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 uttrykk
Merk: Aβ varianter forevise annet relative aggregering propensities på grunn av en mutasjon i en enkelt rester i posisjon 19 (Phe19) av Aβ42 peptid (figur 1A). Disse peptid variantene er merket med grønne fluorescerende protein (GFP), som fungerer som en samling reporter (figur 1)29.
<ol><li>Transformere plasmider koding for 20 Aβ42-GFP varianter i gj...

<ol>
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En rekke sykdommer knyttet til opphopning av misfolded proteiner i mobilnettet innskudd6,7,8,33. Mange forsøk har vært gjort å rakne molekylære mekanismer som utløser utbruddet av disse sykdommer med computational tilnærminger, som ikke tar hensyn til kontoen protein konsentrasjoner, eller i vitro tilnærminger, som protein konsentrasjonen forblir konstant under reaksjonen. Men i...

Proteostasis er en grunnleggende determinant av celle helse og aldring prosesser. I celler vedlikeholdes protein homeostase av sofistikert protein kvalitetskontroll nettverk rettet å sikre den riktige refolding av misfolded protein conformers chaperones og/eller deres målrettede proteolyse med flere godt bevarte mekanismer1 ,2,3,4,5. En rekke studier gir støtte til koblingen mellom utbruddet og utviklingen av en rekke menneskelige sykdommer og svikt i proteostasis, fører til protein misfolding og aggregering. For eksempel tilstedeværelsen av protein innskudd er ansett som en patologisk kjennetegner mange nevrodegenerative sykdommer, som Alzheimers, Parkinsons, og Huntingtons sykdommer6,7,8, prionogenic sykdommer og ikke-degenerative amyloidoses9. Det har blitt foreslått at tidlig oligomeric og protofibrillar samlinger i samling reaksjonen er de viktigste elicitors av cytotoksisitet, etablere avvikende interaksjoner med andre proteiner i overfylte mobilnettet miljø10. I tillegg kan protein Inneslutninger (PI) overføres mellom celler, spre sine toksisk effekt11,12. Derfor kan det være at dannelsen av PI kan faktisk utgjør en detoxifying mekanisme som begrenser tilstedeværelsen av farlig aggregert arter til bestemte steder i cellen, der de kan behandles eller akkumulert uten store bivirkninger 13 , 14.
Standard i vitro biokjemiske tilnærminger har gitt viktig innsikt i de ulike artene som fyller aggregering reaksjoner og deres egenskaper15,16. Men betingelsene i disse analyser er klart forskjellig fra de som forekommer i cellen, og derfor spørsmålet relevansen fysiologiske. På grunn av kjente bevaring av mobilnettet som protein kvalitetskontroll, autophagy eller regulering av mobilnettet redoks staten17,18 blant eukaryoter19,20,21 ,22,23, spirende gjær Saccharomyces cerevisiae (S. cerevisiae) har dukket opp som en privilegert enkel mobilnettet modell å studere molekylære determinanter av protein aggregering og tilknyttede cytotoksiske innvirkning på biologisk relevante miljøer24,25,26.
Protein aggregering tilbøyelighet er en funksjon iboende kodet i primære sekvensen. Dermed dannelsen av amyloid-lignende strukturer kan forutsies basert på identifisering og evaluering av styrken på aggregering-fremme regioner i polypeptides27. Men til tross for bioinformatic algoritmer å forutsi egenskapene for aggregering i vitro protein sekvenser, er de fortsatt langt fra prognoser hvordan disse propensities oversette til i vivo cytotoksiske innvirkning. Studier som tar koblingen mellom et bestemt protein aggregert staten og dens tilknyttede cellular skade på en systematisk måte kan bidra til å omgå denne beregningsorientert begrensningen. Denne tilkoblingen behandles i studien, drar nytte av en rekke varianter av amyloid-β peptid Aβ42 forskjellig fra en enkelt rester, men viser en kontinuerlig rekke aggregering propensities i vivo28. Spesielt er en FC-basert tilnærming til til conformational arten regnskap for oksidative skader brakt frem av aggregering utsatt proteiner i gjærceller beskrevet. Metodikken gir mange fordeler som enkelhet, høy gjennomstrømming evne og nøyaktig kvantitativ måling. Dette gjorde det mulig å bekrefte som PI spiller en beskyttende rolle mot oksidativt 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 misfolding og aggregering i amyloid konformasjonen har vært knyttet til utbruddet og progresjon av flere nevrodegenerative sykdommer. Men det er fortsatt litt informasjon om hvordan uløselig protein aggregater utøve deres toksiske effekter i vivo. Enkel prokaryotic og eukaryotic modell organismer, som bakterier og gjær, har bidratt betydelig til vår nåværende forståelse av mekanismene bak intracellulær amyloid dannelse, aggregater forplantning, og toksisitet. I denne protokollen, er bruk av gjær beskrevet som en modell for å analysere forholdet mellom dannelsen av protein aggregater og deres innvirkning på mobilnettet oksidativt stress. Metoden kombinerer påvisning av intracellulær løselig/samlet status for en amyloidogenic protein med kvantifisering av mobilnettet oksidative skader som følge av sitt uttrykk ved hjelp av flowcytometri (FC). Denne tilnærmingen er enkel, rask og kvantitative. Studien viser teknikken ved å samkjøre mobilnettet oksidativt stress forårsaket av et stort sett av amyloid-β peptid varianter med sine respektive iboende samling propensities.

Denne protokollen beskriver hvordan å ansette en samling av 20 varianter av Aβ42 peptid der Phe19 er mutert til alle naturlige proteinogenic aminosyrer28. De teoretiske aggregering propensities av disse proteinene kan analyseres ved hjelp av to forskjellige bioinformatic algoritmer (AGGRESCAN og TANGO31,32). I begge tilfeller denne analysen gjengir en progressiv gradering av aggregering tendenser, tillegg...

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 av virkningen av Protein Oppsamling på mobilnettet oksidativt Stress i gjær

Protein aggregering utløser mobilnettet oksidativt stress. Denne protokollen beskriver en metode for overvåking intracellulær statene amyloidogenic proteiner og oksidativt stress forbundet med dem, ved hjelp av flowcytometri. Tilnærmingen til å studere atferden til løselig og aggregering utsatt varianter av 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 pattedyrceller

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 elektrospray-ionisering Hydrogen-deuterium Veksling massespektrometri for å studere Protein struktur og dynamikk

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 enkel metode for høy gjennomstrømning kjemiske 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 gjær 2-Hybrid skjerm i Batch sammenligne Protein interaksjoner

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 selv organisere Protein mønstre på støttede Lipid Bilayers

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 av proteiner for ett molekyl Force 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 av proteinfolding, Transport og nedbrytning i levende celler av 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 av protein-protein interaksjoner ved hjelp av en on-membran fordøyelsen Technique

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

Undersøke dynamikken i Cellular vedheft og spredning av epitelceller på Fibronektin under oksidativt 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 av lokalisering av submitochondrialprotein i spirende gjær saccharomyces cerevisiae

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