Name: analysis of scap n-glycosylation and trafficking in human cells
Uploaded: 2016-11-08T14:00:00
Description: Watch this Scientific Journal Video about Analysis of SCAP N-glycosylation and Trafficking in Human Cells at JoVE.com

We are grateful to Drs. Mike S. Brown and Joseph L. Goldstein for their agreement to publish this method according to the methods described in their publications. We appreciate Dr. Peter Espenshade for sharing the GFP-SCAP plasmid. This work was supported by NIH grants NS072838 and NS079701 to D.G., American Cancer Society Research Scholar Grant RSG-14-228-01-CSM to D.G., and OSUCCC Pelotonia Postdoctoral Fellowship to C.C. We also appreciate the support from the Ohio State Neuroscience Core (P30 NS038526) and OSUCCC Translational Therapeutic Program seed grant and start-up funds to D.G.

1. Détection de Endogène SCAP protéine dans les cellules humaines <ol><li> Culture cellulaire et traitement <ol><li> Cellules de graine ~ 1 × 10 6 U87 dans une boîte de 10 cm avec du milieu Eagle modifié par Dulbecco (DMEM) supplémenté avec du sérum de 5% de fœtus bovin (FBS) et incuber les cellules à 37 ° C et 5% de CO 2 pendant 24 heures avant le traitement. </li><li> Laver les cellules une fois avec un tampon phosphate salin (PBS), puis passer les cellules dans un milieu de DMEM fr...

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insig-2,+a+second+endoplasmic+reticulum+protein+that+binds+SCAP+and+blocks+export+of+sterol+regulatory+element-binding+proteins.">Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins.</a> Proc Natl Acad Sci U S A. 99, 12753-12758 (2002).</li><li>Yabe, D., Xia, Z. P., Adams, C. M., Rawson, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+mutations+in+sterol-sensing+domain+of+SCAP+block+interaction+with+insig+and+render+SREBP+cleavage+insensitive+to+sterols.">Three mutations in sterol-sensing domain of SCAP block interaction with insig and render SREBP cleavage insensitive to sterols.</a> Proc Natl Acad Sci U S A. 99, 16672-16677 (2002).</li><li>Espenshade, P. J., Li, W. 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Dans cette étude, nous décrivons un protocole pour l&#39;isolement des fractions membranaires des cellules humaines et pour la préparation des échantillons pour la détection de SCAP N -glycosylation et la protéine totale en utilisant Western Blot. Nous fournissons en outre un procédé pour surveiller le trafic de SCAP en utilisant GFP-étiquetage et la microscopie confocale. La méthode est spécifiquement utilisé pour analyser la protéine de la membrane et est un outil important pour étudier SCAP <em...

La dérégulation du métabolisme des lipides est une caractéristique commune des maladies cancéreuses et métaboliques 1-6. Dans ces procédés, les protéines de régulation du stérol élément de liaison (SREBPs), une famille de facteurs de transcription, jouent un rôle crucial dans le contrôle de l&#39;expression des gènes importants pour l&#39;absorption et la synthèse du cholestérol, des acides gras et des phospholipides 7-10. SREBPs, y compris SREBP-1a, SREBP-1c, et SREBP-2, sont synthétisés sous forme de précurseurs inactifs qui se lient à la membrane du réticulum endoplasmique (RE) en vertu de deux domaines transmembranaires 7. L&#39; extrémité N-terminale de SREBPs contient un domaine de liaison d&#39;ADN pour la transactivation des gènes cibles 11. L&#39;extrémité C-terminale des précurseurs de SREBP se lie à SREBP clivage protéine d&#39; activation (SCAP) 12,13, une protéine membranaire polytopic qui joue un rôle central dans la régulation de la stabilité et de l&#39; activation 14-16 SREBP. La mise enla mise en fonction de la transcription nécessite la translocation du complexe SCAP / SREBP du RE à l&#39;appareil de Golgi, où deux proteases clivent séquentiellement SREBP et la libération de son fragment N-terminal, qui pénètre alors dans le noyau pour la transactivation des gènes lipogenèse 7. Dans ces procédés, les niveaux de stérols dans la membrane du RE contrôlent la sortie du complexe SCAP / SREBP du RE 7,17. Dans des conditions de stérol élevées stérol se lie à SCAP ou ER-résident insuline induite par la protéine du gène-1 (Insig-1) ou 2 (Insig-2), l&#39;amélioration de l&#39;association des SCAP avec Insigs qui maintiennent le complexe SCAP / SREBP dans le ER 18-20. Lorsque les niveaux de stérols diminuent, SCAP dissocie avec Insigs. Cela conduit à un changement conformationnel SCAP, ce qui permet une interaction avec SCAP protéines d&#39;enveloppe communes complexe (COP) II. Le complexe médie l&#39;incorporation du complexe SCAP / SREBP dans les vésicules en herbe et dirige son transport de l&#39;ER au Golgi 21,22. Sur TransLocation du Golgi, SREBPs sont séquentiellement clivés par Site-1 et Site-2 protéases, conduisant à la libération de l&#39;extrémité N-terminale 7,23-29. Protéines SCAP porte trois N oligosaccharides liés en au asparagine (N) positionne N263, N590, N641 et 15. Nous avons récemment révélé que le glucose médiée N -glycosylation de SCAP dans ces sites est une condition préalable à SCAP / SREBP traite de l&#39;ER au Golgi dans des conditions de stérol bas 30-32. Perte de SCAP glycosylation par mutation de tous les trois asparagine en glutamine (NNN à QQQ) désactive le trafic de / complexe et les résultats de SREBP SCAP dans l&#39;instabilité de la protéine et de la réduction de l&#39; activation de SREBP 31 SCAP. Nos données récentes démontrent également que SREBP-1 est fortement activée dans le glioblastome et réglementé par SCAP N -glycosylation 4,31,33,34. Ciblage SCAP / SREBP-1 signalisation est en train de devenir une nouvelle stratégie pour traiter les tumeurs malignes et métaboliques syndromes 1,3,35-38. Par conséquent, il est important de développer une méthode efficace pour analyser les niveaux de protéines SCAP et N -glycosylation et de surveiller son trafic de cellules et de tissus humains de patients. La région luminale des protéines SCAP (acides aminés 540-707) dans le ER contient deux sites de -glycosylation N (N590 et N641) qui sont protégés de la protéolyse lorsque les membranes sont intactes sont traitées avec de la trypsine 15. Ce fragment a luminale a une masse moléculaire de ~ 30 kDa qui est suffisamment petite pour permettre la résolution des variants glycosylés individuels de SCAP par dodécylsulfate de sodium gel de polyacrylamide (SDS-PAGE) 15,31. Ici, nous fournissons une méthode permettant de détecter la N -glycosylation de PAP et la protéine totale dans les cellules humaines. Ce protocole est dérivé du procédé décrit dans les publications du laboratoire Brown et Goldstein 15 et notre publication récente 31. Le protocole peut être utilisé en til étudie des protéines SCAP à partir de cellules de mammifères.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">X-treme GENE HP DNA Transfection Reagent&#160;</td>
			<td style="width:163px;">Roche</td>
			<td style="width:123px;">6366236001</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEMI medium&#160;</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trypsin&#160;</td>
			<td>Sigma</td>
			<td>T6567</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PNGase F&#160;</td>
			<td>Sigma</td>
			<td>P7367</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SCAP (9D5) antibody&#160;&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-13553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP antibody&#160;</td>
			<td>Roche</td>
			<td>11814460001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SREBP-1 antibody (IgG-2A4)</td>
			<td>BD Pharmingen</td>
			<td>557036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDI Antibody (H-17)</td>
			<td>Santa Cruz</td>
			<td style="width:123px;">sc-30932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lamin A Antibody (H-102)</td>
			<td>Santa Cruz</td>
			<td style="width:123px;">sc-20680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SCAP antibody (a.a 450-500)</td>
			<td>Bethyl Laboratories, Inc.</td>
			<td>A303-554A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) without glucose, pyruvate and glutamine&#160;&#160;</td>
			<td>Cellgro</td>
			<td>17-207-CV</td>
			<td>Add 1 Mm Pyravate and 4 mM Glutamine before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22G x 1 1/2 needle&#160;</td>
			<td>BD&#160;</td>
			<td>305156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pepstatin A</td>
			<td>Sigma</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>Sigma</td>
			<td>43819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ALLN</td>
			<td>Sigma</td>
			<td>A6185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose membrane</td>
			<td>GE</td>
			<td>RPN3032D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA Solution 0.5 M PH8.5 100 ml&#160;</td>
			<td>VWR</td>
			<td>82023-102&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA 0.5 M sterile (PH 8.0) 50 ml</td>
			<td>Fisher Scentific</td>
			<td>50255956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HyClone FBS</td>
			<td>Thermo scientific</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol&#160;</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bromophenol blue</td>
			<td>Sigma</td>
			<td>B8026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">prolong gold antifade reagent with dapi</td>
			<td>life technologies</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine (200 mM)</td>
			<td>life technologies</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium pyruvate</td>
			<td>life technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of scap n-glycosylation and trafficking in human cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

La figure 1 montre la détection d&#39; une protéine endogène et SCAP SREBP-1 sous forme nucléaire dans les cellules du glioblastome U87 humains en réponse à une stimulation de glucose en utilisant Western Blot. SCAP protéine membranaire est détectée dans la «fraction membranaire». La forme de noyau (N-terminal) de SREBP-1 est détectée dans les «extraits nucléaires». Le niveau de protéine SCAP (PDI sert de contrôle interne des protéines de la memb...

Watch this Scientific Journal Video about Analysis of SCAP N-glycosylation and Trafficking in Human Cells at JoVE.com

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Nous décrivons un procédé modifié pour l&#39; isolement de la fraction membranaire à partir de cellules humaines et la préparation des échantillons pour la détection de SCAP N -glycosylation et la protéine totale en utilisant Western Blot. Nous présentons en outre un procédé GFP-étiquetage pour surveiller le trafic SCAP utilisant la microscopie confocale. Ce protocole peut être utilisé dans les laboratoires de biologie réguliers.

Analyse des SCAP<em&gt; N</em&gt; -glycosylation Et la traite des cellules humaines

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

Research

JoVE Journal

Biology

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Vidéo analyse bioinformatique des droits de croissance de la tige colonie de cellules embryonnaires

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Recherche

Biologie

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Analyse phénotypique et isolement de cellules souches hématopoïétiques murines et Lineage-engagés progéniteurs

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

ARN-seq Analyse des transcriptomes de thrombine traitée et de contrôle de l&#39;homme des cellules endothéliales microvasculaires pulmonaires

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Induction et analyse des épithéliale à mésenchymateuses transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

Génération efficace d&#39;origine humaine souches pluripotentes à partir de cellules somatiques humaines avec Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Profilage individuel embryonnaires humaines Cellules souches par RT-PCR quantitative

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

Suivi des modifications induites par la drogue dans des récepteurs post-internalisation traite par Analyse Colocalizational

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

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The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

En direct d&#39;imagerie cellulaire et l&#39;analyse 3D des antagonistes des récepteurs de type 1a traite des transfectées embryonnaires humaines cellules rénales Utilisation Microscopie confocale

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Une interface utilisateur graphique pour le suivi assisté par logiciel de la concentration de protéines dans les protrusions cellulaires dynamiques

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...