoryleringssteder 6 – 8. NMR som en analysemetode muliggør identifikation af Tau phosphoryleringssteder i en global måde, visualisering af alle enkelt-site modifikationer i et enkelt eksperiment, og kvantificering af omfanget af phosphat inkorporering. Dette punkt er vigtigt, da selv fosforylering undersøgelser om Tau bugne i litteraturen, har de fleste af dem er udført med antistoffer, hvilket giver en høj grad af usikkerhed om komplet profil af phosphorylering og dermed den sande betydning af de enkelte phosphoryleringsbegivenheder. Rekombinante kinaser herunder PKA, Glycogen-syntase kinase 3β (GSK3p), cyklin-afhængige kinase 2 / cyclin A (CDK2 / CYCA), cyklin-afhængig kinase 5 (CDK5) / p25 handleivator protein, ekstracellulært-signal-reguleret kinase 2 (ERK2) og mikrotubuli-affinitet-regulerende kinase (MARK), som viser fosforylering aktivitet mod Tau, fremstilles i en aktiv form. Desuden er Tau-mutanter, der muliggør frembringelse af specifikke tau-protein-isoformer med velkarakteriserede phosphorylering mønstre anvendes til at dechifrere phosphorylering kode Tau. NMR-spektroskopi anvendes derefter til karakterisering enzymatisk modificerede Tau prøverne 6 – 8. Selvom in vitro-phosphorylering af Tau er mere udfordrende end pseudo-fosforylering såsom ved mutation af udvalgte Ser / Thr til glutaminsyre (Glu) rester, denne tilgang har sine fordele. Faktisk kan hverken de strukturelle virkninger eller interaktion parametre fosforylering altid efterlignes af glutaminsyrer. Et eksempel er den turn motiv observeret omkring phosphoserin 202 (pSer202) / phosphothreonin 205 (pThr205), som ikke er gengivet med Glu mutationer 9.

<p class = "jove_content"> Her fremstilling af isotopmærket Tau til NMR undersøgelser vil blive beskrevet først. Tau protein phosphoryleret med ERK2 ændres på mange websteder, der er beskrevet som patologiske steder af phosphorylering, og repræsenterer således en interessant model af hyperphosphoryleret Tau. En detaljeret protokol af Tau in vitro phosphorylering med rekombinant ERK2 kinase præsenteres. ERK2 aktiveres ved phosphorylering af mitogenaktiveret proteinkinase / ERK-kinase (MEK) 10 – 12 år. Ud over fremstilling af modificerede, isotopisk mærkede Tau-protein, er NMR-strategi, der anvendes til identifikation af PTMS beskrevet.

Protocol

1. Fremstilling af 15N, 13C-Tau (figur 1) Transform pET15b-Tau rekombinant T7-ekspressionsplasmid 13,14 i BL21 (DE3) kompetente Escherichia coli bakterieceller 15. BEMÆRK: cDNA kodende for den længste (441 aminosyrerester) Tau isoform klones mellem Ncol og Xhol-restriktionssteder i pET15b plasmid. Bland forsigtigt 50 pi kompetente BL21 (DE3) -celler, som danner 1-5 x 10 7 kolonier pr ug plasmid-DNA, med 100 ng …

Representative Results

Figur 3A viser en væsentlig absorptionstop ved 280 nm observeret under elueringsgradient. Denne top svarer til oprenset Tau-protein som set på acrylamidgel ovenfor kromatogrammet. Figur 3B viser et godt adskilt absorptionstop ved 280 nm og toppen af ledningsevne, der sikrer, at afsaltning af proteinet er effektiv. Figur 4 viser proteingel-shift observeret ved SDS-PAGE-analyse 16 karakteristisk for multipel proteinphosphorylering (sammenlign bane 2 og 3). Figur 6</strong…

Discussion

Vi har brugt NMR spektroskopi til karakterisering enzymatisk modificerede Tau prøver. Den rekombinante ekspression og oprensning beskrevet her for det humane Tau-protein i fuld længde kan tilsvarende anvendes til fremstilling af mutante Tau eller Tau domæner. Isotopisk er nødvendig beriget protein til NMR-spektroskopi, hvilket nødvendiggør rekombinant ekspression. Identifikation af phosphoryleringssteder kræver resonans opgave og en 15 N, 13C dobbelt mærkede protein. Betragtning af omkostni…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

The NMR facilities were funded by the Région Nord, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry and the University of Sciences and Technologies of Lille. We acknowledge support from the TGE RMN THC (FR-3050, France), FRABio (FR 3688, France) and Lille NMR and RPE Health and Biology core facility. Our research is supported by grants from the LabEx (Laboratory of Excellence) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer’s disease), EU ITN TASPPI and ANR BinAlz.

Materials

pET15B recombinant T7 expression plasmid Novagen 69257 Keep at -20°C
BL21(DE3) transformation competent E.coli bacteria New England Biolabs C2527I Keep at -80°C
Autoclaved LB Broth, Lennox  DIFCO 240210 Bacterial Growth Medium
MEM vitamin complements 100X Sigma 58970C Bacterial Growth Medium Supplement
15N, 13C-ISOGRO complete medium powder Sigma 608297 Bacterial Growth Medium Supplement
15NH4Cl Sigma 299251 Isotope
13C6-Glucose Sigma 389374 Isotope
Protease inhibitor tablets  Roche 5056489001 Keep at 4°C
1 tablet in 1ml is 40X solution that can be kept at -20°C
DNaseI EUROMEDEX 1307 Keep at -20°C
Homogenizer (EmulsiFlex-C3) AVESTIN Lysis is realized at 4°C
Pierce™ Unstained Protein MW Marker Pierce 266109
Active human MEK1 kinase, GST Tagged Sigma M8822 Keep at -80°C
AKTÄ Pure chromatography system GE Healthcare FPLC
HiTrap SP Sepharose FF (5 mL column) GE Healthcare 17-5156-01 Cation exchange chromatography columns
HiPrep 26/10 Desalting GE Healthcare 17-5087-01 Protein Desalting column
PD MidiTrap G-25 GE Healthcare 28-9180-08 Protein Desalting column
Tris D11, 97% D Cortecnet CD4035P5 Deuterated NMR buffer
5 mm Symmetrical Microtube SHIGEMI D2O ( set of 5 inner & outerpipe)  Euriso-top BMS-005B NMR Shigemi Tubes
eVol kit-electronic syringe starter kit Cortecnet 2910000 Pipetting
Bruker 900MHz AvanceIII with a triple resonance cryogenic probehead Bruker NMR spectrometer for data acquisition
Bruker 600MHz DMX600 with a triple resonance cryogenic probehead Bruker NMR spectrometer for data acquisition
TopSpin 3.1 Bruker Acquisition and Processing software for NMR experiments
Sparky 3.114 UCSF (T. D. Goddard and D. G. Kneller) NMR data Analysis software
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Tags

Nuclear Magnetic Resonance SpectroscopyIntrinsically Disordered ProteinsTau ProteinPhosphorylationMolecular RegulationDiseaseProtein InteractionProtein StructureProtein FunctionAPtc VirusTherapyProtein InhibitorsBL21 CellsPlasmid DNALuria Bertani MediumAmpicillinM9 MediumIsotope LabelingRecombinant PlasmidOptical Density
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Danis, C., Despres, C., Bessa, L. M., Malki, I., Merzougui, H., Huvent, I., Qi, H., Lippens, G., Cantrelle, F., Schneider, R., Hanoulle, X., Smet-Nocca, C., Landrieu, I. Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins. J. Vis. Exp. (118), e55001, doi:10.3791/55001 (2016).
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