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. Påvisning af endogene SCAP protein i humane celler <ol><li> Celledyrkning og behandling <ol><li> Frø ~ 1 × 10 6 U87-celler i en 10 cm skål med Dulbeccos modificerede Eagle-medium (DMEM) suppleret med 5% føtalt bovint serum (FBS) og inkuber cellerne ved 37 ° C og 5% CO2 i 24 timer før behandling. </li><li> Vask cellerne én gang med phosphatbufret saltvand (PBS) og derefter skifte celler til frisk DMEM-medium med eller uden glucose (5 mM) i 12 timer. </li></ol></li><li> Fremstilling af C...

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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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+step+in+cholesterol+feedback+localized+to+budding+of+SCAP+from+ER+membranes.">Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes.</a> Cell. 102, 315-323 (2000).</li><li>Sakai, 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=Sterol-regulated+release+of+SREBP-2+from+cell+membranes+requires+two+sequential+cleavages,+one+within+a+transmembrane+segment.">Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment.</a> Cell. 85, 1037-1046 (1996).</li><li>Wang, X., Sato, R., Brown, M. S., Hua, X., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SREBP-1,+a+membrane-bound+transcription+factor+released+by+sterol-regulated+proteolysis.">SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis.</a> Cell. 77, 53-62 (1994).</li><li>Brown, M. S., Goldstein, J. 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+proteolytic+pathway+that+controls+the+cholesterol+content+of+membranes,+cells,+and+blood.">A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.</a> Proceedings of the National Academy of Sciences of the United States of America. 96, 11041-11048 (1999).</li><li>Rawson, R. B., DeBose-Boyd, R., Goldstein, J. L., Brown, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+to+cleave+sterol+regulatory+element-binding+proteins+(SREBPs)+causes+cholesterol+auxotrophy+in+Chinese+hamster+ovary+cells+with+genetic+absence+of+SREBP+cleavage-activating+protein.">Failure to cleave sterol regulatory element-binding proteins (SREBPs) causes cholesterol auxotrophy in Chinese hamster ovary cells with genetic absence of SREBP cleavage-activating protein.</a> J Biol Chem. 274, 28549-28556 (1999).</li><li>Zelenski, N. G., Rawson, R. B., Brown, M. S., Goldstein, J. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocatalytic+processing+of+site-1+protease+removes+propeptide+and+permits+cleavage+of+sterol+regulatory+element-binding+proteins.">Autocatalytic processing of site-1 protease removes propeptide and permits cleavage of sterol regulatory element-binding proteins.</a> The Journal of biological chemistry. 274, 22795-22804 (1999).</li><li>Guo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCAP+links+glucose+to+lipid+metabolism+in+cancer+cells.">SCAP links glucose to lipid metabolism in cancer cells.</a> Mol Cell Oncol. 3, (2016).</li><li>Cheng, 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=Glucose-Mediated+N-glycosylation+of+SCAP+Is+Essential+for+SREBP-1+Activation+and+Tumor+Growth.">Glucose-Mediated N-glycosylation of SCAP Is Essential for SREBP-1 Activation and Tumor Growth.</a> Cancer Cell. 28, 569-581 (2015).</li><li>Shao, W., Espenshade, P. 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I denne undersøgelse beskriver vi en protokol til isolering af membran fraktioner i humane celler og til udarbejdelse af prøverne til påvisning af SCAP N -glycosylation og total protein ved hjælp af Western blot. Vi tilbyder endvidere en metode til at overvåge SCAP handel ved hjælp af GFP-mærkning og konfokal mikroskopi. Den metode anvendes til at analysere membranprotein og er et vigtigt redskab til at undersøge SCAP N -glycosylation og menneskehandel. Sammenlignet...

Deregulering af lipid metabolisme er et fælles kendetegn for kræft og metaboliske sygdomme 1-6. I disse processer sterol regulatorisk element-bindende proteiner (SREBPs), en familie af transkriptionsfaktorer, spiller en kritisk rolle i at kontrollere ekspressionen af gener vigtige for indførelsen og syntesen af cholesterol, fedtsyrer og phospholipider 7-10. SREBPs, herunder SREBP-1a, SREBP-1c, og SREBP-2, syntetiseres som inaktive forstadier, der binder til det endoplasmatiske reticulum (ER) membran i kraft af to transmembrandomæner 7. N-terminalen af SREBPs indeholder et DNA-bindende domæne for transaktivering af målgener 11. Den C-terminale ende af SREBP forstadier bindes til SREBP spaltning-aktiverende protein (SCAP) 12,13, en polytopic membran protein, der spiller en central rolle i reguleringen af SREBP stabilitet og aktivering 14-16. Den gennemførelførelsen af transkriptionel funktion kræver translokation af SCAP / SREBP kompleks fra ER til Golgi, hvor to proteaser sekventielt spalte SREBP og frigive dets N-terminalt fragment, som derefter træder ind i kernen for transaktivering af lipogene gener 7. I disse processer niveauerne af steroler i ER-membranen styre afgangen fra SCAP / SREBP kompleks fra ER 7,17. Under høje sterol betingelser, sterol binder til SCAP eller ER-resident insulin-induceret gen-protein-1 (INSIG-1) eller -2 (INSIG-2), øge associeringen af ​​SCAP med Insigs der bevarer SCAP / SREBP kompleks i ER 18-20. Når sterol niveauet falder, SCAP dissocieres med Insigs. Dette fører til en SCAP konformationel ændring, som tillader SCAP interaktion med fælles kappeproteiner (COP) II-komplekset. Komplekset medierer inkorporeringen af SCAP / SREBP kompleks i den spirende vesikler og dirigerer sin transport fra ER til Golgi 21,22. Ved translocation til Golgi, er SREBPs sekventielt spaltes af anlægsområde 1 og site-2 proteaser, hvilket fører til frigivelse af N-terminalen 7,23-29. SCAP protein bærer tre N-forbundet oligosaccharider på asparagin (N) positioner N263, N590, og N641 15. Vi viste for nylig, at glucose-medieret N -glycosylation af SCAP i disse steder er en forudsætning for SCAP / SREBP handel fra ER til Golgi under lave sterol betingelser 30-32. Tab af SCAP glykosylering via mutation af alle tre asparagin til glutamin (NNN til QQQ) deaktiverer handelen med det SCAP / SREBP kompleks og resulterer i ustabilitet SCAP protein og reduktion af SREBP aktivering 31. Vores seneste data viser også, at SREBP-1 er stærkt aktiveret i glioblastom og reguleret af SCAP N -glycosylation 4,31,33,34. Målretning SCAP / SREBP-1 signalering fremstår som en ny strategi til at behandle maligniteter og metaboliske syndromes 1,3,35-38. Derfor er det vigtigt at udvikle en effektiv metode til at analysere SCAP protein og N -glycosylation niveauer, og overvåge dens handel med humane celler og patientens væv. Den luminale region SCAP protein (aminosyrer 540-707) i ER indeholder to N -glycosylation steder (N590 og N641), der er beskyttet mod proteolyse når intakte membraner behandles med trypsin 15. Denne luminale fragmentet har en molekylvægt på ~ 30 kDa, der er lille nok til at tillade opløsningen af individuelle glycosylerede varianter af SCAP ved natriumdodecylsulfat polyacrylamid gel elektroforese (SDS-PAGE) 15,31. Her giver vi en metode til at påvise N -glycosylation af SCAP og det totale protein i humane celler. Denne protokol er afledt af den beskrevet i publikationer fra Brown og Goldstein laboratorium 15 og vores nylige publikation 31-metoden. Protokollen kan bruges i than studerer af SCAP protein fra pattedyrceller.

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			<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.

Figur 1 viser påvisningen af endogent SCAP protein og SREBP-1 nuklear udgave i humane glioblastom U87 celler som respons på glucose stimulation ved anvendelse af Western-blot. Membranproteinet SCAP detekteres i &quot;membranfraktionen&quot;. Kernen formular (N-terminal) af SREBP-1 detekteres i &quot;kerneekstrakter&quot;. Niveauet af SCAP protein (PDI tjener som en intern kontrol af ER membranproteiner) og SREBP-1 nuklear formular er markant forbedret ved glucose...

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

Vi beskriver en modificeret fremgangsmåde til membranfraktion isoleret fra humane celler og prøveforberedelse til påvisning af SCAP N -glycosylation og totalt protein ved hjælp af Western blot. Vi yderligere indføre en metode GFP-mærkning for at overvåge SCAP menneskehandel hjælp konfokal mikroskopi. Denne protokol kan bruges i almindelige biologiske laboratorier.

Analyse af SCAP<em&gt; N</em&gt; -glycosylation Og handel med humane celler

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

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 ...

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

Fænotypisk analyse og isolering af murine hæmatopoietiske stamceller og Lineage-progenitorer

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

RNA-seq Analyse af Transcriptomes i behandlet Thrombin-og humant kontrol Pulmonal mikrovaskulære endotelceller

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

Induktion og analyse af Epithelial til Mesenchymale 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

Effektiv Generation Menneskelige induceret pluripotente stamceller fra humane somatiske celler med 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

Profilering Individuelle humane embryonale stamceller ved kvantitativ RT-PCR

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

Sporing lægemiddelinduceret Ændringer i Receptor Post-internalisering Trafficking af Colocalizational Analysis

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

Høj opløsning Time-lapse Imaging og Automatiseret analyse af mikrotubulusdynamik i levende menneske navlestrengsveneendotelceller

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

Levende celler og 1a 3D Analyse af angiotensin receptor type handel i transfekterede humane embryoniske nyreceller Brug konfokalmikroskopi

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

Et grafisk brugergrænseflade til softwareassisteret sporing af proteinkoncentration i dynamiske cellulære protrusioner

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