Questo lavoro è supportato da Takasaki Università di salute e del benessere.

1. crescere cellule e aggiunta di antigeni esogeni <ol><li>preparare bOVA utilizzando un contrassegno di biotina-della proteina kit seguendo il produttore &#39; protocollo s. 
	Nota: Normalmente, bOVA contiene biotina di 2 M per 1 M OVA mediamente.</li>
	<li>DC2.4 crescere le cellule in RPMI-1640 completate con 2 mM L-Glutammina, piruvato di sodio di 1 mM, 0,1 mM aminoacidi non essenziali, 100 U/mL penicillina-streptomicina, 55 mM 2-mercaptoetanolo, 10 mM HEPES (pH 7.5) e 10% siero fetale del vitello (d'ora in avanti RPMI) a 37 ° C in 5 % CO 2 in un incubatore (d'ora in avanti senza menzione, le cellule vengono incubate a questa condizione). È anche possibile utilizzare DMEM completati con 10% siero fetale di vitello, 3,7 g/L NaHCO 3 (d'ora in avanti DMEM) al posto di RPMI.</li>
	<li>Un giorno prima di purificazione, dividere le celle in RPMI a 1 x 10 5 cellule/mL in una piastra di coltura del tessuto. Evitare di mantenere le cellule in uno stato confluente. DC2.4 cellule possono altamente incorporare antigeni esogeni fino a uno stato semi-confluente, ma perdono rapidamente la capacità dopo aver raggiunto uno stato di sovra-confluenti.</li>
	<li>Prima the DC2.4 cellule sono semi-confluenti, aggiungere 250 µ g/mL di bOVA per 1 x 10 6 cellule/mL e incubare per 2-4 h. 
	Nota: Durante gli esperimenti a valle, tutti i buffer e i reagenti sono conservati a 4 ° C se non diversamente indicato.</li>
</ol> 2. Preparazione dei microsomi <ol><li>raccolto le cellule DC2.4 di pipettaggio delicato dalla piastra di tessuto in una nuova provetta conica 50 mL.</li>
	<li>Centrifugare a 1.000 x g per 5 min, 4 ° C e sfilare con cautela il mezzo con bOVA aspirazione.</li>
	<li>Lavare le cellule DC2.4 due volte con 40 mL di tampone PBS (1,37 mM NaCl, 8,1 mM Na 2 HPO 4, 2,68 mM KCl, 1,47 mM KH 2 PO 4) mediante centrifugazione a 1.000 x g per 5 min, 4 ° C e scartare il supernatante ogni volta dall'aspirazione.</li>
	<li>Risospendere il pellet in passaggio 2.3 in 1-2 mL (1/20-1/10 di volume di terreno di coltura) del mezzo di omogeneizzazione (0,25 M saccarosio, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7.4]) con volume di 1/1.000 di cocktail di inibitore di proteasi e trasferimento del sedimento DC2.4 di celle in un ghiacciata lisata omogeneizzatore.</li>
	<li>Frantumare le cellule DC2.4 sedimento delicatamente da 10-20 colpi con l'omogeneizzatore lisata ghiacciata. 
	Nota: Durante la fase di rottura, l'omogeneizzatore lisata è raffreddato su Ice.</li>
	<li>Trasferire la sospensione cellulare-interrotto ad un nuovo tubo conico da 15 mL.</li>
	<li>Aggiungere 8-9 mL di terreno di omogeneizzazione a totale di 10 mL e centrifugare la provetta conica per 10 min a 2.000 x g e 4 ° C.</li>
	<li>Trasferire il surnatante in una nuova provetta conica 15 mL per rimuovere cellule ininterrotte e nuclei e centrifugare la provetta conica ancora per 10 min a 2.000 x g e 4 ° C.</li>
	<li>Trasferire il surnatante in un nuovo ultracentrifugazione provetta e centrifugare per 45 min a 100.000 x g e 4 ° C.</li>
	<li>Aspirare il supernatante e risospendere il sedimento con attenzione in 1-2 mL di terreno di omogeneizzazione con volume di 1/1.000 di cocktail di inibitore di proteasi pipettando la soluzione su e giù più volte per rendere una frazione del microsoma per esperimenti a valle.</li>
	<li>Trasferire la frazione del microsoma in una provetta da 5 mL fondo tondo nuovo. 
	Nota: Dopo questo passaggio, è possibile arricchire i microsomi oggettive di centrifugazione in gradiente di densità iodixanol secondo il produttore &#39; protocollo s, rimuovere aspecifici microsomi. Le frazioni di picco per antigeni esogeni sono raccolti e poi sottoposto al passaggio successivo di purificazione (passaggio 3).</li>
</ol> 3. Purificazione dei microsomi con bOVA ERAD che subiscono <ol><li>volume aggiuntivo 1/100 di fresco SA-magnetico perline alla frazione del microsoma di passo 2.11 nei 5 mL fondo tubo tondo. 
	Nota: Prima di questo passaggio, è possibile pre-cancellare la frazione del microsoma di controllo magnetico perline per ridurre contaminazioni dei microsomi aspecifici.</li>
	<li>Delicatamente mescolare bene e ruotare il tubo del fondo tondo lentamente per 30 min a 4 ° C.</li>
	<li>Aggiungere 2-3 mL di terreno di omogeneizzazione a 4 mL totale nella provetta fondo tondo e miscelare bene.</li>
	<li>Mettete il tubo fondo tondo su un supporto magnetico e incubare per 10 min a 4 ° C. Poiché le perline associate alle vescicole sono attratti dal magnete, microsfere marrone si accumulano gradualmente alla parete del tubo più vicina al magnete.</li>
	<li>Con il tubo attentamente restanti nello stand magnetico, scartare i surnatanti di aspirazione per rimuovere le vescicole non associate.</li>
	<li>Lavare i branelli magnetici vincolati alle vescicole due volte con 5 mL di medium di omogeneizzazione magnetico riposare per 10 min a 4 ° C e scartare il surnatante ogni volta accuratamente aspirando. 
	Nota: Senza il supporto magnetico, purificazione tramite le centrifugazioni a 2.000 x g per 10 min, 4 ° C è disponibile anche.</li>
	<li>Risospendere i branelli magnetici associati alle vescicole attentamente nel medium di omogeneizzazione 100 µ l pipettando la soluzione su e giù più volte.</li>
	<li>Trasferire le vescicole sedimento in una microprovetta nuova come il microsoma purificato per esperimenti a valle. 
	Nota: In genere, 50 µ l del microsoma frazione contenente 5-20 µ g proteine da 1 x 10 7 cellule può essere isolato.</li>
</ol> 4. Analisi dei microsomi del purificato perle <ol><li>risospendere il magnetico associati alle vescicole da passo 3.8 da 100-200 µ l di tampone TNE (pH a 20 mM Tris-HCl 7.4, 150 mM NaCl, 0,5 M EDTA, 1% Nonidet P-40) con volume di 1/1.000 di cocktail di inibitore della proteasi invece il mezzo di omogeneizzazione.</li>
	<li>Trasferire il lisato dal punto 4.1 una nuova microprovetta 1,5 mL.</li>
	<li>Determinare la concentrazione di proteina di fase 4.2 utilizzando un kit di test BCA per il produttore &#39; protocollo s. 
	Nota: In genere, 5-10 µ l lisato dalla fase 4.2 è sufficiente per determinare la concentrazione nella proteina.</li>
	<li>Trasferire il lisato dalla fase 4.2 (2 µ g di proteina per la macchiatura d'argento e 10 µ g di proteina per macchiarsi occidentale) in nuove microprovette.</li>
	<li>Mettere le microprovette su un blocco di calore alle proteine 95 ° C e far bollire in 1x tampone di gel-caricamento SDS (100 mM Tris-HCl a pH 6.8, bromofenolo 200 mM dithiothreitol, 4% SDS, 0,2%, 20% glicerolo) per 5 min.</li>
	<li>Centrifugare le microprovette per 10 min a 21.500 x g e 4 ° C. raccogliere i surnatanti in nuove microprovette per rimuovere le frazioni insolubili.</li>
	<li>Analizzare le proteine risolte nel passaggio 4.6 (2 µ g) di SD-pagina standard.</li>
	<li>Visualizzare le bande proteiche dall'argento che macchia usando la macchiatura d'argento kit in seguito il produttore &#39; protocollo s.</li>
	<li>Analizzare le proteine risolte nel passaggio 4.6 (10 µ g) di SD-pagina standard e il Western blotting. Utilizzare il reagente (SA-HRP) per il produttore &#39; s protocollo e visualizzare da fluorography.</li>
</ol> 5. In Vitro Ricostituzione di ERAD ubiquitinazione di bOVA usando purificato microsomi <ol><li>trasferimento purificato microsomi (5-10 µ g di proteine) dal passaggio 3.8 con un 50% del volume del reticulocyte lysato (RL), 1 x tampone di reazione (50 mM Tris-HCl pH 7.4, 3 mM ATP, 0,5 mM MgCl 2) e 12:02 ubiquitina etichetta a bandiera in una nuova microprovetta. Il volume finale di questo esperimento è 20-40 µ l.</li>
	<li>Incubare la provetta per 2 ore a 37 ° C. 
	Nota: Se la quantità di bOVA ubiquitinata nel passaggio 5.12 è troppo piccola per rilevare mediante Western blotting, aggiungere 10 µM di MG132 o 2 µM di lactacistina per inibire l'elaborazione di bOVA l'ubiquitinate da proteosomi.</li>
	<li>Fermare la reazione inserendo nella microprovetta su ghiaccio.</li>
	<li>Risolvere i microsomi aggiungendo 500 µ l di tampone TNE con volume di 1/1.000 di cocktail di inibitore di proteasi.</li>
	<li>Centrifugare la provetta per 10 min a 21,500 x g e a 4 ° C. raccogliere i surnatanti in una nuova microprovetta per rimuovere le frazioni insolubili, che contengono ubiquitinate bOVA in DC2.4 prima del dosaggio di ubiquitinazione in vitro.</li>
	<li>Trasferire il surnatante ad una nuova microprovetta e aggiungere volume 1/100 di nuove perline SA-magnetico (solitamente 5 µ l per 500 µ l di surnatanti).</li>
	<li>Delicatamente mescolare bene e ruotarlo nella microprovetta lentamente per 30 min a 4 ° C.</li>
	<li>Centrifugare la provetta per 10 min a 21.500 x g e 4 ° C. scartare il surnatante dall'aspirazione a recuperare la bOVA e bOVA ubiquitinate associato con i branelli magnetici SA.</li>
	<li>Lavare due volte i branelli magnetici SA raccolti con 1 mL di tampone TNE mediante centrifugazione per 10 min a 21.500 x g e 4 ° C. scartare il supernatante ogni volta dall'aspirazione.</li>
	<li>Bollire i branelli magnetici SA in 1x tampone per 5 min a 95 ° C su un calore-blocco per risolvere le proteine purificate mediante i branelli magnetici SA di caricamento del gel di SDS.</li>
	<li>Centrifugare la provetta per 10 min a 21.500 x g e 4 ° C. raccogliere i surnatanti in una nuova microprovetta per rimuovere le frazioni insolubili.</li>
	<li>Analizzare i branelli magnetici SA vincolati alle proteine standard SD-PAGE e Western blotting. L'uso di anticorpi (Anti-Flag, anti-multi-Ub e anti-mouse IgG-HRP) e il reagente (SA-HRP) è per il produttore &#39; protocollo s e visualizzate da fluorography.</li>
</ol>

<ol>
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L., Kyritsis, C., Tamp&#233;, R., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+phagosomes+in+dendritic+cells+form+a+cellular+compartment+sufficient+for+cross+presentation+of+exogenous+antigens.">Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens.</a> Proc Natl Acad Sci U S A. 100 (22), 12889-12894 (2003).</li><li>Saveanu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRAP+identifies+an+endosomal+compartment+required+for+MHC+class+I+cross-presentation.">IRAP identifies an endosomal compartment required for MHC class I cross-presentation.</a> Science. 325 (5937), 213-217 (2009).</li><li>Zehner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translocon+protein+Sec61+mediates+antigen+transport+from+endosomes+in+the+cytosol+for+cross-presentation+to+CD8(+)+T+cells.">The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.</a> Immunity. 42 (5), 850-863 (2015).</li></ol>

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Negli studi precedenti di CP, gli antigeni esogeni incorporati accumulato nell'area riservata del tardo endosoma o ER da microscopia immunofluorescente16,30,31,32. Si stima che ERAD-come trasporto e la lavorazione di antigeni esogeni sono effettuate in queste aree specialistiche dell'ER o endosoma tardivo, come il compartimento cellulare è stato identificato da saccarosio o centrifugazione su ...

Il MHC sono molecole sono espresse sulla superficie di tutte le cellule nucleate, con brevi peptidi antigenici derivati da antigeni endogeni, che vengono elaborati dal sistema ubiquitina-proteasoma nel cytosol1. Dopo l'elaborazione, peptidi antigenici sono trasportati nel lume del reticolo endoplasmatico (ER) del trasportatore di peptidi TAP. Nel lume dell'ER, una serie di specifiche chaperoni assistere il caricamento del peptide e il corretto ripiegamento del MHC I complessi. Questa serie di molecole è chiamato il complesso peptide-caricamento (PLC), che indica che l'ER è un vano centrale per peptide caricamento su MHC I2. Dopo peptide di caricamento, il MHC sono molecole vengono trasportati alla superficie delle cellule e svolgono un ruolo chiave nel sistema immunitario adattativo come auto-marcatori e consente il CD8+ linfociti T citotossici (CTL) per rilevare le cellule tumorali o agenti infettivi di antigenica peptidi da proteine non-sé3.
In cellule (APC) presentanti l'antigene, peptidi antigenici da antigeni esogeni sono anche presentata al MHC I4,5,6,7,8 tramite CP, che è trasportato principalmente fuori da DCs9,10,11. CP è essenziale sia per l'attivazione di ingenuo CD8+ T cellule e memoria CD8+ T cells in anti-infettivi e anti-tumorali CTLs12,13e nel mantenimento della tolleranza immunitaria inattivando di self-acting cellule T naive14,15. Il CP svolge molti ruoli importanti nel sistema immunitario adattativo, tuttavia i meccanismi molecolari della CP devono ancora essere descritti dettagliatamente. Gli studi precedenti di CP ha rivelato che antigeni esogeni sono stati localizzati in ER e l'endosoma e sono stati elaborati dal ERAD, suggerendo che antigeni esogeni sono trasportati dall'endosoma al pronto soccorso per ERAD-come elaborazione e peptide caricamento16 . Tuttavia, raccogliendo la prova indica che il caricamento del peptide di CP è effettuato non in ER, ma piuttosto in compartimenti endocitosi non classici, che hanno anche caratteristiche distintive del ER (Figura 1)17,18 ,19,20,21. Per evitare la degradazione dei precursori del peptide antigenico dall'alta attività di aminopeptidasi22 nel citosol, elaborazione e caricamento in CP peptide si verifica nella zona prossimale di questi comparti endocitosi non classici (Figura 1). Anche se le caratterizzazioni di questi comparti endocitosi sono controverse, non ci sono nessun esistenti specifiche molecole diverse da antigeni esogeni in questo comparto.
ERAD è una via cellulare, che in particolare rimuove le proteine misfolded dall'ER. Nella via ERAD, proteine misfolded vengono retrogradely trasportate attraverso la membrana ER al citoplasma ed elaborate dal sistema ubiquitina-proteasoma23,24,25. Quando le molecole di grandi dimensioni, quali le proteine, vengono trasportate attraverso il doppio strato lipidico, queste molecole passano attraverso un apparato molecolare chiamato complesso in un Traslocone, come il complesso di Sec61 e Derlin complesso l'ER26, e il complesso Tom e Tim il i mitocondri27. Quando esogenicamente aggiunto gli antigeni vengono trasportati attraverso la membrana dell'ER, essi devono penetrare bilayer del lipido in complesso con translocons, come il complesso di Sec61. Il metodo qui descritto purificato la vescicola mirata utilizzando queste molecole di membrana-penetrante come marcatori per i compartimenti endocitosi.
Il metodo qui descritto è un nuovo protocollo di purificazione di vescicola utilizzando il DC-come cella linea DC2.428 e biotinilati ovoalbumina (bOVA) come un antigene esogeno. I compartimenti endocitosi sono stati purificati da streptavidina (SA)-biglie magnetiche utilizzando il bOVA membrana-penetrante come un creatore. In questo microsoma purificato, alcuni esogenicamente aggiunto bOVA era ancora conservata nelle frazioni della membrana ma sono stato trasportato all'esterno del microsoma e poi ubiquitinate e trattato in vitro29. Questo microsoma purificata contenuta non solo proteine endocitiche vano, ma anche proteine ER-residente per ERAD e il peptide caricamento complesso; suggerendo che il compartimento cellulare è il vano endocitico prospettico per CP29. Questo protocollo non è dipenda dal tipo di antigeni esogeni e vale anche per altri sottoinsiemi di DC e altri tipi di cellule, quali i macrofagi, linfociti B e cellule endoteliali, per chiarire il meccanismo molecolare preciso di DCs per CP abile.

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			<td></td>
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			<td style="width:239px;">gibco by life technologies</td>
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			<td></td>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">HEPES</td>
			<td style="width:239px;">gibco by life technologies</td>
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			<td></td>
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		<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>
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			<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>
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		<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>
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			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:239px;">gibco by life technologies</td>
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			<td height="21" style="height:21px;">OVA</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">A5503</td>
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		<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>
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			<td height="21" style="height:21px;width:272px;">lactacystin&#160;</td>
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		<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>
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			<td height="21" style="height:21px;width:272px;">protease inhibitor cocktails&#160;</td>
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			<td style="width:119px;">P8340</td>
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			<td style="width:119px;">201270</td>
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			<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>
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		<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>
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		<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>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:272px;">ant-multi-ubiquitin (mouse)</td>
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			<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>
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		<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>
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purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation

Le cellule dendritiche (DCs) sono altamente capace di elaborare e presentare antigeni esogeni interiorizzati su classe di istocompatibilità (MHC) sono molecole conosciuto anche come cross-presentazione (CP). CP svolge un ruolo importante non solo nella stimolazione dell'ingenuo CD8+ T cellule e memoria CD8+ T cellule per infettive e immunità del tumore ma anche nell'inattivazione di self-acting le cellule T naive di anergia delle cellule T o l'eliminazione delle cellule T. Sebbene il meccanismo molecolare critico del CP rimane essere delucidato, raccogliendo la prova indica che antigeni esogeni vengono elaborati tramite associati al reticolo endoplasmatico degradazione (ERAD) dopo l'esportazione da Tronchetti endocitosi scomparti. Fino a poco tempo, caratterizzazioni di questi comparti endocitosi sono stati limitati perché non c'erano specifici marcatori molecolari diversi da antigeni esogeni. Il metodo qui descritto è un nuovo protocollo di isolamento della vescicola, che permette per la purificazione di questi comparti endocitosi. Utilizzando questo microsoma purificato, abbiamo ricostituito il ERAD-come trasporto, ubiquitinazione e l'elaborazione del antigene esogeno in vitro, suggerendo che il sistema ubiquitina-proteasoma elaborato l'antigene esogeno dopo l'esportazione da questo compartimento cellulare. Questo protocollo possa essere applicato ad altri tipi di cella per chiarire il meccanismo molecolare di CP.

Per delucidare il meccanismo molecolare di CP, è necessario identificare i compartimenti cellulari, dove antigeni esogeni subiscono ERAD-come trasporto e la lavorazione. Mentre le osservazioni da microscopia di immunofluorescenza o da microscopia elettronica identificato il compartimento cellulare dove antigeni esogeni accumulato16,17,18,19</s...

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

Il metodo qui descritto è un nuovo protocollo di isolamento della vescicola, che permette per la purificazione dei compartimenti cellulari dove antigeni esogeni vengono elaborati dal reticolo endoplasmatico-collegata di degradazione in cross-presentazione.

Purificazione del compartimento di membrana per la degradazione del reticolo endoplasmatico-collegata di antigeni esogeni in Cross-presentazione

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

purification-membrane-compartment-for-endoplasmic-reticulum

Research

JoVE Journal

Immunology and Infection

8.2K Views.  Takasaki University of Health and Welfare. Il metodo qui descritto è un nuovo protocollo di isolamento della vescicola, che permette per la purificazione dei compartimenti cellulari dove antigeni esogeni vengono elaborati dal reticolo endoplasmatico-collegata di degradazione in cross-presentazione.

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

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Spettrometria di massa, di antigeni glicosfingolipide

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

Ricerca

Immunologia e infezioni

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched 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 anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Gli antigeni Protetto funzionali globuli rossi dalla membrana innesto di Compact Poligliceroli Hyperbranched

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

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

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

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Monitoraggio dei cambiamenti di polarità membrana, membrana integrità e intracellulare di ioni concentrazioni in<em&gt; Streptococcus pneumoniae</em&gt; Utilizzo di tinture fluorescenti

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

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA Purificazione da nella cellula Grown<em&gt; Listeria monocytogenes</em&gt; In macrofagi

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

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 surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

Utilizzando cristallografia a raggi X, Biofisica, e saggi funzionali per determinare i meccanismi di amministrazione delle cellule T del recettore Riconoscimento di cancro antigeni

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

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 cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Purificazione di DNA virale per l'identificazione delle proteine virali e cellulari associate

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

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 protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

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

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Espressione di antigeni esogeni nel Mycobacterium bovis BCG vaccino via genetica decorazione superficiale con il sistema avidina-biotina

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

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Sovrapressione e purificazione dell&#39;uomo<em&gt; Cis</em&gt; -preniltransferasi in<emEscherichia 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)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

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

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Dissezione multiproteici segnalazione di purificazione di affinità di complementazione bimolecolare (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

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 integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

Assemblaggio e purificazione del prototipo Virus schiumoso 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 ...

Purificazione del compartimento di membrana per la degradazione del reticolo endoplasmatico-collegata di antigeni esogeni in Cross-presentazione

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