Name: evaluation of the impact of protein aggregation on cellular oxidative stress in yeast
Uploaded: 2018-06-23T13:00:00
Description: Watch this Scientific Journal Video about Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast at JoVE.com

1. S. cerevisiae kulturer og Protein udtryk
Bemærk: Aβ varianter udviser forskellige relative sammenlægning tilbøjeligheder på grund af en mutation i et enkelt rester på holdning 19 (Phe19) af Aβ42 peptid (figur 1A). Disse peptid varianter er markeret med grøn fluorescerende proteiner (NGL), der fungerer som en sammenlægning reporter (figur 1)29.
<ol><li>Omdanne plasmider kodning for 20 Aβ4...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+power+of+yeast+to+unravel+the+molecular+basis+of+neurodegeneration.">Harnessing the power of yeast to unravel the molecular basis of neurodegeneration.</a> Journal of Neurochemistry. 127, 438-452 (2013).</li><li>Yang, J., Hao, X., Cao, X., Liu, B., Nystr&#246;m, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+sequestration+and+detoxification+of+huntingtin+by+the+ribosome+quality+control+complex.">Spatial sequestration and detoxification of huntingtin by the ribosome quality control complex.</a> eLife. 5, (2016).</li><li>Braun, R. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+blocks+ER-Golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313, 324-328 (2006).</li><li>Johnson, B. S., McCaffery, J. M., Lindquist, S., Gitler, A. 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En bred vifte af sygdomme er forbundet med ophobning af fejlfoldede proteiner i cellulære indskud6,7,8,33. Har gjort mange anstrengelser at optrævle de molekylære mekanismer, der udløser udbrud af disse sygdomme ved hjælp af beregningsmæssige metoder, som ikke tager hensyn protein fusioner, eller in vitro- metoder, hvor proteinkoncentration forbliver konstante under reaktionen. M...

Proteostasis er en grundlæggende faktor for celle fitness og aldersfordelte processer. I celler vedligeholdes protein homøostase af sofistikerede protein kvalitetskontrol netværk har til formål at sikre den korrekte hvorved man genfolder af fejlfoldede protein konformere af chaperoneproteiner og/eller deres målrettede proteolyse med flere godt bevarede mekanismer1 ,2,3,4,5. Et stort antal undersøgelser understøtte forbindelsen mellem symptomdebut og progression af en bred vifte af sygdomme hos mennesker og manglende proteostasis, fører til protein misfoldning og sammenlægning. For eksempel, betragtes tilstedeværelsen af protein indskud som en patologisk kendetegnende for mange neurodegenerative sygdomme som Alzheimers, Parkinsons, og Huntingtons sygdomme6,7,8, prionogenic sygdomme, og ikke-degenerative amyloidoses9. Det er blevet foreslået, at tidlig oligomere og protofibrillar forsamlinger i sammenlægning reaktion er de vigtigste elicitors af cytotoksicitet, etablere afvigende interaktioner med andre proteiner i overfyldte cellulære miljø10. Desuden kan protein indeslutninger (PI) overføres mellem celler, formerings deres giftvirkning11,12. Det kunne derfor være, at dannelsen af PI faktisk kan udgøre en afgiftende mekanisme, der begrænser forekomsten af farlige aggregerede arter til bestemte steder i cellen, hvor de kan behandles eller akkumuleret uden større bivirkninger 13 , 14.
Standard in vitro- biokemiske metoder har givet vigtige indsigter i de forskellige fiskearter, der befolker sammenlægning reaktioner og deres egenskaber15,16. Men de betingelser, der anvendes i disse assays er klart forskellige fra dem, der forekommer inden for cellen, og derfor sætte spørgsmålstegn ved deres fysiologiske betydning. På grund af den bemærkelsesværdige bevarelse af cellulære veje som protein kvalitetskontrol, autophagy eller regulering af cellulære redox stat17,18 blandt eukaryoter19,20,21 ,22,23, spirende gær Saccharomyces cerevisiae (S. cerevisiae) fremstod som en privilegeret simpel cellulære model til at studere de molekylære determinanter for protein sammenlægning og dens tilknyttede cytotoksiske effekt i biologisk relevante miljøer24,25,26.
Protein sammenlægning tilbøjelighed er en funktion i sagens natur kodet i den primære sekvens. Dannelsen af amyloid-lignende strukturer kan således forudsiges baseret på identificering og evaluering af styrken af fremme af sammenlægning regioner i polypeptider27. Men trods succesen med bioinformatic algoritmer til at forudsige in vitro- sammenlægning egenskaberne af protein sekvenser, de er stadig langt fra forecasting, hvordan disse tilbøjeligheder omsættes i vivo cytotoksiske effekt. Undersøgelser, der vedrører forbindelsen mellem den aggregerede stat af et bestemt protein og dens tilknyttede cellulære skader på en systematisk måde kan bidrage til at omgå denne beregningsmæssige begrænsning. Denne forbindelse behandles i den foreliggende undersøgelse, at drage fordel af en lang række varianter af amyloid-β peptid Aβ42 adskiller sig kun i en enkelt rester, men viser en kontinuerlig række sammenlægning tilbøjeligheder i vivo28. Især er en FC-baseret tilgang til at identificere de konformationelle arter tegner sig for den oxidative skader fremkaldes ved sammenlægning-tilbøjelige proteiner i gærceller beskrevet. Metoden, der giver mange fordele såsom enkelhed, høj overførselshastighed kapacitet og præcis kvantitativ måling. Denne fremgangsmåde gjorde det muligt at bekræfte, at PI spiller en beskyttende rolle mod oxidativt stress.

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	<tbody>
		<tr>
			<td>Yeast cells BY4741&#160;</td>
			<td>ATCC</td>
			<td>201388</td>
			<td>Genotype: MATa his3&#916;1 leu2&#916;0 met15&#916;0 ura3&#916;0</td>
		</tr>
		<tr>
			<td>pESC(-Ura) plasmid&#160;</td>
			<td>Agilent Genomics</td>
			<td>217454</td>
			<td>Yeast expression plasmid with a Gal promotor. Selectable marker URA3</td>
		</tr>
		<tr>
			<td>Yeast Synthetic Drop-out Medium Supplements</td>
			<td>Sigma</td>
			<td>Y1501</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base Without Amino Acids</td>
			<td>Sigma</td>
			<td>Y0626</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Raffinose</td>
			<td>Sigma</td>
			<td>R7630</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>Sigma</td>
			<td>G0750</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP3991&#160;</td>
			<td>Solution 10X</td>
		</tr>
		<tr>
			<td>CellROX Deep Red Reagent&#160;</td>
			<td>Life Technologies</td>
			<td>C10422</td>
			<td>Free radical cell-permeant fluorescent sensor, non-fluorescent while in a reduced state, and exhibits bright fluorescence upon oxidation by reactive oxygen species (ROS), with absorption/emission maxima at 644/665 nm.&#160;</td>
		</tr>
		<tr>
			<td>Y-PER protein extraction reagent&#160;</td>
			<td>Thermo Scientific</td>
			<td>78990</td>
			<td>Liquid cell lysis buffer</td>
		</tr>
		<tr>
			<td>Acrylamide/Bis-acrylamide</td>
			<td>Sigma</td>
			<td>A6050</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Bradford dye reagent</td>
			<td>Bio-Rad&#160;</td>
			<td>5000205</td>
			<td>Dye reagent for one-step determination of protein concentration</td>
		</tr>
		<tr>
			<td>&#946;-amyloid antibody 6E10&#160;</td>
			<td>BioLegend</td>
			<td>803001</td>
			<td>Mouse IgG1. The epitope lies within amino acids 3-8 of beta amyloid (EFRHDS).</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-HRP conjugate&#160;</td>
			<td>Bio-Rad</td>
			<td>1721011</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Immobilon-P, PVDF</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminata forte</td>
			<td>Merk</td>
			<td>WBLUF0100</td>
			<td>Premixed, ready to use chemiluminescent HRP detection reagent</td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride solution (PMSF)</td>
			<td>Sigma</td>
			<td>93482</td>
			<td>Protease inhibitor. Dissolved at 0.1 M in ethanol</td>
		</tr>
		<tr>
			<td>FACSCanto flow cytometer&#160;</td>
			<td>BD Biosciences</td>
			<td>657338</td>
			<td>Equipped with a 488&#160;nm blue laser for the detection of GFP, and 635&#160;nm red laser / 530/30 nm BP filter and 660/20 BP filter</td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Electrophoresis Transfer cell</td>
			<td>Bio-Rad</td>
			<td>1703930</td>
			<td>Protein transference system</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Handcast Systems</td>
			<td>Bio-Rad</td>
			<td>1658000FC</td>
			<td>Electrophoresis system</td>
		</tr>
	</tbody>
</table>

evaluation of the impact of protein aggregation on cellular oxidative stress in yeast

Protein misfoldning og sammenlægning i amyloid konformationer har været relateret til debut og progression af flere neurodegenerative sygdomme. Der er dog stadig lidt information om hvordan uopløselige protein aggregater udøve deres toksiske virkninger in vivo. Simpel prokaryote og eukaryote modelorganismer, såsom bakterier og gær, har bidraget væsentligt til vores nuværende forståelse af mekanismerne bag intracellulære amyloid dannelse, aggregater formering og toksicitet. I denne protokol, er brugen af gær beskrevet som en model til at dissekere forholdet mellem dannelsen af protein aggregater og deres indvirkning på cellulære oxidativt stress. Metoden kombinerer påvisning af intracellulære opløselige/aggregerede status for en amyloidogenic protein med kvantificeringen af cellulære oxidative skader som følge af dens udtryk ved hjælp af flowcytometri (FC). Denne fremgangsmåde er enkel, hurtig og kvantitative. Undersøgelsen viser teknikken ved at sammenholde de cellulære oxidativt stress forårsaget af et stort sæt af amyloid-β peptid varianter med deres respektive iboende sammenlægning tilbøjeligheder.

Denne protokol beskriver hvordan til at ansætte en samling af 20 varianter af den Aβ42 peptid hvor Phe19 har været muteret til alle naturlige proteinogeniske aminosyrer28. De teoretiske sammenlægning tilbøjeligheder af disse proteiner kan analyseres ved hjælp af to forskellige bioinformatic algoritmer (AGGRESCAN og TANGO31,32). I begge tilfælde, gør denne analyse en progressiv graduering af sammenl?...

Watch this Scientific Journal Video about Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast at JoVE.com

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Evaluering af virkningen af Protein aggregering på cellulært Oxidative Stress i gær

Protein sammenlægning fremkalder cellulære oxidativt stress. Denne protokol beskriver en metode til overvågning af de intracellulære stater af amyloidogenic proteiner og den oxidative stress i forbindelse med dem, ved hjælp af flowcytometri. Metoden bruges til at studere funktionen af opløselige og sammenlægning-tilbøjelige varianter af amyloid-β-peptid.

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Protein Complex Affinity Capture fra Cryomilled pattedyrsceller

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Time-løst elektrosprayioniserings Brint-deuterium Exchange-massespektrometri til at studere proteiners struktur og Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

En simpel metode til High Throughput kemisk Screening i Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

En gær 2-Hybrid skærm i Batch for at sammenligne Protein interaktioner

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

In Vitro Rekonstituering af selvstændige organisering Protein mønstre på understøttede Lipid dobbeltlag

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

Kovalente immobilisering af proteiner for enkelt molekyle kraft spektroskopi

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Analyse af proteinfoldning, Transport og nedbrydning i levende celler af radioaktivt puls Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Evaluation of ProteinÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Protein Interactions using an On-Membrane Digestion Technique

Evaluering af protein-protein interaktioner ved hjælp af en on-membran fordøjelse teknik

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

Undersøge dynamikken i cellulære vedhæftning og spredning af Epiteliale celler på Fibronectin under oxidativ stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Vurdering af indsendeokondrial protein lokalisering i spirende gær saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...