Diese Arbeit wird von der Takasaki Hochschule für Gesundheit und Soziales unterstützt.

1. wachsenden Zellen und Ergänzung der exogene Antigene <ol><li>Prepare bOVA mit einem Biotin-Protein-Kennzeichnung kit nach dem Hersteller &#39; s Protokoll. 
	Hinweis: Normalerweise bOVA enthält 2 M Biotin pro 1 M OVA durchschnittlich.</li>
	<li>Wachsen DC2.4 Zellen RPMI-1640 ergänzt mit 2 mM L-Glutamin, 1 mM Natrium Pyruvat, 0,1 mM nichtessentiellen Aminosäuren, 100 U/mL Penicillin-Streptomycin, 55 mM 2-Mercaptoethanol, 10 mM HEPES (pH 7,5) und 10 % fetalen Kälberserum (im folgenden RPMI) bei 37 ° C in 5 % CO 2 in einem befeuchteten Inkubator (im folgenden ohne Erwähnung, Zellen sind inkubiert in diesem Zustand). Es ist auch möglich, verwenden Sie DMEM mit 10 % fetalen Kälberserum, 3,7 g/L Nahco3 3 (im folgenden DMEM) anstelle von RPMI ergänzt.</li>
	<li>Einen Tag vor der Reinigung, die Zellen in RPMI bis 1 x 10 5 Zellen/mL in einer Gewebekultur Platte aufgeteilt. Vermeiden Sie die Zellen an einem konfluierende Zustand zu halten. DC2.4 Zellen können exogene Antigene hoch bis semi-konfluierende Zustand zu übernehmen, aber schnell verlieren die Fähigkeit nach einer übermäßigen Zusammenfluss Zustand zu erreichen.</li>
	<li>Vor der DC2.4 Zellen sind semi-konfluierende, fügen Sie 250 µg/mL bOVA für 1 x 10 6 Zellen/mL und inkubieren Sie für 2-4 h 
	Hinweis: Während der nachfolgenden Experimente alle Puffer und Reagenzien sind gehalten bei 4 ° C sofern nicht anders angegeben.</li>
</ol> 2. Vorbereitung der Microsomen <ol><li>die DC2.4 Zellen durch sanfte Pipettieren von der Gewebe-Platte in ein neues 50 mL konische Röhrchen ernten.</li>
	<li>Zentrifuge bei 1.000 x g für 5 min, 4 ° C und entfernen Sie vorsichtig das Medium mit bOVA durch Aspiration.</li>
	<li>Die DC2.4 Zellen zweimal mit 40 mL PBS-Puffer (1,37 mM NaCl, 8,1 mM Na 2 HPO 4, 2,68 mM KCl, 1,47 mM KH 2 PO 4) durch Zentrifugation bei 1.000 x g für 5 min, 4 ° C zu waschen und entsorgen des Überstands jedesmal durch Absaugen.</li>
	<li>Aufschwemmen das Pellet in Schritt 2.3 in 1-2 mL (1/20-1/10 Volumen Kulturmedium) Homogenisierung Medium (0,25 M Saccharose, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7.4]) mit 1/1.000 Volumen der Protease-Hemmer Cocktails und Übertragung der resuspendierte DC2.4 Zellen in einer eiskalte Dounce-Homogenisator.</li>
	<li>Stören die resuspendierte DC2.4 Zellen sanft von 10-20 Schläge mit dem eiskalten Dounce-Homogenisator. 
	Hinweis: Während der Unterbrechung Schritt Dounce-Homogenisator wird gekühlt auf Eis</li>
	<li>Übertragen die Zelle gestört Aussetzung zu einem neuen 15 mL konische Schlauch.</li>
	<li>10 mL insgesamt 8-9 mL Homogenisierung Medium hinzu und Zentrifugieren die konische Röhre für 10 min bei 2.000 x g und 4 ° c</li>
	<li>Übertragen den Überstand auf eine neue 15 mL konische Rohr, ungebrochene Zellen und Kerne entfernen und Zentrifugieren die konische Röhre wieder für 10 min bei 2.000 x g und 4 ° c</li>
	<li>Übertragen den Überstand in ein neues Ultrazentrifugation Rohr und Zentrifuge für 45 Minuten bei 100.000 x g und 4 ° c</li>
	<li>Aspirieren überstand und Aufschwemmen der Pellet sorgfältig in 1-2 mL Homogenisierung Medium mit 1/1.000 Volumen der Protease-Hemmer Cocktails durch pipettieren die Lösung nach oben und unten mehrmals in Sekundenbruchteilen Microsome für nachgeschaltete Experimente machen.</li>
	<li>Übertragen die Microsome Bruchteil in ein neues Röhrchen 5 mL Rundboden. 
	Hinweis: Nach diesem Schritt ist es möglich, Objektive Microsomen durch Iodixanol Dichte Gradienten Zentrifugation laut Hersteller bereichern &#39; s-Protokoll, um unspezifische Microsomen zu entfernen. Die Peak-Fraktionen für exogene Antigene werden gesammelt und dann mit dem nächsten Schritt der Reinigung (Schritt 3) unterworfen.</li>
</ol> 3. Reinigung von Microsomen mit bOVA unterziehen ERAD <ol><li>Add 1/100 Volumen von frischen SA-magnetischen Kügelchen auf den Microsome Bruchteil Schritt 2.11 in der 5 mL Runde Unterrohr. 
	Hinweis: Bevor Sie diesen Schritt, es ist möglich, Microsome Bruchteil von Kontrolle-magnetische Beads zu Verunreinigungen der unspezifischen Microsomen vorab klar.</li>
	<li>Sanft vermischen und drehen die Rundboden-Röhre langsam für 30 min bei 4 ° c</li>
	<li>Insgesamt 4 mL in die Rundboden Röhrchen ca. 2-3 mL Homogenisierung Medium hinzufügen und vorsichtig vermengen.</li>
	<li>Ein Magnetstativ Rundboden Rohr aufsetzen und 10 min bei 4 ° c inkubieren Da die Perlen an die Vesikel gebunden der Magnet angezogen sind, braune Mikro-Perlen werden allmählich reichern sich an der Rohrwand am nächsten an den Magneten.</li>
	<li>Mit dem Rohr in der Magnetstativ verbleibenden sorgfältig verwerfen die Überstände durch Absaugen, die ungebundenen Vesikel zu entfernen.</li>
	<li>Waschen die magnetische Beads die Vesikel zweimal mit 5 mL Medium Homogenisierung durch die magnetische verpflichtet stehen für 10 min bei 4 ° C und entsorgen des Überstands jedesmal durch sorgfältig absaugen. 
	Hinweis: Ohne Magnetstativ, Reinigung von Centrifugations bei 2.000 x g für 10 min, 4 ° C ist auch verfügbar.</li>
	<li>Aufschwemmen der magnetischen Kügelchen durch pipettieren die Lösung nach oben und unten mehrmals an die Vesikel sorgfältig in 100 µL Homogenisierung Medium gebunden.</li>
	<li>Übertragen die resuspendierte Vesikel in eine neue Reaktionscup als die gereinigten Microsome für nachgeschaltete Experimente. 
	Hinweis: In der Regel 50 µL Microsome Bruch mit 5-20 µg Proteinen aus 1 x 10 7 Zellen isoliert werden kann.</li>
</ol> 4. Analyse der gereinigt Microsomen <ol><li>Aufschwemmen der magnetischen Perlen verpflichtet die Vesikel aus Schritt 3,8 für 100-200 µL Puffer TNE (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0,5 M EDTA, 1 % Nonidet p-40) mit 1/1.000 Volumen der Protease-Hemmer Cocktails anstelle der Homogenisierung Medium.</li>
	<li>Übertragen die lysate aus Schritt 4.1 auf eine neue 1,5-mL-Reaktionscup.</li>
	<li>Bestimmen die Proteinkonzentration Schritt 4.2 mithilfe einer BCA Assay Kit pro Hersteller &#39; s Protokoll. 
	Hinweis: In der Regel 5-10 µL lysate aus Schritt 4.2 genügt, um festzustellen, die Proteinkonzentration.</li>
	<li>Übertragen die lysate aus Schritt 4.2 (2 µg Protein für silberne Färbung und 10 µg Protein für das westliche Beflecken) in neue Mikroröhrchen.</li>
	<li>Setzen die Mikroröhrchen auf einem Heizblock bei 95 ° C und Kochen Proteine in 1 x SDS-Gel-laden-Puffer (100 mM Tris-HCl pH 6,8, 200 mM Dithiothreitol, 4 % SDS, 0,2 % Bromophenol Blue, 20 % Glycerin) für 5 min.</li>
	<li>Zentrifugieren Mikroröhrchen für 10 min bei 21.500 x g und 4 ° C. sammeln die Überstände in neue Mikroröhrchen die unlöslichen Anteile entfernen.</li>
	<li>Die aufgelöste Proteine in Schritt 4.6 (2 µg) von standard SD-Seite zu analysieren.</li>
	<li>Visualisieren die Protein-Bands durch die silberne Färbung mit silbernen Färbung nach dem Hersteller Kit &#39; s Protokoll.</li>
	<li>Analysieren die aufgelöste Proteine in Schritt 4.6 (10 µg) von standard SD-PAGE und Western-Blot. Verwenden Sie das Reagenz (SA-HRP) pro Hersteller &#39; s-Protokoll und visualisieren von Fluorography.</li>
</ol> 5. In-vitro- Rekonstitution des ERAD Ubiquitination von bOVA Verwendung gereinigt Microsomen <ol><li>Übertragung der gereinigten Microsomen (5-10 µg Protein) von 3,8 Schritt mit einem Volumen von 50 % der Retikulozytenzahl lysate (RL), 1 x Reaktion Puffer (50 mM Tris pH 7.4, 3 mM ATP, 0,5 mM MgCl (2), und 12:02-Flag gekennzeichnet Ubiquitin in eine neue Reaktionscup. Der letzte Band dieses Experiments ist 20-40 µL.</li>
	<li>Inkubation die Reaktionscup für 2 h bei 37 ° c 
	Hinweis: Wenn die Menge an Ubiquitinated bOVA in Schritt 5.12 zu klein, durch westliche Beflecken zu erkennen ist, fügen Sie 10 µM MG132 oder 2 µM Lactacystin, die Verarbeitung von Ubiquitinated bOVA durch Proteasomen hemmen.</li>
	<li>Stoppen Sie die Reaktion indem man die Reaktionscup auf Eis.</li>
	<li>Lösen die Microsomen durch Zugabe von 500 µL TNE Puffer mit 1/1.000 Volumen der Protease-Hemmer Cocktails.</li>
	<li>Die Reaktionscup Zentrifuge für 10 min bei 21,500 x g und 4 ° C. sammeln die Überstände in eine neue Reaktionscup die unlöslichen Brüche zu entfernen, die Ubiquitinated bOVA in DC2.4 vor der in-vitro- Ubiquitination Assay enthalten.</li>
	<li>Den Überstand auf eine neue Reaktionscup übertragen und Volumen 1/100 der neuen SA-magnetische Beads (in der Regel 5 µL für 500 µL Überstände).</li>
	<li>Sanft vermischen und drehen die Reaktionscup langsam für 30 min bei 4 ° c</li>
	<li>Zentrifugieren Sie die Reaktionscup für 10 min bei 21.500 x g und 4 ° C. verwerfen der Überstand durch Absaugen, bOVA und Ubiquitinated bOVA wiederherzustellen mit der SA-magnetische Beads gebunden.</li>
	<li>Waschen zweimal die gesammelten SA-magnetische Beads mit 1 mL TNE Puffer durch Zentrifugation für 10 min bei 21.500 x g und 4 ° C. verwerfen des Überstands jedesmal durch Aspiration.</li>
	<li>Kochen der SA-magnetischen Kügelchen in 1 x SDS-Gel Ladepuffer für 5 min bei 95 ° C auf einem Hitze-Block, der gereinigten Proteine durch die SA-magnetische Beads zu lösen.</li>
	<li>Zentrifugieren Sie die Reaktionscup für 10 min bei 21.500 x g und 4 ° c in eine neue Reaktionscup, die unlöslichen Anteile zu entfernen die Überstände sammeln.</li>
	<li>Analysieren die SA-magnetische Beads durch standard SD-Seite und Western-Blot-an die Proteine gebunden. Der Einsatz von Antikörpern (Anti-Flag, Anti-Multi-Ub und Anti-Maus IgG-HRP) und das Reagenz (SA-HRP) ist pro Hersteller &#39; s-Protokoll und visualisiert durch Fluorography.</li>
</ol>

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<li>Zehner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+translocon+protein+Sec61+mediates+antigen+transport+from+endosomes+in+the+cytosol+for+cross-presentation+to+CD8%28%2B%29+T+cells%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">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>

Die Autoren haben nichts preisgeben.

In früheren Studien von CP eingearbeitete exogene Antigene in den geschützten Bereich des späten Endosom oder ER durch immunofluorescent Mikroskopie16,30,31,32angesammelt. Es wird geschätzt, dass ERAD-wie Transport und Verarbeitung von exogenen Antigenen in diesen spezialisierten Bereichen ER oder späten Endosom durchgeführt werden, wie das zelluläre Fach von Saccharose oder Iodixanol Di...

Die MHC ich Moleküle auf der Oberfläche aller kernhaltigen Zellen, mit kurzen Antigene Peptide abgeleitet endogene Antigene, die durch das Ubiquitin-Proteasom-System in der Zellflüssigkeit1verarbeitet werden ausgedrückt werden. Nach der Bearbeitung werden Antigene Peptide in das endoplasmatische Retikulum (ER)-Lumen von Peptid-Transporter TAP transportiert. Das ER-Lumen eine Reihe von spezifischen Chaperone unterstützen das Peptid-laden und die korrekte Faltung des MHC ich komplexe. Diese Reihe von Molekülen nennt man die Peptid-laden-Komplex (PLC), darauf hinweist, dass ER ein zentrales Fach für Peptid laden auf MHC ich2. Nach Peptid laden, MHC ich Moleküle an der Zelloberfläche transportiert und spielen eine Schlüsselrolle bei der adaptiven Immunsystems als selbständige Marker und ermöglicht die CD8+ zytotoxische T-Lymphozyten (CTLs), Krebszellen oder Krankheitserreger erkennen von Antigenen Peptide aus Nichtselbst Proteine3.
Antigenpräsentierende Zellen (APC), Antigene Peptide aus exogene Antigene sind auch auf MHC habe ich4,5,6,7,8 über CP, das hauptsächlich getragen wird vorgestellt von DCs9,10,11. CP ist wichtig sowohl für die Aktivierung von naiven CD8+ T Zellen und Gedächtnis CD8+ T-Zellen gegen Infektionen und Anti-Tumor CTLs12,13, und bei der Aufrechterhaltung der Immuntoleranz durch die Inaktivierung der selbsttätige naive T-Zellen14,15. Der CP spielt viele wichtige Rollen in das adaptive Immunsystem jedoch die molekularen Mechanismen der CP müssen noch im Detail beschrieben werden. Frühere Studien der CP ergab, dass exogene Antigene, in der ER und das Endosom lokalisiert wurden und wurden von ERAD, was darauf hindeutet, dass exogene Antigene, die ER für ERAD-wie Verarbeitung und laden16 Peptid aus dem Endosom transportiert werden verarbeitet . Sammeln Beweise deutet jedoch darauf hin, dass das Peptid-Laden von CP durchgeführt wird, nicht in der Notaufnahme sondern im nicht-klassischen endocytic Fächer, die auch Besonderheiten der ER (Abbildung 1)17,18 ,19,20,21. Zur Vermeidung von Abbau der Antigen Peptid Vorläufer von der hohen Aktivität des Aminopeptidase tritt22 im Zytosol, Verarbeitung und Peptid im CP laden im proximalen Bereich dieser nichtklassischen endocytic Abteilungen (Abbildung 1). Obwohl die Charakterisierungen dieser endocytic Abteilungen umstritten sind, gibt es keine bestehende spezifische Moleküle als exogene Antigene in diesem Fach.
ERAD ist ein zellulärer Weg, der speziell fehlgefaltete Proteine aus dem ER entfernt. In dem ERAD Weg fehlgefaltete Proteine Doppelthebel durch die ER-Membran, das Zytoplasma transportiert und verarbeitet von den Ubiquitin-Proteasom System23,24,25. Beim Transport von großer Molekülen wie Proteinen, durch die Lipid-Bilayer durchlaufen diese Moleküle ein molekularer Apparat namens ein Translocon, wie der Sec61 komplex und Delrin Komplex in die ER26, und die komplexe Tom und Tim Komplex in der Mitochondrien-27. Wenn exogen hinzugefügt Antigene durch die ER-Membran transportiert werden, müssen sie die Lipid-Bilayer im Komplex mit Translocons, wie den Sec61-Komplex eindringen. Die hier beschriebene Methode gereinigt die gezielte Blase durch die Nutzung dieser Membran durchdringen Moleküle als Marker für die endocytic Fächer.
Die hier beschriebene Methode ist ein neues Vesikel-Reinigung-Protokoll mit der DC-ähnliche Zelle Zeile DC2.428 und biotinylierte Ovalbumin (bOVA) als eine exogene Antigen. Die endocytic Fächer wurden gereinigt, indem Streptavidin (SA)-magnetische Beads, die mit der Membran durchdringen bOVA als Hersteller. In diesem gereinigten Microsome hinzugefügt einige exogen bOVA blieb noch in Bruchteilen Membran aber waren an der Außenseite des Microsome und dann Ubiquitinated transportiert und verarbeitet in-vitro-29. Dieses gereinigte Microsome enthielt nicht nur endocytic Fach-spezifische Proteine, sondern auch ER ansässigen Proteine für ERAD und das Peptid Komplex laden; darauf hindeutet, dass das zelluläre Fach das prospektive endocytic Fach für CP29. Dieses Protokoll ist nicht abhängig von der Art der exogene Antigene und gilt auch für andere DC-Subsets und andere Zelltypen, z. B. Endothelzellen, Makrophagen und B-Zellen, den genaue molekularen Mechanismus der DCs für kompetente CP zu klären.

<table><tbody><tr><td>RPMI 1640</td><td>gibco by life technologies</td><td>11875-093</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Equitech bio</td><td>SFB30</td><td></td></tr><tr><td>Sodium pyruvate</td><td>gibco by life technologies</td><td>11360-070</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>gibco by life technologies</td><td>11140-050</td><td></td></tr><tr><td>HEPES</td><td>gibco by life technologies</td><td>15630-080</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>gibco by life technologies</td><td>21985-023</td><td></td></tr><tr><td>L-glutamine</td><td>gibco by life technologies</td><td>25030-164</td><td></td></tr><tr><td>Penisicillin-Sreptomycin</td><td>gibco by life technologies</td><td>15140-122</td><td></td></tr><tr><td>DMEM</td><td>gibco by life technologies</td><td>12100-46</td><td></td></tr><tr><td>OVA</td><td>SIGMA</td><td>A5503</td><td></td></tr><tr><td>Biotin-protein labelling kit</td><td>Thermo Fisher Scientific</td><td>F6347</td><td></td></tr><tr><td>MG-132</td><td>Santa Cruz Biotechnology</td><td>201270</td><td></td></tr><tr><td>lactacystin</td><td>SIGMA</td><td>L6785</td><td></td></tr><tr><td>Dounce homogenizer</td><td>IUCHI</td><td>131703</td><td></td></tr><tr><td>protease inhibitor cocktails</td><td>SIGMA</td><td>P8340</td><td></td></tr><tr><td>iodixanol</td><td>Cosmo bio</td><td>1114542</td><td></td></tr><tr><td>SA-magnetic beads</td><td>New England Biolabs</td><td>201270</td><td></td></tr><tr><td>control magnetic beads</td><td>Chemagen</td><td>M-PVA012</td><td></td></tr><tr><td>magnetic stand</td><td>BD Biosciences</td><td>552311</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>silver staining kits</td><td>Cosmo bio</td><td>423413</td><td></td></tr><tr><td>Reticulocyte Lysate</td><td>Promega</td><td>1730714</td><td></td></tr><tr><td>Flag-tagged ubiquitin</td><td>SIGMA</td><td>U5382</td><td></td></tr><tr><td>anti-ovalbumin (OVA,mouse)</td><td>Antibody Shop</td><td>HYB 094-06</td><td></td></tr><tr><td>ant-multi-ubiquitin (mouse)</td><td>MBL</td><td>D058&amp;minus;3</td><td></td></tr><tr><td>anti-Flag (mouse)</td><td>SIGMA</td><td>F3165</td><td></td></tr><tr><td>trypsin</td><td>SIGMA</td><td>85450C</td><td></td></tr></tbody></table>

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

Dendritische Zellen (DCs) sind leistungsfähige Verarbeitung und verinnerlichten exogene Antigene auf großen Histocompatibility-Klasse (MHC) zu präsentieren ich Moleküle auch bekannt als Kreuz-Präsentation (CP). CP spielt eine wichtige Rolle nicht nur in der Stimulation der naiven CD8+ T Zellen und Gedächtnis CD8+ T-Zellen für infektiöse und Tumor Immunität sondern auch bei der Inaktivierung von selbsttätige naive T-Zellen durch T-Zell-Anergie oder T-Zell-Löschung. Wenn auch der kritischen molekularen Mechanismus der CP geklärt werden noch, gibt es sammeln Hinweise darauf, dass exogene Antigene durch endoplasmatische Retikulum verbundenen Abbau (ERAD) nach dem Export aus nichtklassischen endocytic verarbeitet werden Fächer. Bis vor kurzem waren Charakterisierungen dieser endocytic Abteilungen beschränkt, denn es keine spezifische molekulare Marker als exogene Antigene gab. Die hier beschriebene Methode ist ein neues Protokoll Vesikel Isolierung, die für die Reinigung dieser endocytic Abteilungen ermöglicht. Mit diesem gereinigten Microsome, rekonstituiert wir ERAD-wie Transport, Ubiquitination und Verarbeitung des exogene Antigen in Vitro, was darauf hindeutet, dass das Ubiquitin-Proteasom-System die exogene Antigen nach dem Export aus dieser verarbeitet Handy-Fach. Dieses Protokoll kann weiter auf andere Zelltypen zu klären, den molekularen Mechanismus der CP angewendet werden.

Um den molekularen Mechanismus der CP zu erhellen, ist es notwendig, die zellulare Fächer zu identifizieren, wo exogene Antigene ERAD-wie Transport und Verarbeitung unterzogen werden. Während Beobachtungen durch immunofluorescent Mikroskopie oder durch Elektronenmikroskopie der zelluläre Fach wo exogene Antigene16,17,18,19angesamm...

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

Die hier beschriebene Methode ist eine neue Vesikel Isolierung Protokoll, das für die Reinigung von den zellulären Kompartimenten ermöglicht dem exogene Antigenen durch endoplasmatische Retikulum verbundenen Abbau in Kreuz-Präsentation verarbeitet werden.

Reinigung der Membran-Fach für endoplasmatische Retikulum-assoziierten Abbau von exogenen Antigenen in Kreuz-Präsentation

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 Aufrufe.  Takasaki University of Health and Welfare. Die hier beschriebene Methode ist eine neue Vesikel Isolierung Protokoll, das für die Reinigung von den zellulären Kompartimenten ermöglicht dem exogene Antigenen durch endoplasmatische Retikulum verbundenen Abbau in Kreuz-Präsentation verarbeitet werden.

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

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Reinigung und Aggregation des Amyloid Precursor Protein intrazelluläre Domäne

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Forschung

Medizin

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Mitochondrien-assoziierte ER Membranen (MAMs) und Glycosphingolipid Angereichert Mikrodomänen (GEM): Isolation aus Mäusehirn

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Neurowissenschaften

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

Rekonstitution der Msp1-Extraktionsaktivität mit vollständig gereinigten Komponenten

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Biochemie

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

Spontane Bildung und Neuordnung der künstlichen Lipid Nanotube Netze als Bottom-Up-Modell für endoplasmatische Retikulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Biotechnik

Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific physicochemical conditions during purification to obtain them with high purity and structural quality and in a catalytically active form. The disaccharide trehalose is a compatible solute that is synthesized and accumulated in high concentrations in the yeast cytoplasm to stabilize the membranes and proteins. The use of trehalose as an additive in the protocol for the purification of plasma membrane H+-ATPase results in a high-quality preparation, the hexameric structure of which is shown by biochemical analytical methods. Trehalose can, therefore, be used as a stabilizing additive for the purification of membrane proteins (P-ATPases). This protocol describes the modification of the classical protocol for SERCA purification by subjecting SERCA to centrifugation on a trehalose concentration gradient. The inclusion of this carbohydrate led to the purification of SERCA in a catalytically active form with high purity and, importantly, in a stable form. Partial biochemical characterization of the purified SERCA (SDS-PAGE, enzyme kinetics, FITC labeling, circular dichroism spectroscopy) showed that the enzyme is suitable for functional and structural studies. The use of trehalose in the purification protocol of P-type ATPases and other labile membrane (and cytosolic) proteins is suggested.

This protocol describes an improved SERCA purification method, which includes the disaccharide trehalose in the final centrifugation step. This carbohydrate stabilizes proteins under harsh conditions. The purified SERCA was catalytically active and displayed high purity, making it suitable for structural and functional studies.

Aufreinigung des Sarko-endoplasmatischen Retikulums Ca2+-ATPase aus dem Kaninchenmuskel

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualisierung von endoplasmatischen Retikulum lokalisierte mRNAs in Säugerzellen

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Biologie

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualisierung des endoplasmatischen Reticulum Subdomains in kultivierten Zellen

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

Cycloheximid Chase-Analyse von Proteinabbau in<em&gt; Saccharomyces cerevisiae</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

The Endoplasmic Reticulum

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary protein and lipid synthesis factory for most cell organelles, such as the Golgi apparatus, lysosomes, secretory vesicles, and the plasma membrane. Despite being the most extensive and functionally complex subcellular organelle, ER was the last to be discovered. After years of deliberation, Keith Porter and George Palade in the year 1954, produced the first high-resolution electron microscope images to affirm the presence of ER in the eukaryotic cell.
The ER membranes were identified as sites for protein synthesis and crucial for intracellular transport using radio-labeled and fluorescent-labeled amino acids. The isolation of ER is a tricky task as it forms an intricate mesh with other subcellular organelles. However, on cell homogenization, the disrupted ER membranes reseal into small closed vesicles called microsomes. These vesicles form a microsystem capable of sustaining all ER-related functions like protein and lipid synthesis, calcium signaling, and glycosylation. Subcellular fractionation is the best and often-used technique for the purification of these membranes. When separating using a sucrose gradient, the rough ER microsomes sediments at a higher density than smooth ER microsomes.
The ER network in a cell is dynamic. It is constantly shape-shifting along with the cytoskeleton to bolster mechanical support for the cell structure. Despite the distinct cisternae and tubule morphologies of the ER membrane, interconversion between the two is possible and is governed by the expression of membrane proteins. The ER network rearranges by tubule growth, retraction, and fusion of adjacent ER-ER membranes.
As mentioned above, the ER network is required for optimal overall cellular health. Disruption of ER morphology is linked to pathological conditions, including neurological disorders like Alzheimer&#39;s disease, hereditary spastic paraplegia, and viral infections like hepatitis C virus, and dengue virus.

Das endoplasmatische Retikulum

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary ...

Lehre

JoVE Core

Zellbiologie

Unit 1

Transmembrantransport im endoplasmatischen Retikulum und in Peroxisomen

SCHLÜSSELBEGRIFFE UND KONZEPTE

ER Retrieval Pathway

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct location. Occasionally, misfolded proteins and incorrect proteins escape their original compartments, and a retrieval pathway is used to return the escaped proteins to their original compartment.
The ER uses many checkpoints to prevent the entry of incorrectly folded or a resident protein as cargo onto a transport vesicle. These mechanisms include cargo selectivity of the receptors, aggregation of functionally similar proteins, and retention sequences on resident proteins.
Biochemical studies characterizing the cargo-sorting receptors have shown that they are ligand-specific and bind only at specific pH (pH 5.0 in yeast and mammals). Functionally similar ER-resident can proteins aggregate to form large complexes that cannot be loaded onto transport vesicles, thus avoiding the accidental escape of these proteins from the organelle. Additionally, the membrane-spanning domain of several ER and Golgi proteins contain a retention sequence that marks these proteins for staying back in the ER, so receptors do not bind them.
If an ER-resident protein escapes these checkpoints and gets loaded onto a Golgi-bound vesicle, a specific amino acid sequence called a retrieval signal helps the protein get recognized and transported back to the ER. The receptors involved in retrieval mechanisms recognize the retrieval signals at the carboxyl terminus of soluble proteins of the ER and in the cytoplasmic domain of some ER and Golgi membrane proteins. The receptors selectively capture such proteins and package them in vesicles that transport them back to the ER.
Two well-characterized retrieval signals are the carboxy-terminal tetrapeptide KDEL and KKXX found in many ER lumen resident proteins. BiP, a molecular chaperone, plays a vital role in preventing the aggregation of misfolded proteins. Interaction between BiP and unfolded proteins is mediated by a substrate-binding domain and a nucleotide-binding domain for ATPase activity. KDEL receptor in the post-ER compartments such as ERGIC and Golgi binds with the carboxyl-terminal retrieval signal of BiP and returns BiP to the ER via COPI vesicles.

ER Abrufweg

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct ...

Reinigung der Membran-Fach für endoplasmatische Retikulum-assoziierten Abbau von exogenen Antigenen in Kreuz-Präsentation

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