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 av Endogen SCAP Protein i humane celler <ol><li> Cellekultur og behandling <ol><li> Seed ~ 1 x 10 6 U87-celler i en 10 cm skål med Dulbeccos modifiserte Eagle-medium (DMEM) supplert med 5% føtalt bovint serum (FBS) og inkubere cellene ved 37 ° C og 5% CO2 i 24 timer før behandling. </li><li> Vask cellene en gang med fosfat-bufret saltvann (PBS), deretter skifte cellene til friskt DMEM-medium med eller uten glukose (5 mM) i 12 timer. </li></ol></li><li> Utarbeidelse av cellemembr...

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I denne studien, beskriver vi en protokoll for isolering av membranfraksjoner i humane celler og for fremstilling av prøver for påvisning av SCAP N -glycosylation og totalt protein ved hjelp av western blot. Vi gir videre en metode for å overvåke SCAP trafficking ved hjelp av GFP-merking og konfokalmikroskopi. Metoden er spesielt brukt til å analysere membran protein og er et viktig verktøy for å undersøke SCAP N -glycosylation og trafficking. Sammenlignet med metod...

Deregulering av lipid metabolisme er et fellestrekk for kreft og metabolske sykdommer 1-6. I disse prosesser sterol regulerende element-bindende proteiner (SREBPs), en familie av transkripsjonsfaktorer, spiller en kritisk rolle i å kontrollere ekspresjonen av gener som er viktige for opptak og syntese av kolesterol, fettsyrer, fosfolipider og 7-10. SREBPs, inkludert SREBP-1a, SREBP-1c, og SREBP-2, syntetiseres som inaktive forløpere som binder seg til det endoplasmatiske retikulum (ER) membran i kraft av to transmembranområdene 7. Den N-terminale ende av SREBPs inneholder et DNA-bindende domene for transaktivering av målgener 11. C-terminalen av SREBP forløpere binder seg til SREBP cleavage aktiverende protein (SCAP) 12,13, en polytopic membran protein som spiller en sentral rolle i reguleringen av SREBP stabilitet og aktivering 14-16. det iverktering av transkripsjonen funksjon krever translokasjon av den SCAP / SREBP komplekset fra ER til Golgi, hvor to proteaser sekvensielt spalter SREBP og slipper sin N-terminale fragment, som deretter trer inn i kjernen for transaktivering av lipogenic gener 7. I disse prosessene, nivåene av steroler i ER membranen kontrollere avkjørselen til SCAP / SREBP kompleks fra ER 7,17. Under høye sterol forhold, binder sterol å SCAP eller ER-resident insulin-indusert gen protein-1 (Insig-1) eller 2 (Insig-2), styrke foreningen av SCAP med Insigs som beholder SCAP / SREBP kompleks i ER 18-20. Når sterol nivåer reduseres, dissosierer SCAP med Insigs. Dette fører til en SCAP konformasjonsendring som gjør det mulig SCAP interaksjon med vanlige kappeproteiner (COP) ll-komplekset. Komplekset formidler inkorporering av SCAP / SREBP komplekset i spirende vesikler og retter sin transport fra ER til Golgi 21,22. Ved translocasjon til Golgi er SREBPs sekvensielt spaltes av Site-en og Site-2 proteaser, som fører til utgivelsen av den N-terminale 7,23-29. SCAP protein bærer tre N -bundne oligosakkarider på asparagin (N) posisjonerer N263, N590 og N641 15. Vi har nylig avdekket at glukose-mediert N -glycosylation av SCAP i disse områdene er en forutsetning for SCAP / SREBP menneskehandel fra ER til Golgi under lave sterol forhold 30-32. Tap av SCAP glykosylering via mutasjon av alle tre asparagin til glutamin (NNN til qqq) deaktiverer trafficking av SCAP / SREBP kompleks og resulterer i ustabilitet SCAP protein og reduksjon av SREBP aktivering 31. Våre nyeste dataene viser også at SREBP-en er sterkt aktivert i glioblastom og regulert av SCAP N -glycosylation 4,31,33,34. Målrette SCAP / SREBP-en signalfremstår som en ny strategi for å behandle ondartede sykdommer og metabolske syndromes 1,3,35-38. Derfor er det viktig å utvikle en effektiv metode for å analysere SCAP protein og N -glycosylation nivåer og overvåke handel med menneskeceller og pasient vev. Den luminale delen av SCAP protein (aminosyrene 540-707) i ER inneholder to N -glycosylation steder (N590 og N641) som er beskyttet fra proteolyse når intakte membraner blir behandlet med trypsin 15. Denne luminal fragmentet har en molekylvekt på ~ 30 kDa som er liten nok til å tillate oppløsning av individuelle glykosylerte varianter av SCAP av natriumdodecylsulfat polyakrylamidgel-elektroforese (SDS-PAGE) 15,31. Her gir vi en metode for å påvise N -glycosylation av SCAP og det totale protein i humane celler. Denne protokollen er avledet fra fremgangsmåten som er beskrevet i publikasjonene fra Brown og Goldstein laboratorium 15 og vår nylig publikasjon 31. Protokollen kan brukes i tHan studerer for SCAP protein fra pattedyrceller.

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			<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.

Figur 1 viser påvisningen av endogent SCAP protein og SREBP-en kjerneform i human glioblastoma U87-celler i respons til glukose stimulering ved hjelp av western blot. Den membranprotein SCAP detekteres i &quot;membranfraksjonen&quot;. Kjernen form (N-terminal) av SREBP-en er oppdaget i &quot;kjerneekstrakter&quot;. Nivået av SCAP protein (PDI tjener som en intern kontroll av ER membranproteiner) og SREBP-en kjerneform markert forbedret med glukose stimulering. Di...

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 modifisert metode for membranfraksjon isolert fra humane celler og prøvepreparering for påvisning av SCAP N -glycosylation og totalt protein ved hjelp av western blot. Vi introduserer ytterligere en GFP-merking metode for å overvåke SCAP trafficking ved hjelp konfokalmikroskopi. Denne protokollen kan brukes i vanlige biologiske laboratorier.

Analyse av 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

Fenotypeanalyser og Isolering av murine hematopoetiske stamceller og Lineage-engasjerte stamfedre

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 av Transcriptomes i Thrombin-behandlet og Kontroll Menneskelig Pulmonal mikrovaskulær 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

Induksjon og analyse av Epiteliale 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 Menneskelig indusert pluripotent stamceller fra menneskelige 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 Individuell 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 Drug-induced Endringer i Receptor Post-intern Trafficking etter 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

Høy oppløsning Time-lapse Imaging og automatisk analyse av mikrotubulidynamikk i levende menneske navleveneendotel Cells

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

Live-Cell Imaging og 3D Analyse av angiotensin reseptor type 1a Trafficking i transfekterte humane embryonale nyreceller ved hjelp 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 brukergrensesnitt for programvareassistert sporing av proteinkonsentrasjon i dynamiske cellulære protrusjoner

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