Name: assaying proteasomal degradation in a cell-free system in plants
Uploaded: 2014-03-26T17:45:00
Description: Watch this Scientific Journal Video about Assaying Proteasomal Degradation in a Cell-free System in Plants at JoVE.com

Det arbejde, der fører til denne publikation har modtaget støtte fra Marie Curie COFUND program &quot;U-mobilitet&quot;, medfinansieret af University of Malaga og Den Europæiske Union 7. rammeprogram (FP7/2007-2013) under GA nr. 246.550. Arbejdet i vores laboratorium er støttet af tilskud fra NIH, USDA / NIFA, NSF, Bard, og BSF til VC

1.. Proteinekspression <ol><li> Valg af ekspressionssystem Vælg system, dvs, vektorer og vektor-levering metode bedst egnet til ekspression af proteinet af interesse i den specifikke model organisme / celle. Bemærk, at vores analyse kræver udtryk af de testede proteiner i let detekterbare mængder, hvilket bedst opnås ved forbigående transformation af et stort antal celler. I planter, for eksempel, er dette bedst opnås ved hjælp af binære plasmider ekspressionsvektorer og Agrobacterium ...

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Denne analyse bygger på ekspressionen af ​​de testede proteiner i plantevæv, og dermed den potentielle proteasomalaktivitet nedbrydningsprocessen naturligvis sker allerede inden for de levende væv. Vi assay protein destabilisering, dog kun i ekstrakterne med tiden nul prøve tjener som det første referencepunkt. Derfor har vi definere det som et cellefrit assay. Et vigtigt aspekt for succes i dette assay er det korrekte valg af ekspressionsvektoren, hvorfra det testede protein (er) v...

Den ubiquitin/26S proteasom vej fremstår som en udbredt mekanisme til styring af forskellige biologiske reaktioner, herunder transkriptionel regulering, cellecyklusfremadskriden og signaltransduktion, receptor nedregulering eller endocytose, blandt andre processer 1-4. I denne vej, er målproteinet tagget af ubiquitinenheder som først fastgjort via en thiolesterbinding til ubiquitin-aktiverende enzym E1 og derefter translokeres til en cystein aminosyrerest i ubiquitin-konjugation enzym E2, og endelig E2 interagerer med ubiquitin-ligase E3 , hvilket resulterer i polyubiquitination af proteinsubstratet. I sidste ende er de polyubiquitinated proteiner anerkendt og nedbrydes af 26S proteasomet. I denne mekanisme, E3 enzym angiver underlaget og fungerer som den centrale regulerende element i ubiquitin/26S proteasom systemet (UPS). E3 ligaser kan handle uafhængigt, såsom RING domæne ligaser, eller som del af en multisubunit SCF (Skp1/Cullin/F-box protein)-kompleks, såsom F-box domæne ligaser. SCF-medierede proteasomalaktivitet nedbrydningsveje er involveret i reguleringen af transskription, cellecyklus, signaltransduktion 5-10 og mange andre store cellulære funktioner. Udover disse kritiske roller i reguleringen af ​​cellulære processer, UPS tager den centrale scene i mange plante-patogen interaktioner. For eksempel flere beviser tyder på, at flere plante patogener, herunder Agrobacterium tumefaciens, stole på værten UPS for at lette infektionen processen 11.. Agrobacterium fremkalder neoplastiske vækster på planter, som repræsenterer dets naturlige værter, og det kan også omdanne en lang række andre eukaryoter, fra svampe 1,2 til humane celler 12,13. , Agrobacterium eksporterer løbet af sin infektion et DNA-element (T-DNA) og flere virulens (Vir) proteiner i værtscellen 12-13. Et af disse proteiner er VirF den første F-box-protein fundetat være kodet af en prokaryotisk genom 14. Som en del af SCF ubiquitinligase kompleks, VirF og dets funktionelle vært homolog VBF 15, lette Agrobacterium infektion via UPS-medieret proteinnedbrydning, hvilket formentlig letter virionkappe af invaderende bakterie T-DNA fra de ledsagende bakteriel og værtsproteiner, VirE2 og VIP1 henholdsvis 16,17. Interessant mange F-box proteiner, herunder VirF er uløseligt ustabile på grund af deres egen proteolyse, som medieres af autoubiquitination aktivitet 18,19 eller andre E3-ligaser, som F-box-proteiner kan tjene som substrater 20-23. Når man studerer biokemiske aktiviteter F-box-proteiner, andre ubiquitin ligaser, og / eller deres substrater, ville det være meget nyttigt at anvende en enkel og pålidelig analyse for proteasomalaktivitet nedbrydning. Her beskriver vi en sådan protokol for analyse af protein stabilitet i en celle-Free system. I dette assay er stabiliteten af ​​UPS substrat analyseret i nærvær eller fravær af en af ​​de væsentlige dele af proteasomalaktivitet nedbrydningsvej, såsom en F-box-protein i et cellefrit system. Generelt udtrykker vi den testede protein (r) i plantevæv, forberede celle-fri ekstrakter fra disse væv og overvåge mængder af proteinet (r) af interesse ved western blotting. UPS-afhængige mekanisme af protein nedbrydning påvises ved inddragelse af specifikke proteasomalaktivitet hæmmere og / eller ved hjælp coexpression af dominant negativ form af en SCF-komponent, Cullin. Mens vi illustrere dette assay under anvendelse af proteasomalaktivitet nedbrydning af Arabidopsis VIP1 17 protein ved F-box-protein VBF 15, kan den anvendes til at undersøge stabiliteten af andre proteasomalaktivitet substrater.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Protein assay kit</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">500-0001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Proteinase inhibitor cocktail&#160;</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mini-Protean system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">165-8000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Semi-dry western blotting SD electrotransfer system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">170-3940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Affinity Purified Rabbit Anti-Ha</td>
			<td style="width:129px;">icllab</td>
			<td>RHGT-45A-Z</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Goat anti-Rabbit IgG Peroxidase Conjugate</td>
			<td style="width:129px;">Thermo Scientific</td>
			<td style="width:119px;">31460</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">BioTrace, NT nitrocellulose transfer membrane</td>
			<td style="width:129px;">Pall Corportation</td>
			<td style="width:119px;">27377-000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Immobilon western chemiluminescent HRP substrate</td>
			<td style="width:129px;">EMD Millipore</td>
			<td style="width:119px;">WBKL S0 050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MG132</td>
			<td style="width:129px;">EMD Millipore</td>
			<td>474790-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Lactacystin</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td>L6785</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Thermo Scientific Pierce Fast Western Blot Kit, ECL Substrate</td>
			<td style="width:129px;">Pierce</td>
			<td style="width:119px;">35055</td>
			<td></td>
		</tr>
	</tbody>
</table>

assaying proteasomal degradation in a cell-free system in plants

Den ubiquitinproteasomvejen for proteinnedbrydning dukket op som en af ​​de vigtigste mekanismer til regulering af et bredt spektrum af cellefunktioner i praktisk taget alle eukaryote organismer. Specifikt i planter, det ubiquitin/26S proteasom systemet (UPS) regulerer proteinnedbrydning og bidrager væsentligt til udviklingen af ​​en lang række processer, herunder immunrespons, udvikling og programmeret celledød. Desuden øger tyder på, at mange plante-patogener, såsom Agrobacterium, udnytte værten UPS til effektiv infektion, understreger betydningen af UPS i plante-patogen interaktioner. Underlaget specificitet UPS opnås ved E3 ubiquitinligase der handler i forståelse med E1 og E2 ligase til at genkende og markere bestemte proteinmolekyler bestemt til nedbrydning ved at knytte til dem kæder af ubiquitin-molekyler. En klasse af E3 ligaser er SCF (SKP1 / CUllin / F-box protein) kompleks, som specifikt genkender UPS substrater og målretter dem til ubiquitinering via sin F-box protein komponent. At undersøge en mulig rolle UPS i en biologisk proces af interesse, er det vigtigt at udvikle en enkel og pålidelig analyse for UPS-medieret proteinnedbrydning. Her beskriver vi en sådan assay under anvendelse af en plante cellefrit system. Denne analyse kan tilpasses til undersøgelser af roller reguleret protein nedbrydning i forskellige cellulære processer, med særlig fokus på F-box protein-substrat interaktioner.

Figur 1, tilpasset fra Zaltsman et al. 17 illustrerer repræsentative forsøg til påvisning af proteasomalaktivitet nedbrydning i et cellefrit system. Konkret har vi demonstrere destabilisering af en plante forsvar-relateret protein VIP1 af VBF F-box protein via SCF VBF vej i N. benthamiana. Arabidopsis VBF og HA-mærkede VIP1 (HA-VIP1) proteiner blev transient coudtrykt, og HA-VIP1 proteinindhold inden uddrag af udtrykkende blade blev analyseret ved wes...

Watch this Scientific Journal Video about Assaying Proteasomal Degradation in a Cell-free System in Plants at JoVE.com

Assaying Proteasomal Degradation in a Cell-free System in Plants

Analysering proteasomalaktivitet Nedbrydning i en celle-fri system i planter

Målrettet protein nedbrydning udgør en stor reguleringsmekanisme for celle funktion. Det sker via en konserveret ubiquitinproteasomvejen, som lægger multiubiquitinkæder til målproteinet som derefter tjene som molekylære &quot;tags&quot; for 26S proteasom. Her beskriver vi en enkel og pålidelig cellefrit assay for proteasomalaktivitet nedbrydning af proteiner.

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Assaying the Kinase Activity of LRRK2 in vitro

Analysering af kinase aktivitet af LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Reconstitution Of ÃƒÅ½Ã‚Â²-catenin Degradation In Xenopus Egg Extract

Rekonstituering af β-catenin nedbrydning i<em&gt; Xenopus</em&gt; Æggeekstrakt

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux

Måling af Døgnrytmer i Autophagic og Proteasomal flux

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

En simpel protokol til kortlægning af plantens rodsystemarkitekturegenskaber

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration (Video) | JoVE 

Decellulariseringsbaseret kvantificering af skeletmuskulatur fedtinfiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Embedding CFPS Reactions in a Variety of Hydrogel Matrices

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

A Novel Approach for Embedding Cell-Free Protein Synthesis Reactions in Hydrogels (Video) | JoVE

Metoder til indlejring af cellefrie proteinsyntesereaktioner i hydrogeler i makroskala

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. ...

Quantitative Analysis of Mitophagy in Murine Pancreatic &#946;-Cells

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Assessment of Mitophagy Flux in Pancreatic Î²-Cells Using Effective and Robust Complementary Approaches (Video) | JoVE

Komplementære tilgange til at forhøre mitofagiflux i β-celler i bugspytkirtlen

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

Cell-Free Protein Synthesis in Rehydrated Lyophilized Hydrogels

Cell-Free Protein Synthesis in Rehydrated Lyophilized Hydrogels (Video) | JoVE 

Cellefri proteinsyntese i rehydrerede frysetørrede hydrogeler