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

Dette arbeidet er støttet av Takasaki universitetet av helse og velferd.

1. voksende celler og tillegg av eksogene antigener <ol><li>forberede bOVA bruker en biotin protein merking kit etter produsenten &#39; s protokollen. 
	Merk: Vanligvis bOVA inneholder 2 M biotin per 1 M OVA gjennomsnittlig.</li>
	<li>Vokser DC2.4 celler i RPMI-1640 supplert med 2 mM L-glutamin, 1 mM natrium pyruvate, 0,1 mM unødvendige aminosyrer, 100 U/mL penicillin-streptomycin, 55 mM 2-mercaptoethanol, 10 mM HEPES (pH 7.5) og 10% fosterets kalv serum (senere RPMI) på 37 ° C i 5 % CO 2 i en fukte...

<ol>
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I tidligere studier av CP akkumulert innarbeidet eksogene antigener i begrensede området av sen endosome eller ER ved immunofluorescent mikroskopi16,30,31,32. Det er anslått at ERAD-lignende transport og behandling av eksogene antigener er gjennomført i disse spesialiserte områder ER eller sene endosome, som cellular rommet ble identifisert av sukrose eller iodixanol tetthet gradert sentrif...

MHC jeg molekyler er uttrykt på overflaten av alle kjerne celler, med kort antigen peptider fra endogene antigener, som behandles av ubiquitin-proteasome systemet i stoffer1. Etter prosessering transporteres antigen peptider i det endoplasmatiske retikulum (ER)-lumen med den peptid transporter trykk. I ER lumen, en rekke spesifikke chaperones hjelper peptid lasting og riktig folding av MHC jeg komplekse. Denne serien av molekyler kalles peptid-lasting komplekset (PLC), indikerer at ER en sentral avlukke for peptid lasting på MHC jeg2. Etter peptid lasting, MHC jeg molekyler transporteres til celleoverflaten og spiller en viktig rolle i det adaptive immunsystemet som selv markører og aktiverer CD8+ cytotoksiske T-lymfocytter (CTLer) å oppdage kreftceller eller smittsomme stoffer av antigen peptider fra ikke-selv proteiner3.
I antigen presentere celler (APC), antigen peptider fra eksogene antigener er også presentert på MHC jeg4,5,6,7,8 via CP, som gjennomføres hovedsakelig ut av DCs9,10,11. CP er viktig både for aktivering av naiv CD8+ T celler og minne CD8+ T celler til anti-smittsomme og anti-tumoral CTLer12,13, og i vedlikehold av immun toleranse av inaktivere av selv fungerende naiv T celler14,15. CP spiller mange viktige roller i det adaptive immunsystemet, men de molekylære mekanismene av CP har ennå å bli beskrevet i detalj. Tidligere studier av CP avslørte at eksogene antigener ble lokalisert både ER og endosome og ble behandlet av ERAD, antyder at eksogene antigener transporteres fra endosome til ER for ERAD-lignende behandling og peptid lasting16 . Imidlertid indikerer samler bevis at peptid lasting av CP utføres ikke i ER men i ikke-klassiske endocytic avdelinger, som også har karakteristiske trekk ved de ER (figur 1)17,18 ,19,20,21. For å unngå nedbrytning av antigen peptid forløpere av den høye aktiviteten til aminopeptidase oppstår22 i stoffer, behandling og peptid lasting i CP i det proksimale området av ikke-klassiske endocytic seksjonene (figur 1). Karakteristikkene av endocytic seksjonene er kontroversielle, finnes det ingen eksisterende bestemt molekyler enn eksogene antigener i dette rommet.
ERAD er en mobilnettet sti, som spesielt fjerner misfolded proteiner fra ER. I ERAD veien, misfolded proteiner retrogradely transportert gjennom ER membran til cytoplasma og behandles av ubiquitin-proteasome systemet23,24,25. Når store molekyler, som proteiner, transporteres gjennom lipid bilayer, disse molekylene passere en molekylær apparater kalt en translocon, som Sec61 kompleks og Derlin i ER26, og Tom komplekse og Tim kompleks i den mitokondrier27. Når exogenously-lagt antigener transporteres gjennom ER membran, må de trenge lipid bilayer i kompleks med translocons, som Sec61 komplekset. Metoden beskrevet her renset målrettet vesicle ved å benytte disse membran-gjennomtrengende molekyler som markører for endocytic seksjonene.
Metoden beskrevet her er en ny vesicle rensing protokollen bruker i DC-like cellen linje DC2.428 og biotinylated ovalbumin (bOVA) som et eksogene antigen. Endocytic seksjonene ble renset ved streptavidin (SA)-magnetiske perler med membran-gjennomtrengende bOVA som en maker. I denne renset microsome, noen exogenously lagt bOVA ble bevart i membranen fraksjoner, men ble transportert til utsiden av microsome, og deretter ubiquitinated og behandlet i vitro29. Denne renset microsome inneholdt ikke bare endocytic rom-spesifikke proteiner, men også ER-resident proteiner ERAD og peptid lasting sammensatt; antyder at mobilnettet rommet er potensielle endocytic batterirommet CP29. Denne protokollen er ikke avhengig av hva slags eksogene antigener, og gjelder også for andre DC delsett og andre celletyper, som makrofager, B-celler og endotelceller, å avklare presise molekylære mekanismen av DCs for dyktige 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

Dendrittiske celler (DCs) er svært kan presentere internalisert eksogene antigener ved store histocompatibility klasse (MHC) jeg molekyler også kjent som cross-presentasjon (CP). CP spiller en viktig rolle ikke bare i stimulering av naiv CD8+ T celler og minne CD8+ T celler smittsomme og svulst immunitet men også i inaktivering av selvstendig fungerende naiv T celler av T celle anergy eller T celle sletting. Selv om kritiske molekylær mekanisme CP gjenstår å bli belyst, indikerer samler bevis at eksogene antigener behandles gjennom endoplasmatiske retikulum-assosiert degradering (ERAD) etter eksport fra ikke-klassiske endocytic rom. Inntil nylig var characterizations av endocytic seksjonene begrenset fordi det var ingen spesifikke molekylære markører enn eksogene antigener. Metoden beskrevet her er en ny vesicle isolasjon protokoll, som gjør det mulig for rensing av endocytic seksjonene. Bruker denne renset microsome, rekonstituert vi ERAD som transport, ubiquitination, og behandlingen av ytre antigen i vitro, antyder at ubiquitin-proteasome systemet behandlet eksogene antigen etter eksport fra dette mobilnettet rommet. Denne protokollen kan brukes videre til andre celletyper å avklare molekylær mekanisme CP.

For å belyse molekylær mekanismen av CP, er det nødvendig å identifisere mobilnettet seksjonene, der eksogene antigener gjennomgå ERAD-lignende transport og behandling. Mens observasjoner av immunofluorescent mikroskopi eller elektronmikroskop identifiserte mobilnettet rommet hvor eksogene antigener akkumulert16,17,18,19, ...

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 vesicle isolasjon protokoll, som gjør det mulig for rensing av mobilnettet seksjonene der eksogene antigener behandles av endoplasmatiske retikulum-assosiert degradering av kryss-presentasjon.

Rensing av membran kupé for endoplasmatiske retikulum-assosiert nedbrytning av eksogene antigener i Cross-presentasjon

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 av glykosphingolipid 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 Funksjonell røde blodceller av membranen Pode av kompakt hyperforgrenede Polyglycerols

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

Overvåking Endringer i Membrane polaritet, Membran Integrity, og intracellulær ionekonsentrasjoner i<em&gt; Streptococcus pneumoniae</em&gt; Ved hjelp av fluorescerende fargestoffer

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 Purification 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

Ved hjelp av røntgenkrystallografi, biofysikk og funksjonelle analyser fastslår mekanismer som styrer T-celle reseptor Anerkjennelse av kreft 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

Rensing av Viral DNA for identifikasjon av tilknyttet 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

Uttrykk for eksogene antigener i Mycobacterium bovis BCG vaksinen via ikke-genetiske overflaten dekorasjon med Avidin-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

Overekspresjon og rensing av menneske<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 signalnettverk komplekser av resultatet av dette Complementation affinitet rensing (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 og rensing av 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 ...