Este trabalho é apoiado pela Universidade de Takasaki da saúde e bem-estar.

1. cultivo de células e adição de antígenos exógenos <ol><li>preparar bOVA usando uma rotulagem de biotina-proteína kit seguindo o fabricante &#39; protocolo s. 
	Nota: Normalmente, bOVA contém biotina 2 M por 1 M de óvulos em média.</li>
	<li>DC2.4 crescer células em RPMI-1640 suplementado com 2 mM L-glutamina, piruvato de sódio 1 mM, aminoácidos não essenciais de 0,1 mM, 100 U/mL de penicilina-estreptomicina, 55 mM 2-Mercaptoetanol, 10 mM HEPES (pH 7,5) e soro fetal bezerro de 10% (doravante RPMI) a 37 ° C em 5 % CO 2 em uma incubadora umidificada (doravante sem menção, as células são incubadas a essa condição). Também é possível usar DMEM suplementado com 10% de soro fetal bezerro, 3,7 g/L NaHCO 3 (doravante DMEM) no lugar de RPMI.</li>
	<li>Um dia antes da purificação, dividir as células em RPMI a 1 x 10 5 células/mL em uma placa de cultura de tecido. Evite manter as células em um estado confluente. DC2.4 células podem altamente incorporar antígenos exógenos até um estado semi confluente, mas rapidamente perdem a capacidade depois de atingir um estado de excesso confluente.</li>
	<li>Antes do DC2.4 células são semi confluentes, adicionar 250 µ g/mL de bOVA para 1 x 10 6 células/mL e incubar durante 2-4 h. 
	Nota: Durante a jusante experimentos, todos os buffers e os reagentes são mantidos a 4 ° C a menos que indicado de outra forma.</li>
</ol> 2. Preparação de Microsomes <ol><li>colher as DC2.4 células pipetando suave da placa de tecido em um novo tubo cónico de 50 mL.</li>
	<li>Centrifugar 1.000 x g por 5 min, 4 ° C e remova cuidadosamente o meio com bOVA por aspiração.</li>
	<li>Lavar as células DC2.4 duas vezes com 40 mL de tampão PBS (1,37 mM NaCl, 8,1 mM Na 2 HPO 4, 2,68 mM KCl, 1,47 mM KH 2 PO 4) por centrifugação a 1.000 x g, durante 5 min, 4 ° C e descartar o sobrenadante de cada vez por aspiração.</li>
	<li>Ressuspender o pellet em passo 2.3 em 1-2 mL (1/20-1/10 volume de meio de cultura) do meio de homogeneização (0,25 M de sacarose, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7,4]) com volume de 1/1.000 de coquetéis de inibidor de protease e transferência a ressuspensão DC2.4 células em um gelada homogenizacao homogenizador.</li>
	<li>Interromper as células DC2.4 ressuspensa suavemente por 10-20 pancadas com o gelado homogenizacao homogenizador. 
	Nota: Durante a etapa de perturbação, a homogenizacao homogeneizador é refrigerado no gelo</li>
	<li>Transferir a suspensão celular-interrompido para um novo tubo cônico de 15 mL.</li>
	<li>Adicionar 8-9 mL de meio de homogeneização total 10 mL e centrifugar o tubo cônico durante 10 minutos a 2.000 x g e 4 ° C.</li>
	<li>Transferir o sobrenadante para um novo tubo cônico de 15 mL para remover células ininterrupta e núcleos e centrifugar o tubo cônico novamente por 10 min a 2.000 x g e 4 ° C.</li>
	<li>Transferir o sobrenadante para um novo tubo de ultracentrifugação e centrifugar por 45 min em 100.000 x g e 4 ° C.</li>
	<li>Aspirar o sobrenadante e ressuspender o cuidadosamente em 1-2 mL de meio de homogeneização com volume de 1/1.000 de cocktails de inibidor de protease pipetando a solução acima e para baixo várias vezes para fazer uma fração do microsome para jusante experiências.</li>
	<li>Transferir a fração do microsome para um novo tubo de fundo redondo de 5 mL. 
	Nota: Após esta etapa, é possível enriquecer microssomas objetivas por iodixanol centrifugação gradiente de densidade de acordo com o fabricante &#39; protocolo de s, remover microssomas específico. As frações de pico para antígenos exógenos são recolhidas e em seguida, submetidas à etapa seguinte de purificação (etapa 3).</li>
</ol> 3. Purificação de Microsomes com bOVA ERAD passando por <ol><li>volume de adicionar 1/100 de fresco grânulos SA-magnético para a fração do microsome da etapa 2.11 no 5 mL tubo de fundo redondo. 
	Nota: Antes desta etapa, é possível pre-limpar a fração do microsome por grânulos controle magnético para reduzir contaminações de específico microsomes.</li>
	<li>Delicadamente, misture bem e gire o tubo de fundo redondo, lentamente por 30 min a 4 ° C.</li>
	<li>Adicionar 2-3 mL de meio de homogeneização total 4 ml para o tubo de fundo redondo e misture suavemente bem.</li>
	<li>Colocar o tubo de fundo redondo num suporte magnético e incubar durante 10 minutos a 4 ° C. Desde que os grânulos ligados para as vesículas são atraídos para o ímã, microgrânulos marrons gradualmente acumularão na parede do tubo mais próximo ao ímã.</li>
	<li>Com o tubo restante no suporte magnético, cuidadosamente descartar os sobrenadantes por aspiração para remover as vesículas desacopladas.</li>
	<li>Lavagem os grânulos magnéticos vinculados as vesículas duas vezes com 5 mL de meio de homogeneização, a magnética defende a 10 min a 4 ° C e descartar o sobrenadante cada vez por cuidadosamente aspiração. 
	Nota: Sem o suporte magnético, purificação por centrifugações a 2.000 x g por 10 min, 4 ° C também está disponível.</li>
	<li>Ressuspender os grânulos magnéticos vinculados as vesículas cuidadosamente em meio de homogeneização 100 µ l a pipetagem a solução acima e para baixo várias vezes.</li>
	<li>Transferir as ressuspensa vesículas em um microtubo de novo como do microsome purificado para jusante experiências. 
	Nota: Normalmente, fração do microsome 50 µ l contendo 5-20 µ g proteínas de 1 x 10 7 células pode ser isolados.</li>
</ol> 4. Análise do Microsomes purificado grânulos <ol><li>Ressuspender o magnético ligados para as vesículas de passo 3.8 a 100-200 µ l de tampão TNE (pH de 20 mM Tris-HCl 7.4, 150 mM de NaCl, EDTA 0,5 M, 1% Nonidet P-40) com volume de 1/1.000 de cocktails de inibidor de protease em vez de meio de homogeneização.</li>
	<li>Transferir o lisado de passo 4.1 para um novo microtubo de 1,5 mL.</li>
	<li>Determinar a concentração de proteína da etapa 4.2 usando um kit de ensaio do BCA pelo fabricante &#39; protocolo de s. 
	Nota: Normalmente, 5-10 µ l lisado da etapa 4.2 é suficiente para determinar a concentração de proteína.</li>
	<li>Transferir o lisado de passo 4.2 (2 µ g de proteína para coloração de prata e 10 µ g de proteína para a mancha ocidental) em microtubos novos.</li>
	<li>Colocar os microtubos em um bloco de calor em proteínas de 95 ° C e ferver em tampão de carregamento de gel de SDS 1x (pH 100 mM Tris-HCl 6,8, azul de bromofenol 200mm ditiotreitol, SDS de 4%, 0,2%, 20% de glicerol) 5 min.</li>
	<li>Centrifugar os microtubos para 10 min a 21.500 x g e 4 ° C. coletar os sobrenadantes em microtubos novos para remover as frações insolúveis.</li>
	<li>Analisar as proteínas resolvidas na etapa 4.6 (2 µ g) por padrão SD-PAGE.</li>
	<li>Visualize as bandas de proteína pela prata coloração usando coloração prata kit após o fabricante &#39; protocolo de s.</li>
	<li>Analisar as proteínas resolvidas na etapa 4.6 (10 µ g) por padrão SD-PAGE e Western Bloting. Use o reagente (SA-HRP) pelo fabricante &#39; s de protocolo e Visualizar por fluorography.</li>
</ol> 5. In Vitro Reconstituição da ERAD de bOVA usando purificado Microsomes <ol><li>transferência o purificado microssomas (5-10 µ g de proteína) da etapa 3.8 com um volume de 50% de reticulócitos lisado (RL), em tampão de reação (50 mM Tris pH 7,4, 3mm ATP, 0.5 mM MgCl 1x 2) e 12:02 bandeira-tag ubiquitin em um microtubo de novo. O volume final deste experimento é 20-40 µ l.</li>
	<li>Incubar o microtubo para 2 h a 37 ° C. 
	Nota: Se a quantidade de ubiquitinated bOVA na etapa 5.12 é muito pequena para detectar pela mancha ocidental, adicionar 10 µ m de MG132 ou 2 µM de lactacystin para inibir o processamento do bOVA ubiquitinated por proteasomes.</li>
	<li>Parar a reação, colocando o microtubo na Ice.</li>
	<li>Resolver as microssomas adicionando buffer de TNE 500 µ l com volume de 1/1.000 de cocktails de inibidor de protease.</li>
	<li>o microtubo de centrifugação durante 10 minutos a 21,500 x g e 4 ° C. recolher os sobrenadantes para um microtubo novo para remover as frações insolúveis, que contêm ubiquitinated bOVA em DC2.4 antes do ensaio em vitro ubiquitination.</li>
	<li>Transferir o sobrenadante para um microtubo de novo e adicionar o volume de 1/100 de novas contas SA-magnético (geralmente 5 µ l para 500 µ l de sobrenadantes).</li>
	<li>Delicadamente, misture bem e girar o microtubo lentamente por 30 min a 4 ° C.</li>
	<li>Centrifugar o microtubo para 10 min a 21.500 x g e 4 ° C. descartar o sobrenadante por aspiração para recuperar o bOVA e ubiquitinated bOVA vinculado com os grânulos SA-magnético.</li>
	<li>Lavar duas vezes os grânulos SA-magnética coletados com 1 mL de tampão TNE por centrifugação durante 10 min a 21.500 x g e 4 ° C. descartar o sobrenadante de cada vez por aspiração.</li>
	<li>Ferver os grânulos SA-magnético em 1x do gel de SDS carregando buffer para min 5 a 95 ° C, em um bloco de calor para resolver as proteínas purified pelos grânulos magnético-SA.</li>
	<li>Centrifugar o microtubo para 10 min a 21.500 x g e 4 ° C. recolher os sobrenadantes para um microtubo novo para remover as frações insolúveis.</li>
	<li>Analisar as contas SA-magnéticos ligadas às proteínas por padrão SD-PAGE e Western Bloting. O uso de anticorpos (anti-Flag, anti-multi-Ub e anti-mouse IgG HRP) e o reagente (SA-HRP) é pelo fabricante &#39; protocolo s e visualizado por fluorography.</li>
</ol>

<ol>
	<li>van Endert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Providing+ligands+for+MHC+class+I+molecules.">Providing ligands for MHC class I molecules.</a> Cell Mol Life Sci. 68 (9), 1467-1469 (2011).</li><li>Janeway, C., Travers, P., Walport, M., Shlomchik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunobiology: The Immune System in Health and Disease. , (2001).</li><li>McDevitt, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+the+role+of+the+major+histocompatibility+complex+in+the+immune+response.">Discovering the role of the major histocompatibility complex in the immune response.</a> Annu Rev Immunol. 18 (1), 1-17 (2000).</li><li>Bedoui, S., 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=Cross-presentation+of+viral+and+self+antigens+by+skin-derived+CD103++dendritic+cells.">Cross-presentation of viral and self antigens by skin-derived CD103+ dendritic cells.</a> Nat Immunol. 10 (5), 488-495 (2009).</li><li>Segura, E., Villadangos, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+presentation+by+dendritic+cells+in+vivo.">Antigen presentation by dendritic cells in vivo.</a> Curr Opin Immunol. 21 (1), 105-110 (2009).</li><li>Cheong, C., 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=Microbial+stimulation+fully+differentiates+monocytes+to+DC-SIGN/CD209(+)+dendritic+cells+for+immune+T+cell+areas.">Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas.</a> Cell. 143 (3), 416-429 (2010).</li><li>Henri, S., 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=CD207++CD103++dermal+dendritic+cells+cross-present+keratinocyte-derived+antigens+irrespective+of+the+presence+of+Langerhans+cells.">CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-derived antigens irrespective of the presence of Langerhans cells.</a> J Exp Med. 207 (2), 189-206 (2010).</li><li>Shortman, K., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD8++dendritic+cell+subset.">The CD8+ dendritic cell subset.</a> Immunol. Rev. 234 (1), 18-31 (2010).</li><li>Carbone, F. R., Kurts, C., Bennett, S. R., Miller, J. F., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-presentation:+a+general+mechanism+for+CTL+immunity+and+tolerance.">Cross-presentation: a general mechanism for CTL immunity and tolerance.</a> Immunol Today. 19 (8), 368-373 (1998).</li><li>Nair-Gupta, P., Blander, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+updated+view+of+the+intracellular+mechanisms+regulating+cross-presentation.">An updated view of the intracellular mechanisms regulating cross-presentation.</a> Front Immunol. , (2013).</li><li>Joffre, O. P., Segura, E., Savina, A., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-presentation+by+dendritic+cells.">Cross-presentation by dendritic cells.</a> Nat Rev Immunol. 12 (8), 557-569 (2012).</li><li>Kurts, C., 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=Constitutive+class+I-restricted+exogenous+presentation+of+self+antigens+in+vivo.">Constitutive class I-restricted exogenous presentation of self antigens in vivo.</a> J Exp Med. 184 (3), 923-930 (1996).</li><li>Kurts, C., Kosaka, H., Carbone, F. R., Miller, J. F., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+I-restricted+cross-presentation+of+exogenous+self-antigens+leads+to+deletion+of+autoreactive+CD8(+)+T+cells.">Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells.</a> J Exp Med. 186 (2), 239-245 (1997).</li><li>Bonifaz, 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=Efficient+targeting+of+protein+antigen+to+the+dendritic+cell+receptor+DEC-205+in+the+steady+state+leads+to+antigen+presentation+on+major+histocompatibility+complex+class+I+products+and+peripheral+CD8++T+cell+tolerance.">Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance.</a> J Exp Med. 196 (12), 1627-1638 (2002).</li><li>Heath, W. R., Carbone, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-presentation+in+viral+immunity+and+self-tolerance.">Cross-presentation in viral immunity and self-tolerance.</a> Nat Rev Immunol. 1 (2), 126-134 (2001).</li><li>Imai, J., Hasegawa, H., Maruya, M., Koyasu, S., Yahara, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+antigens+are+processed+through+the+endoplasmic+reticulum-associated+degradation+(ERAD)+in+cross-presentation+by+dendritic+cells.">Exogenous antigens are processed through the endoplasmic reticulum-associated degradation (ERAD) in cross-presentation by dendritic cells.</a> Int Immunol. 17 (1), 45-53 (2005).</li><li>Houde, 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=Phagosomes+are+competent+organelles+for+antigen+cross-presentation.">Phagosomes are competent organelles for antigen cross-presentation.</a> Nature. 425 (6956), 402-406 (2003).</li><li>Guermonprez, P., 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=ER-phagosome+fusion+defines+an+MHC+class+I+cross-presentation+compartment+in+dendritic+cells.">ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells.</a> Nature. 425 (6956), 397-402 (2003).</li><li>Burgdorf, S., Scholz, C., Kautz, A., Tampe, R., Kurts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+mechanistic+separation+of+cross-presentation+and+endogenous+antigen+presentation.">Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation.</a> Nat Immunol. 9 (5), 558-566 (2008).</li><li>Liz&#233;e, G., 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=Control+of+dendritic+cell+cross-presentation+by+the+major+histocompatibility+complex+class+I+cytoplasmic+domain.">Control of dendritic cell cross-presentation by the major histocompatibility complex class I cytoplasmic domain.</a> Nat Immunol. 4 (11), 1065-1073 (2003).</li><li>Basha, G., 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=A+CD74-dependent+MHC+class+I+endolysosomal+cross-presentation+pathway.">A CD74-dependent MHC class I endolysosomal cross-presentation pathway.</a> Nat Immunol. 13 (3), 237-245 (2012).</li><li>Saveanu, L., Carroll, O., Hassainya, Y., van Endert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity,+contradictions,+and+conundrums:+studying+post-proteasomal+proteolysis+in+HLA+class+I+antigen+presentation.">Complexity, contradictions, and conundrums: studying post-proteasomal proteolysis in HLA class I antigen presentation.</a> Immunol Rev. 207 (1), 42-59 (2005).</li><li>Wiertz, E. J., 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=Sec61-mediated+transfer+of+a+membrane+protein+from+the+endoplasmic+reticulum+to+the+proteasome+for+destruction.">Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction.</a> Nature. 384 (6608), 432-438 (1996).</li><li>Hampton, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER-associated+degradation+in+protein+quality+control+and+cellular+regulation.">ER-associated degradation in protein quality control and cellular regulation.</a> Curr Opin Cell Biol. 14 (4), 476-482 (2002).</li><li>Tsai, B., Ye, Y., Rapoport, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-translocation+of+proteins+from+the+endoplasmic+reticulum+into+the+cytosol.">Retro-translocation of proteins from the endoplasmic reticulum into the cytosol.</a> Nat Rev Mol Cell Biol. 3 (4), 246-255 (2002).</li><li>Nyathi, Y., Wilkinson, B. M., Pool, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-translational+targeting+and+translocation+of+proteins+to+the+endoplasmic+reticulum.">Co-translational targeting and translocation of proteins to the endoplasmic reticulum.</a> Biochim Biophys Acta. 1833 (11), 2392-2402 (2013).</li><li>Varabyova, A., Stojanovski, D., Chacinska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein+homeostasis.">Mitochondrial protein homeostasis.</a> IUBMB Life. 65 (3), 191-201 (2013).</li><li>Shen, Z., Reznikoff, G., Dranoff, G., Rock, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloned+dendritic+cells+can+present+exogenous+antigens+on+both+MHC+class+I+and+class+II+molecules.">Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules.</a> J Immunol. 158 (6), 2723-2730 (1997).</li><li>Imai, J., Otani, M., Sakai, T., Hatta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+subcellular+compartment+in+which+exogenous+antigens+undergo+endoplasmic+reticulum-associated+degradation+from+dendritic+cells.">Purification of the subcellular compartment in which exogenous antigens undergo endoplasmic reticulum-associated degradation from dendritic cells.</a> Heliyon. , (2016).</li><li>Kovacsovics-Bankowski, M., Rock, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phagosome-to-cytosol+pathway+for+exogenous+antigens+presented+on+MHC+class+I+molecules.">A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules.</a> Science. 267 (5195), 243-246 (1995).</li><li>Ackerman, A. L., Giodini, A., 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=A+role+for+the+endoplasmic+reticulum+protein+retro+translocation+machinery+during+cross+presentation+by+dendritic+cells.">A role for the endoplasmic reticulum protein retro translocation machinery during cross presentation by dendritic cells.</a> Immunity. 25 (4), 607-617 (2006).</li><li>Ackerman, A. 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>

Os autores não têm nada para divulgar.

Em estudos anteriores de CP, os antígenos exógenos incorporados acumularam na área restrita do atrasado endosome ou ER por microscopia de imunofluorescência16,30,31,32. Estima-se que ERAD, como transporte e processamento de antígenos exógenos são realizados nestas áreas especializadas da ER ou endossomo atrasado, como o compartimento celular foi identificado pela sacarose ou iodixanol u...

O MHC moléculas são expressas na superfície de todas as células nucleadas, com curtos peptídeos antigênicos derivados de antígenos endógenos, que são processados pelo sistema ubiquitina-Proteassoma no citosol1. Após o processamento, peptídeos antigênicos são transportados para o lúmen do retículo endoplasmático (ER) pelo transportador peptídeo TAP. No lúmen do ER, uma série de acompanhantes específicos auxiliar o carregamento do peptide e o dobramento correto do MHC complexo eu. Esta série de moléculas é chamado o complexo peptídeo-carregamento (PLC), indicando que o ER é um compartimento central para peptídeo carregando em cima do MHC eu2. Depois de peptídeo de carregamento, o MHC moléculas são transportadas à superfície da célula e desempenhar um papel fundamental no sistema imune adaptativo como marcadores auto e habilita o CD8+ linfócitos T citotóxicos (CTLs) para detectar células cancerosas ou agentes infecciosos por antigênica peptídeos de proteínas não-auto3.
Em células apresentadoras de (APC), peptídeos antigênicos de antígenos exógenos são também apresentados em MHC eu4,5,6,7,8 através do CP, que é realizada principalmente por DCs9,10,11. CP é essencial tanto para a ativação do ingênuo CD8+ T memória CD8 e células+ T células em anti-infecciosos e anti-tumoral CTLs12,13e na manutenção da tolerância imunológica pela inactivar de auto interino ingênuo T células14,15. O CP desempenha muitos papéis importantes no sistema imune adaptativo, porém os mecanismos moleculares de CP tem ainda que ser descrita em pormenor. Estudos anteriores de CP revelaram que antígenos exógenos foram localizados no pronto-socorro e o endossomo e foram processados por ERAD, sugerindo que antígenos exógenos são transportados desde o endossomo ao pronto-socorro para a ERAD, como processamento e peptídeo carregando16 . No entanto, acumula evidências indicam que o carregamento do peptide do CP é realizado, não no PS, mas sim em compartimentos endocítica não-clássica, que também têm características distintivas do ER (Figura 1)17,18 ,19,20,21. Para evitar a degradação dos precursores de peptídeo antigênico pela alta atividade de aminopeptidase22 no citosol, processamento e peptídeo carregando no CP ocorre na região proximal desses compartimentos endocítica não-clássica (Figura 1). Embora as caracterizações desses compartimentos endocítica são controversas, não há nenhuma molécula de específica existente além de antígenos exógenos neste compartimento.
ERAD é uma via celular, que especificamente remove proteínas misfolded da emergência. Na via da ERAD, misfolded proteínas retrogradely são transportadas através da membrana de ER para o citoplasma e processadas pelo sistema da ubiquitina-Proteassoma23,24,25. Quando as grandes moléculas, tais como proteínas, são transportadas através da bicamada lipídica, essas moléculas passam por um aparato molecular chamado um translocon, tais como o complexo de Sec61 e Derlin complexo em ER26e o complexo de Tom e Tim complexo na mitocôndrias,27. Quando adicionado exogenamente antígenos são transportados através da membrana de ER, eles devem penetrar a bicamada lipídica em complexo com translocons, tais como o complexo de Sec61. O método descrito aqui purificado da vesícula alvo utilizando estas moléculas de membrana-penetrante como marcadores para os compartimentos endocítica.
O método descrito aqui é um novo protocolo de purificação de vesículas usando o DC, como célula linha DC2.428 e biotinilado ovalbumina (bOVA) como um antígeno exógeno. Os compartimentos endocítica foram purificados por streptavidin (SA)-grânulos magnéticos usando o bOVA penetrar a membrana como um criador. Neste do microsome purificado, alguns adicionados exogenamente bOVA ainda foi preservado em frações de membrana, mas foram transportado para a parte externa do microsome e então ubiquitinated e processada em vitro29. Este microsome purificado contidos não só endocítica compartimento específico proteínas, mas também proteínas ER-residente para ERAD e o peptídeo carregamento complexo; sugerindo que o compartimento celular é o compartimento endocítica prospectivo para CP29. Este protocolo não é dependente do tipo de antígenos exógenos e também é aplicável para outros subconjuntos de DC e outros tipos de células, como macrófagos, células B e células endoteliais, para esclarecer o mecanismo molecular preciso de DCs para CP proficiente.

<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

Células dendríticas (DCs) são altamente capazes de processar e apresentar antígenos exógenos interiorizados a classe principal de histocompatibilidade (MHC) moléculas também conhecido como Cruz-apresentação (CP). CP desempenha um papel importante não só na estimulação de ingênuo CD8+ T memória CD8 e células+ T células para infecciosas e imunidade de tumor, mas também na inativação de auto-agente ingênuo T células por células T anergy ou supressão de células T. Embora o mecanismo molecular crítico da CP continua a ser elucidado, acumula evidências indicam que antígenos exógenos são processados através de retículo endoplasmático associado degradação (ERAD) após a exportação do não-clássica endocítica compartimentos. Até recentemente, caracterizações desses compartimentos endocítica limitavam-se porque não havia nenhum marcadores moleculares específicos além de antígenos exógenos. O método descrito aqui é um novo protocolo de isolamento de vesículas, que permite a purificação desses compartimentos endocítica. Usando esta purificada do microsome, nós reconstituído a ERAD, como transporte, ubiquitination e processamento do antígeno exógeno em vitro, sugerindo que o sistema ubiquitina-Proteassoma processado o antígeno exógeno após exportação deste compartimento celular. Este protocolo pode ser mais aplicado a outros tipos de células para esclarecer o mecanismo molecular da CP.

Para elucidar o mecanismo molecular da CP, é necessário identificar os compartimentos celulares, onde se submeter a antígenos exógenos ERAD, como transporte e processamento. Enquanto observações por microscopia de imunofluorescência ou microscopia eletrônica identificaram o compartimento celular onde antígenos exógenos acumularam16,17,18,1<sup class="x...

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

O método descrito aqui é um novo protocolo de isolamento de vesículas, que permite a purificação dos compartimentos celulares onde os antígenos exógenos são processados pelo retículo endoplasmático associado a degradação em cruz-apresentação.

Purificação do compartimento da membrana retículo endoplasmático associado a degradação de antígenos exógenos em cruz-apresentação

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 visualizações.  Takasaki University of Health and Welfare. O método descrito aqui é um novo protocolo de isolamento de vesículas, que permite a purificação dos compartimentos celulares onde os antígenos exógenos são processados pelo retículo endoplasmático associado a degradação em cruz-apresentação.

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

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Grande Escala da Purificação de suínos ou bovinos fotorreceptores Outer Segmentos para a fagocitose Ensaios sobre Retinal Pigment células epiteliais

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Imunologia e Infecção

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.

Reconstituição da Atividade de Extração de Msp1 com Componentes Totalmente Purificados

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

Bioquímica

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Um método de rendimento eficiente e alta para o isolamento de subpopulações de células dendríticas Rato

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento de Salmonella typhimurium-contendo Phagosomes de macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

Estudando a dinâmica organelle em células B durante a formação de sinapse imune

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

Isolamento e captação de células dendríticas de pequenas vesículas extracelulares de Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Intralumenal Vesicles and Multivesicular Bodies

Intraluminal vesicles (ILVs) are small vesicles 50-80 nm in diameter formed during the maturation of early endosomes. A specialized endosome containing numerous ILVs is called a multivesicular body (MVB). ILVs contain internalized molecules such as antigens, nucleic acids, proteins, and metabolites. Some of these molecules are released from the MVBs inside exosomes and are transported to other cells. Other MVBs contain molecules that are retained in the ILVs and are later degraded within the cell.
MVBs were first observed in neuronal cells in 1955 using electron microscopy. MVBs have a diameter of 400-500 nm, are spherical, and were initially thought to be a form of the late endosome; however, recent studies have shown that MVBs are an intermediate form of these organelles between the early and the late endosomes.
ILV formation takes place when a ubiquitin-tagged protein enters the cell in an endocytic vesicle and fuses with the early endosome. As the early endosome matures, the endosomal membrane bends inwards to form ILVs. An ESCRT protein complex mediates ILV formation. Phosphatidylinositol 3-phosphate (PtdIns3P) is also required for ILV formation. PtdIns3P acts as a docking site for ESCRTs for the bending of the endosomal membrane.
The ESCRT complex and the ATPase, Vps4, required for ILV formation can be hijacked by enveloped retroviruses that infect host cells. Normally, ILVs are formed inside maturing endosomes with the help of ESCRTs within the cell, and the ubiquitin-tagged proteins are recognized and sorted into ILVs. In contrast, retroviruses use the ESCRTs to be released outside the cell. For example, the human immunodeficiency virus (HIV) codes for proteins, such as Gag, NC-p2, and Tat, that require ubiquitination for export. This ubiquitination is essential because ESCRTs and Vps4 recognize and sort only ubiquitin-tagged proteins into viral buds. Once the viral particles bud off from an infected cell, they are released into the extracellular space to infect other host cells.

Vesículas Intralumenais e Corpos Multivesiculares

Intraluminal vesicles (ILVs) are small vesicles 50-80 nm in diameter formed during the maturation of early endosomes. A specialized endosome containing ...

Education

JoVE Core

Biologia Celular

Unidade 1

Endocitose e Exocitose

TERMOS E CONCEITOS CHAVE

Receptor Downregulation in MVBs

Multivesicular bodies (MVBs) are mature endosomes that sort ubiquitinated proteins and then fuse with lysosomes to degrade the sorted proteins. Epidermal growth factor (EGF) and its receptor (EGFR) form a complex that can be internalized through endocytosis, sorted into an MVB, and later degraded.
The EGFR can initiate signaling pathways that&#160; lead to cell proliferation, migration, and differentiation. Overexpression of EGFR&#160; stimulates cells to proliferate. Excessive&#160; EGFR activation may result in cancer; therefore, EGFR expression must be tightly regulated. The degradation of the EGF-EGFR complex through the endocytic pathway is a means of downregulating EGFR and preventing its overexpression.
EGF binding to EGFR induces EGFR dimerization. EGFR is ubiquitinated by a ubiquitin ligase. Ubiquitination triggers the endocytosis of the dimerized EGF-EGFR complex. Once inside the cell, the complex is first sorted into the early endosome. As the early endosome matures, ESCRT proteins, or endosomal sorting complexes required for transport, translocate&#160; the EGFR-EGF complex into intraluminal vesicles (ILVs). Several ILVs are formed inside the maturing endosome, eventually becoming multivesicular bodies (MVBs). MVBs containing the EGF-EGFR complex then fuses with a lysosome for degradation of the EGFR-EGF complex.
Similar to EGFR, cytokine receptors are also internalized and downregulated by MVBs. Ubiquitinated receptor-cytokine complexes are endocytosed and later delivered to MVBs for degradation. The degradation weakens the cytokine signaling. In the case of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF&#593;) or interleukin-1 (IL-1), any defect in ubiquitination, endocytosis, or degradation of the receptor-cytokine complex through MVB can cause enhanced or sustained signaling. This may lead to inflammatory diseases like Crohn&#39;s disease or rheumatoid arthritis.

Regulação Negativa do Receptor em MVBs

Multivesicular bodies (MVBs) are mature endosomes that sort ubiquitinated proteins and then fuse with lysosomes to degrade the sorted proteins. Epidermal ...

Export of Misfolded Proteins out of the ER

After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for transport to their final destination, while misfolded proteins are held back in the ER lumen. The ER chaperones attempt to unfold and refold the misfolded proteins but sometimes fail to achieve the correct native conformation. Such terminally misfolded proteins are then exported to the cytosol by ER-associated degradation or ERAD pathway for degradation.
Different modes of ERAD pathways
Hrd1 is a ubiquitin ligase and a retro-translocation channel for misfolded polypeptides. It complexes with another membrane protein, Hrd3, to form the core ERAD machinery. The other players associated with these core proteins depend upon the topology of the misfolded protein. The retrotranslocation complex depends on the luminal lectin chaperones to identify misfolded proteins. Studies in the yeast system suggest the presence of ERAD-C, ERAD-L, and ERAD-M pathways to export misfolded proteins from various parts of the ER. The target proteins of the ERAD-L and ERAD-M pathways use the Hrd1 channel to exit the ER lumen and membrane, respectively, while the ERAD-C pathway utilizes the Doa10p ubiquitin ligase complex to degrade the cytoplasmic domains of the ER membrane proteins. Sometimes, these pathways can overlap as some misfolded proteins need translocation machinery from two different ERAD pathways to exit the ER. The mammalian ERAD machinery is more complex than yeast and has multiple proteins that export and mark proteins for proteasomal degradation. Eventually, all ERAD pathways converge due to their dependence on a cytoplasmic AAA-ATPase to provide the mechanical force to extract proteins through the membrane.
Degradation of non-glycosylated proteins
The ER has another lectin-independent system that monitors and clears the non-glycosylated proteins from ER. It consists of the resident ER chaperone, BiP protein, and the ER-localized DnaJ family members (ERdjs). ER stress induces the production of HERP, a ubiquitin-like membrane protein, which assembles with other ERAD proteins like the Hrd1 and AAA-ATPase, forming a complex. BiP uses some ERdj proteins as cofactors to recruit non-glycosylated misfolded proteins to the HERP complex for retrotranslocation and ubiquitination of the misfolded protein. HERP is associated with the proteasome on the cytosolic side, where it delivers the misfolded proteins for degradation.

Exportação de Proteínas Mal Dobradas para Fora do ER

After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for ...

Transporte Transmembranar no Retículo Endoplasmático e Peroxissomos

Antigen Presenting Cells

The immune system is a complex network of cells and molecules that protects the body from foreign invaders. T cells, a type of white blood cell, play a crucial role in this process. They recognize and attack foreign substances, such as pathogens, that enter the body.
T cells require the help of antigen-presenting cells (APCs), which process foreign antigens into smaller fragments that can be recognized by T cells. These APCs are highly specialized cells that efficiently internalize antigens through phagocytosis or receptor-mediated endocytosis.
Dendritic cells, a type of professional APC, are particularly efficient at antigen processing and presentation. They are highly mobile and can be found in tissues first exposed to foreign antigens, such as the skin, mucosal surfaces, and lymphoid organs.
Macrophages may also function as APCs, and are found in all tissues. They can engulf and process foreign antigens and release cytokines, which activate T cells and other immune cells, thereby coordinating the immune response.
B cells are another type of APC that can present antigens to T cells. When an antigen is presented to a B cell, it activates the B cell to produce antibodies against the antigen. This activation process involves the binding of the antigen to the B cell receptor, followed by a series of molecular signaling events that produce antibodies.
When an APC engulfs an antigen, it breaks into smaller fragments and combines them with major histocompatibility complex (MHC) molecules. The antigen-MHC complex is then transported to the APC&#39;s plasma membrane, where it is presented to a T cell.
The appropriate T cell receptor recognizes the processed antigen complex, triggering an immune response against the foreign invader. This process is crucial for protecting the body from infection and disease.

Células Apresentadoras de Antígenos

Antigen-presenting cells, also known as APCs, process foreign antigens, such as fragments from pathogens, for recognition by T cells.
When an APC engulfs ...