Name: coupled assays for monitoring protein refolding in saccharomyces cerevisiae
Uploaded: 2013-07-09T12:00:00
Description: Watch this Scientific Journal Video about Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae at JoVE.com

Dette arbejde blev støttet af en bevilling fra National Institutes of Health (NIGMS-074.696) til KAM og en American Society of Microbiology Robert D. Watkins Graduate Student Research Fellowship til JLA Vi takker også John Glover på University of Toronto for at give FFL -GFP kilde plasmid.

1.. Konstruktion af stammer, som indeholder FFL-GFP plasmid For denne undersøgelse blev Saccharomyces cerevisiae stamme BY4741 (MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0), der anvendes sammen med en HSP104 deletionsstammen fra gæren knockout kollektionen (Open Biosystems / Thermo Scientific). Sletningen blev bekræftet ved hjælp af en Hsp104-specifikt antistof til Western blot-analyse. FFL-GFP blev udtrykt fra p426MET...

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I denne artikel modelprotein FFL-GFP blev brugt til at vise, at gæren disaggregase, Hsp104 bidrager til protein re-solubilisering og reparation. De enzymatiske assays og mikroskopi differentielt forhørt status samme substratproteinet at bestemme refoldning effektivitet og udbytte. Resultaterne af de enzymatiske opsving assays tyder på, at ikke blot er den maksimale opsving i hsp104D mutantstamme ineffektiv, men den oprindelige størrelse udfoldelsen stress var større i mutantstammen (figur 2).</stro...

Hos mennesker neurodegenerative sygdomme, herunder Alzheimers, Parkinsons og Huntingtons sygdomme har været knyttet til protein misfoldning og aggregering 10.. Celler anvender molekylære chaperoner at forhindre kinetisk indfangning af cellulære proteiner i fejlfoldede inaktive strukturer 6.. Chaperones deltager i indviklede interaktionsnetværk i cellen, men det er ikke helt forstået, hvordan summen af ​​disse interaktioner bidrager til organismal proteostasis. En af de vigtigste chaperoner er ansvarlige for størstedelen af cytosolprotein foldning er den 70 kD heat shock protein (Hsp70) familie 19.. Det har vist sig, at i gær tab af Hsp70 falder evnen til at folde spirende heterologt udtrykt FFL og refolde det endogene protein, ornithin transcarbamoylase in vivo 18, 7. Evnen til at analysere folde med nær-tidstro opløsning vil lette forståelsen hvordan ekstra cellulære faktorer contribute til denne Hsp70-afhængig proces. Desuden kan folde / genfoldning reaktioner ikke være helt afhængige af disse bidrager proteiner, så assays skal være følsom nok til at påvise både store og små ændringer i kinetik og effektivitet. Gærcellen disaggregase, Hsp104, spiller en afgørende rolle i at reparere aggregerede fejlfoldede proteiner. Selvom Hsp104 homologer er blevet identificeret i svampe og planter, denne familie synes at være fraværende i metazoans. Det er blevet foreslået, at andre chaperoner såsom dem af Hsp110 familien udføre nogle af de kendte Hsp104 aktiviteter i pattedyr 16. Hsp104 er en AAA +, hexamert protein kompleks, der fungerer i gær at omforme protein aggregater, der bidrager til refoldningen og reparation 13. Hsp104, sammen med gær Hsp70, Ssa1 og gær Hsp40, Ydj1, er nødvendig for inddrivelse af denatureret FFL i gærceller 5, 8. Den lille varmechokprotein, Hsp26, har også vist sig at være vandskravenerød for Hsp104-medieret opdele FFL 2.. FFL er et to-domæne protein, der binder substratet luciferin i det aktive site og efter en konformationsændring, som kræver ATP og oxygen, decarboxylates substratet og frigør oxyluciferin, kuldioxid (CO 2), adenosinmonophosphat (AMP) og lys 11. 9, 3. Den kommercielt tilgængelige FFL substrat, D-luciferin, resulterer i lysemission mellem 550-570 nm, der kan registreres ved hjælp af et luminometer 15. FFL er udsøgt følsom over for denaturering fra kemiske eller varme behandlinger og aggregater hurtigt upon udfoldning. Når de udsættes for temperaturer mellem 39-45 ° C FFL er reversibelt udfoldet og inaktiveret 12.. I modsætning hertil er GFP og dets derivater meget modstandsdygtig over for proteinudfoldning spændinger 14. Derfor fusion af disse to proteiner giver FFL at fungere som et eksperimentelt labil del kan målrette funktionel GFP til intracellulær aflejringer, kan visualiseres ved hjælp af fluorescens mikroskopi på både befolkning og encellede niveauer. Anvendelse af den enzymatiske assay i en semi-automatiseret multimode pladeaflæser kombineret med automatiseret mikroskopi tillader fortilfælde samtidig vurdering af kinetik og udbytte af genfoldning reaktioner. Desuden giver de facile molekylære genetik af modellen eukaryote Saccharomyces cerevisiae både præcis manipulation af proteinet kvalitetskontrol netværk og mulighed for opdagelsen tilgange til at identificere hidtil ukendte spillere bidrager til cellulære stress respons og proteostasis. I denne undersøgelse er vildtype (WT) og HSP104 sletning stammer, der udtrykker FFL-GFP underlagt proteindenaturering varmechok. FFL-GFP refoldningen overvåges gennem både en enzymatisk assay og mikroskopi som en proxy udlæsning til reparation af det udtrykte proteom over en restitutionstid kursus. Sammenlignet med WT celler, viser vi the Hsp104 deletionsstammen er ~ 60% mindre effektive til genfoldning FFL-GFP, støtte tidligere resultater oprettelse en rolle for Hsp104 i reaktivering af denatureret FFL 2..

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>Synergy MX Microplate Reader</td>
 <td>BioTek</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Olympus IX81-ZDC Confocal Inverted Microscope</td>
 <td>Olympus, Tokyo Japan</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Lumitrac 200 white 96-well plates</td>
 <td>USA Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>SlideBook 5.0 digital microscopy software</td>
 <td>Intelligent Imaging Innovations, Inc. Denver, CO, USA</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>Fisher</td>
 <td>BP152-1</td>
 <td></td>
 </tr>
 <tr>
 <td>(LiAc) Lithium Acetate</td>
 <td>Sigma</td>
 <td>L6883</td>
 <td></td>
 </tr>
 <tr>
 <td>PEG (polyethelene glycol)</td>
 <td>Fisher</td>
 <td>BP233-1</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Fisher</td>
 <td>BP120-1</td>
 <td></td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Malinckrodt</td>
 <td>4948</td>
 <td></td>
 </tr>
 <tr>
 <td>SC</td>
 <td>Sunrise</td>
 <td>1300-030</td>
 <td></td>
 </tr>
 <tr>
 <td>SC-URA</td>
 <td>Sunrise</td>
 <td>1306-030</td>
 <td></td>
 </tr>
 <tr>
 <td>cycloheximide</td>
 <td>Acros Organics</td>
 <td>357420050</td>
 <td></td>
 </tr>
 <tr>
 <td>D-luciferin</td>
 <td>Sigma</td>
 <td>L9504</td>
 <td></td>
 </tr>
 <tr>
 <td>low melt agarose</td>
 <td>NuSieve</td>
 <td>50082</td>
 <td></td>
 </tr>
 <tr>
 <td>immersion oil</td>
 <td>Cargille</td>
 <td>16484</td>
 <td></td>
 </tr>
 </tbody>
</table>

coupled assays for monitoring protein refolding in saccharomyces cerevisiae

Proteostasis, defineret som de kombinerede processer proteinfoldning / biogenese, refoldning / reparation, og nedbrydning, er en delikat cellulære balance, der skal opretholdes for at undgå skadelige konsekvenser 1.. Eksterne eller interne faktorer, der forstyrrer denne balance kan føre til proteinaggregering, toksicitet og celledød. Hos mennesker er dette en væsentlig medvirkende faktor til de symptomer forbundet med neurodegenerative lidelser såsom Huntingtons, Parkinsons og Alzheimers sygdom 10.. Det er derfor vigtigt, at de proteiner involveret i opretholdelsen af ​​proteostasis identificeres med henblik på at udvikle behandlinger for disse invaliderende sygdomme. Denne artikel beskriver teknikker til overvågning in vivo proteinfoldning ved nær-realtid opløsning ved hjælp af modellen protein ildflueluciferase fusioneret til grønt fluorescerende protein (FFL-GFP). FFL-GFP er en unik model kimære protein som FFL delen er yderst følsomt over for stress-induceret misfolding og aggregering, der inaktiverer enzymet 12.. Luciferaseaktivitet overvåges ved hjælp af en enzymatisk assay og GFP-delen tilvejebringer en fremgangsmåde til visualisering af opløselige eller aggregerede FFL hjælp automatiseret mikroskopi. Disse koblede metoder indarbejde to parallelle og teknisk uafhængige tilgange til at analysere både refoldningen og funktionel reaktivering af et enzym efter stress. Aktivitet opsving kan være direkte korreleret med kinetik for opdeling og re-solubilisering til bedre at forstå hvordan protein kvalitetskontrol faktorer såsom protein chaperoner samarbejder om at udføre disse funktioner. Desuden kan gendeletioner eller mutationer bruges til at teste bidrag af specifikke proteiner eller protein-underenheder til denne proces. I denne artikel vil vi gennemgå bidragene af proteinet disaggregase Hsp104 13, kendt for at samarbejde med den Hsp40/70/nucleotide udveksling faktor (NEF) genfoldning system 5 til Proteingenfoldningstrin at validere denne fremgangsmåde.

Gær er afhængig af disaggregase, at Hsp104 effektivt genfolde varme-denaturerede proteiner. Aktiviteten af FFL-GFP blev overvåget efter en 25 min varmechok, ved hjælp af en luminescens flash assay Figur 1.. Resultaterne af denne automatiserede assay er vist i figur 2 viste en trinvis stigning i aktivitet i løbet af 90 minutter, der i sidste ende førte til en&gt; 80% genvinding i WT celler. Den hsp104Δ stammen genvundet 19% af den oprindelige aktivitet i den samme tidsram...

Watch this Scientific Journal Video about Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae at JoVE.com

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Denne artikel beskriver anvendelsen af ​​en ildflue luciferase-GFP-fusionsproteinet til at undersøge<em&gt; In vivo</em&gt; Proteinfoldning i<em&gt; Saccharomyces cerevisiae</em&gt;. Brug af denne reagens refoldning af en model varme-denaturerede protein kan monitoreres samtidigt ved fluorescensmikroskopi og en enzymatisk assay til at undersøge roller proteostasis netværkskomponenter i protein kvalitetskontrol.

Koblede Analyser for Monitoring Proteingenfoldningstrin i<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

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