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

Dette arbejde støttes af Takasaki Universitet for sundhed og velfærd.

1. vokser cellerne og tilsætning af eksogene antigener <ol><li>Forbered bOVA ved hjælp af et biotin-protein mærkning kit efter fabrikanten &#39; s protokol. 
	Bemærk: Normalt bOVA indeholder 2 M biotin pr. 1 M æg gennemsnitligt.</li>
	<li>Grow DC2.4 celler i RPMI-1640 suppleret med 2 mM L-glutamin, 1 mM natrium pyruvat, 0.1 mM uvÃ¦sentlige aminosyrer, 100 U/mL penicillin-streptomycin, 55 mM 2-mercaptoethanol, 10 mM HEPES (pH 7,5) og 10% føtalt kalveserum (herefter RPMI) ved 37 ° C i 5 % CO 2 i...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloned+dendritic+cells+can+present+exogenous+antigens+on+both+MHC+class+I+and+class+II+molecules.">Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules.</a> J Immunol. 158 (6), 2723-2730 (1997).</li><li>Imai, J., Otani, M., Sakai, T., Hatta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+subcellular+compartment+in+which+exogenous+antigens+undergo+endoplasmic+reticulum-associated+degradation+from+dendritic+cells.">Purification of the subcellular compartment in which exogenous antigens undergo endoplasmic reticulum-associated degradation from dendritic cells.</a> Heliyon. , (2016).</li><li>Kovacsovics-Bankowski, M., Rock, K. 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I tidligere undersøgelser af CP akkumuleret indarbejdet eksogene antigener begrænset inden for de sene endosome eller ER af immunfluorescent mikroskopi16,30,31,32. Det anslås, at ERAD-lignende transport og forarbejdning af eksogene antigener foretages i disse specialiserede områder af ER eller sene endosome, da det cellulære rum blev identificeret af saccharose eller iodixanol tæthed grad...

MHC jeg molekyler udtrykkes på overfladen af alle nucleated celler, med korte antigene peptider afledt af endogene antigener, som behandles af ubiquitin-proteasom system i cytosol1. Efter forarbejdning transporteres antigene peptider til det endoplasmatiske reticulum (ER) lumen af peptid transportør TAP. I lumen ER en række specifikke chaperoner hjælpe peptid lastning og den korrekte foldning af MHC jeg komplekse. Denne serie af molekyler kaldes peptid-loading komplekset (PLC), der angiver at Skadestuen er en centrale rum for peptid indlæsning på MHC2. Efter peptid lastning, MHC jeg molekyler transporteres til cellens overflade og spille en central rolle i det adaptive immunsystem som selvstændig markører, og gør CD8+ cytotoksiske T-lymfocytter (CTL) hen til opdager kræftceller eller infektiøse agenser af antigene peptider fra ikke-selv proteiner3.
I antigen præsentere celler (APC), antigene peptider fra eksogene antigener er også præsenteret på MHC jeg4,5,6,7,8 via CP, som hovedsagelig transporteres ud af DCs9,10,11. CP er vigtigt både for aktivering af naive CD8+ T celler og hukommelse CD8+ T celler ind i anti-infektiøs og anti-tumorsygdomme CTLs12,13, og i vedligeholdelsen af immuntolerance ved uvirksomme af selvvirkende naive T celler14,15. CP spiller mange vigtige roller i den adaptive immunsystem, men de molekylære mekanismer af CP har endnu at blive beskrevet i detaljer. Tidligere undersøgelser af CP afslørede at eksogene antigener er lokaliseret både i Skadestuen og endosome og blev behandlet af ERAD, tyder på, at eksogene antigener transporteres fra endosome til Skadestuen for ERAD-lignende behandling og peptid lastning16 . Akkumulere beviser tyder imidlertid på, at peptid lastning af CP er udføres ikke på Skadestuen, men snarere i ikke-klassisk endocytic rum, som også har særlige kendetegn ER (figur 1)17,18 ,19,20,21. For at undgå nedbrydning af antigene peptid prækursorer af den høje aktivitet af aminopeptidase opstår22 i cytosol, forarbejdning og peptid lastning i CP i den proksimale område af disse ikke-klassisk endocytic rum (figur 1). Selvom beskrivelser af disse endocytic rum er kontroversielle, er der ingen eksisterende specifikke molekyler end eksogene antigener i dette rum.
ERAD er et cellulært pathway, som specifikt fjerner fejlfoldede proteiner fra Skadestuen. I ERAD pathway, fejlfoldede proteiner retrogradely transporteres gennem ER membran til cytoplasma og behandles af ubiquitin-proteasom system23,24,25. Når store molekyler som proteiner, der transporteres gennem lipid tolagede, disse molekyler passere gennem en molekylær apparat kaldet en translocon, Sec61 complex og Derlin komplekse ER26, og Tom komplekse og Tim komplekse i den mitokondrier27. Når eksogent tilføjet antigener transporteres gennem ER membran, skal de trænge lipid tolagede i kompleks med translocons, som Sec61 kompleks. Metoden beskrevet her renset den målrettede vesikel ved at udnytte disse membran-gennemtrængende molekyler som markører for de endocytic rum.
Metoden beskrevet her er en ny vesikel rensning protokol bruger DC-lignende celle line DC2.428 og biotinylated ægalbumin (bOVA) som en udefrakommende antigen. De endocytic rum blev renset af streptavidin (SA)-magnetiske perler med membran-gennemtrængende bOVA som en maker. I denne renset microsome, nogle eksogent tilføjet bOVA blev bevaret i membranen fraktioner men blev transporteret til ydersiden af microsome, og derefter ubiquitinated og forarbejdet in vitro-29. Denne renset microsome indeholdt ikke kun endocytic rum-specifikke proteiner, men også ER hjemmehørende proteiner til ERAD og peptid lastning kompleks; tyder på at det cellulære rum er den potentielle endocytic rum til CP29. Denne protokol er ikke afhængig af slags eksogene antigener, og gælder også for andre DC delmængder og andre celletyper såsom makrofager, B-celler og endotelceller, at klarlægge det præcise molekylære mekanisme af DCs for dygtige CP.

<table border="0" cellpadding="0" cellspacing="0" width="871">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">RPMI 1640</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">11875-093</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fetal bovine serum</td>
			<td style="width:239px;">Equitech bio</td>
			<td style="width:119px;">SFB30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sodium pyruvate</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">11360-070</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:272px;">MEM non-essential amino acids</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">HEPES</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">2-mercaptoethanol</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">L-glutamine</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">25030-164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Penisicillin-Sreptomycin</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">12100-46</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OVA</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Biotin-protein labelling kit</td>
			<td style="width:239px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F6347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MG-132&#160;</td>
			<td style="width:239px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">201270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">lactacystin&#160;</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">L6785</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Dounce homogenizer</td>
			<td style="width:239px;">IUCHI</td>
			<td style="width:119px;">131703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">protease inhibitor cocktails&#160;</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">iodixanol&#160;</td>
			<td style="width:239px;">Cosmo bio</td>
			<td style="width:119px;">1114542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">SA-magnetic beads&#160;</td>
			<td style="width:239px;">New England Biolabs</td>
			<td style="width:119px;">201270</td>
			<td></td>
		</tr>
		<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

Dendritiske celler (DCs) er meget i stand til at behandle og præsentere internaliseret eksogene antigener på store histocompatibility klasse (MHC) jeg molekyler også kendt som cross-præsentation (CP). CP spiller en vigtig rolle ikke kun i stimulation af naive CD8+ T celler og hukommelse CD8+ T celler smitsomme og tumor immunitet men også i inaktivering af selvvirkende naive T celler af T celle anergy eller T-celle sletning. Selvom den kritiske molekylære mekanisme af CP mangler at blive belyst, indikerer akkumulere beviser, at eksogene antigener behandles gennem endoplasmatiske reticulum-associerede nedbrydning (ERAD) efter eksport fra ikke-klassisk endocytic rum. Indtil for nylig, var beskrivelser af disse endocytic rum begrænsede, fordi der var ingen specifikke molekylære markører end eksogene antigener. Metoden beskrevet her er en ny vesikel isolation protokol, som giver mulighed for rensning af disse endocytic rum. Brug denne renset microsome, rekonstitueres vi ERAD-lignende transport, ubiquitination og forarbejdning af eksogene antigen i vitro, tyder på, at ordningen ubiquitin-proteasom behandlet eksogene antigenet efter eksport fra dette cellulære rum. Denne protokol kan yderligere anvendes til andre celletyper til at præcisere den molekylære mekanisme af CP.

For at belyse de molekylære mekanisme af CP, er det nødvendigt at identificere de cellulære rum, hvor udefrakommende antigener gennemgå ERAD-lignende transport og forarbejdning. Mens betragtninger af immunfluorescent mikroskopi eller ved elektronmikroskopi identificeret den cellulære rum hvor eksogene antigener akkumuleret16,17,18,19...

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

Metoden beskrevet her er en ny vesikel isolation protokol, som giver mulighed for rensning af de cellulære rum hvor eksogene antigener behandles af endoplasmatiske reticulum-associerede nedbrydning i cross-præsentation.

Rensning af membran til kabinen for endoplasmatiske Reticulum-associerede nedbrydning af eksogene antigener i Cross-præsentation

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

Massespektrometrisk analyse af glycosphingolipid 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 Beskyttet Funktionelle røde blodlegemer ved The Membrane podning af Compact hyperforgrenet 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

Overvåge ændringer i Membrane polaritet, Membrane Integritet, og intracellulære Ion Koncentrationerne i<em&gt; Streptococcus pneumoniae</em&gt; Brug fluorescerende farvestoffer

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 Oprensning fra Intracellulært 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

Brug røntgenkrystallografi, Biofysik, og funktionelle assays til Bestem Mekanismer Governing T-celle receptor Anerkendelse af kræft antigener

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

Rensning af Viral DNA til identifikation af tilknyttede Viral og cellulær 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

Udtryk af eksogene antigener i Mycobacterium bovis BCG Vaccine via ikke-genetiske overflade dekoration med begærlighed-biotin System

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

Overekspression og oprensning af mennesket<em&gt; Cis</em&gt; -prenyltransferase 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

Dissekere multi protein Signaling komplekser af Bimolecular komplementering affinitet rensning (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

Forsamling og rensning af Prototype skummende 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 ...