Name: purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation
Uploaded: 2017-08-21T14:00:00
Description: Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

Detta arbete stöds av Takasaki universitet av hälsa och välbefinnande.

1. växande celler och tillägg av exogena antigen <ol><li>Förbered bOVA använder en biotin-protein märkning kit efter tillverkaren &#39; s protokollet. 
	Obs: Normalt, bOVA innehåller 2 M biotin per 1 M ägg genomsnitt.</li>
	<li>Växer DC2.4 celler i RPMI-1640 kompletteras med 2 mM L-glutamin, 1 mM natrium pyruvat, 0,1 mM onödiga aminosyror, 100 U/mL penicillin-streptomycin, 55 mM 2-merkaptoetanol, 10 mM HEPES (pH 7,5) och 10% fetalt kalvserum (härefter RPMI) vid 37 ° C i 5 % CO 2 i en fuktad ...

<ol>
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I tidigare studier av CP ackumulerade de bolagiserade exogena antigenerna i det begränsade området av sena endosome eller ER av Immunofluorescerande mikroskopi16,30,31,32. Det uppskattas att ERAD-liknande transporter och bearbetning av exogena antigen genomförs i dessa specialiserade områden av ER eller sent endosome, som mobilfack identifierades av sackaros eller iodixanol täthet lutning ...

MHC jag molekyler som uttrycks på ytan av alla kärnförsedda celler, med korta antigena peptider som härrör från endogena antigener, som behandlas av den ubiquitin-proteasomsystemet i cytosolen1. Efter bearbetning transporteras antigena peptider i endoplasmatiska nätverket (ER) lumen av peptid transportören TAP. En rad specifika chaperones bistå ER lumen, peptid lastning och den rätta vikningen av MHC I komplexa. Denna serie av molekyler kallas peptid-lastning komplexet (PLC), vilket indikerar att akuten är ett centralt kompartment för peptid lastning på MHC jag2. Efter lastning, peptiden till MHC jag molekyler transporteras till cellytan och spela en nyckelroll i det adaptiva immunsystemet som själv markörer och möjliggör CD8+ cytotoxiska T-lymfocyter (CTL) att upptäcka cancerceller eller infektiösa agens av antigen peptider från icke-själv proteiner3.
I antigenpresenterande celler (APC), antigen peptider från exogena antigen är också presenteras på MHC jag4,5,6,7,8 via CP, som bärs främst ut av DCs9,10,11. CP är viktigt både för aktivering av naiva CD8+ T celler och minne CD8+ T celler in i anti-infektiösa och anti-tumoral CTL12,13, och i underhållet av immuntolerans av det inactivating av själv tillförordnade naiva T-celler14,15. CP spelar många viktiga roller i det adaptiva immunsystemet, men molekylära mekanismer för CP har ännu inte beskrivas i detalj. Tidigare studier av CP avslöjade att exogena antigen var lokaliserade både ER och endosome och bearbetades av ERAD, vilket tyder på att exogena antigen transporteras från endosome till ER för ERAD-liknande bearbetning och peptid som laddar16 . Ackumulerande bevis tyder dock på att peptid lastning av CP utförs inte i ER utan snarare i icke-klassisk endocytic fack, som också har särdrag av ER (figur 1)17,18 ,19,20,21. För att undvika nedbrytning av antigena peptid prekursorer av hög aktivitet av aminopeptidase uppstår22 i cytosolen, bearbetning och peptid lastning i CP i området proximalt i dessa icke-klassisk endocytic fack (figur 1). Även karakteriseringar av dessa endocytic fack är kontroversiell, finns det inga befintliga specifika molekyler än exogena antigen i detta fack.
ERAD är en cellulär väg, som specifikt tar bort felveckning proteiner från ER. I ERAD väg, felveckning proteiner retrogradely transporteras genom ER membranet till cytoplasman och bearbetas av ubiquitin-proteasom systemet23,24,25. När stora molekyler, såsom proteiner, transporteras genom den lipid lipidens, dessa molekyler passera en molekylär apparat som kallas en translocon, såsom Sec61 komplex och Derlin komplex i den ER26, och Tom komplexa och Tim komplex i den mitokondrier27. När exogent-lade antigener transporteras genom ER membranet, måste de tränger den lipid lipidens i komplex med translocons, såsom Sec61 komplexet. Den metod som beskrivs här renas det riktade vesikler genom att utnyttja dessa membran-penetrerande molekyler som markörer för endocytic facken.
Den metod som beskrivs här är ett nytt vesikler rening protokoll med hjälp av DC-liknande cell linje DC2.428 och biotinylerade äggalbumin (bOVA) som exogena antigen. Endocytic facken var renas genom streptividin (SA)-magnetiska pärlor med den membran-penetrerande bOVA som en maker. I denna renat levermikrosom, lite extra exogent bOVA var fortfarande finns bevarade i membran fraktioner men var transporteras till utsidan av levermikrosom, och sedan ubiquitineras och bearbetas i vitro29. Denna renade levermikrosom innehöll inte bara endocytic fack-specifika proteiner, utan också ER boförälder proteiner för ERAD och peptiden lastning komplex; tyder på att mobilfack är det blivande endocytic facket för CP29. Detta protokoll är inte beroende av vilken typ av exogena antigen, och är även tillämplig för andra DC underdelar och andra celltyper, såsom makrofager, B-celler och endotelceller, att klarlägga den exakta molekylära mekanismen av DCs för skickliga CP.

<table border="0" cellpadding="0" cellspacing="0" width="871">
	<tbody>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">control magnetic beads</td>
			<td style="width:239px;">Chemagen</td>
			<td style="width:119px;">M-PVA012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">magnetic stand</td>
			<td style="width:239px;">BD Biosciences</td>
			<td style="width:119px;">552311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BCA protein assay kit</td>
			<td style="width:239px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">silver staining kits</td>
			<td style="width:239px;">Cosmo bio</td>
			<td style="width:119px;">423413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Reticulocyte Lysate</td>
			<td style="width:239px;">Promega</td>
			<td style="width:119px;">1730714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Flag-tagged ubiquitin&#160;</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">U5382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-ovalbumin (OVA,mouse)</td>
			<td style="width:239px;">Antibody Shop</td>
			<td style="width:119px;">HYB 094-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">ant-multi-ubiquitin (mouse)</td>
			<td style="width:239px;">MBL</td>
			<td style="width:119px;">D058&#8722;3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-Flag (mouse)</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">trypsin</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">85450C</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation

Dendritiska celler (DCs) är mycket kapabel för bearbetning och presentation av internaliserade exogena antigen vid större histocompatibility klass (MHC) jag molekyler kallas även cross-presentation (CP). CP spelar en viktig roll inte bara i stimulering av naiva CD8+ T celler och minne CD8+ T-celler för smittsamma och tumör immunitet men också i inaktivering av självverkande naiva T-celler T cell anergi eller T cell radering. Även om den kritiska molekylära mekanismen av CP återstår belysas, indikerar ackumulerande bevis att exogena antigen bearbetas via endoplasmatiska nätverket-associerade nedbrytning (ERAD) efter export från icke-klassisk endocytic fack. Tills nyligen, var karakteriseringar av dessa endocytic avdelningar begränsade eftersom det fanns inga specifika molekylära markörer än exogena antigen. Den metod som beskrivs här är en ny vesikler isolering protokoll, vilket tillåter för rening av dessa endocytic fack. Använder denna renat levermikrosom, beredas vi den ERAD-liknande transporter, ubikvitinering och bearbetning av exogena antigen i vitrotyder på att den ubiquitin-proteasomsystemet bearbetas den exogena antigenen efter export från denna mobilfack. Detta protokoll kan tillämpas vidare till andra celltyper att klargöra den molekylära mekanismen av CP.

För att belysa den molekylära mekanismen av CP, är det nödvändigt att identifiera de cellulära fack, där exogena antigen genomgår ERAD-liknande transporter och bearbetning. Samtidigt observationer av Immunofluorescerande mikroskopi eller elektronmikroskopi identifieras de mobilfack där exogena antigen ackumulerade16,17,18,19...

Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Den metod som beskrivs här är en ny vesikler isolering protokoll, vilket tillåter för rening av de cellulära fack där exogena antigen bearbetas av endoplasmatiska nätverket-associerade nedbrytning i cross-presentation.

Rening av membran facket för endoplasmatiska nätverket-associerade nedbrytningen av exogena antigen i Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Research

JoVE Journal

Immunology and Infection

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Masspektrometrisk analys av glykosfingolipid antigener

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Antigener skyddade funktionella röda blodkroppar av membranet Ympning av kompakt Hyperförgrenade Polyglyceroler

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Övervaknings Förändringar i Membrane polaritet, Membrane Integritet och intracellulära jonkoncentrationer i<em&gt; Streptococcus pneumoniae</em&gt; Med hjälp av fluorescerande färger

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA Rening från Intracellulärt Grown<em&gt; Listeria monocytogenes</em&gt; I makrofagceller

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Med användning av röntgenkristallografi, Biophysics, och Funktionella analyser för att bestämma de mekanismer som styr T-cellsreceptorigenkänning of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

Rening av virus-DNA för identifiering av associerade Viral och cellulära proteiner

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Uttryck av exogena antigen i Mycobacterium bovis BCG vaccinet via icke-genetiska yta dekoration med metoden-biotin systemet

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overuttryck och rening av människa<em&gt; Cis</em&gt; -prenyltransferas i<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

Dissekera multi signalering proteinkomplex av Bimolecular komplettering affinitet rening (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Assembly and Purification of Prototype Foamy Virus Intasomes

Montering och rening av prototyp skummande Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...