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Abstract
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Biology

SCAP分析<em>ñ</em> -glycosylation和贩卖人体细胞

Published: November 08, 2016
doi:
10.3791/54709
, , , , , ,
1Department of Radiation Oncology,The Ohio State University Comprehensive Cancer Center and College of Medicine

Summary

我们通过使用蛋白质印迹描述了从人类细胞的膜级分的分离和样品制备用于检测SCAPÑ-glycosylation和总蛋白的改进方法。我们还引进了GFP标记法使用共聚焦显微镜监测SCAP贩卖。这种协议可以在常规生物学实验室使用。

Abstract

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.

Introduction

脂质代谢的失调是癌症和代谢性疾病1-6的一个共同特点。在这些方法中，固醇调节元件结合蛋白（SREBPs），一个家族的转录因子，在控制基因的摄取和胆固醇，脂肪酸的合成，和磷脂7-10重要的表达中起关键作用。

SREBPs，包括SREBP-1a中，SREBP-1c和SREBP-2，合成为凭借两个跨膜结构域7的结合于内质网（ER）膜活性的前体。 SREBPs的N-末端包含靶基因11的转录的DNA结合结构域。 SREBP前体的C末端结合至SREBP裂解激活蛋白（SCAP）12,13，即起着SREBP稳定性和活化14-16的调节中发挥关键作用一个多面体膜蛋白。

该implemen转录功能的塔季翁需要从ER到高尔基体，其中两个蛋白酶顺序裂解SREBP并释放其N-末端片段，然后进入细胞核为生脂基因7的反式激活的SCAP / SREBP复合物的转运。在这些过程中，甾醇的在ER膜的水平从ER 7,17控制SCAP / SREBP复合物的出口。下高固醇的条件下，固醇结合SCAP或ER驻留胰岛素诱导的基因蛋白-1（INSIG-1）或-2（INSIG-2），从而增强与保留SCAP / SREBP复杂的Insigs SCAP的关联ER 18-20。当固醇水平降低，SCAP解离与Insigs。这导致了SCAP构象变化，这允许具有共同的外壳蛋白（COP）的二复杂SCAP相互作用。复合介导SCAP / SREBP复合物的掺入出芽囊泡和从ER到高尔基体21,22指示其传输。当transloc通货膨胀到高尔基体，SREBPs依次由站点1和站点2蛋白酶裂解，导致N末端7,23-29的释放。

SCAP蛋白携带三个N天冬酰胺-连接低聚糖（N）位置N263，N590，N641和15。最近，我们发现，在这些网站SCAP的葡萄糖介导ñ-glycosylation是从低固醇条件下30-32内质网向高尔基SCAP / SREBP贩卖的先决条件。通过以谷氨酰胺（NNN到QQQ）所有三个天冬酰胺的突变SCAP糖基化的损失禁用SCAP / SREBP复杂并且导致SCAP蛋白和减少SREBP活化31的不稳定的贩卖。我们最近的数据还表明，SREBP-1在胶质母细胞瘤是高度激活和SCAPñ-glycosylation 4,31,33,34调节。针对SCAP / SREBP-1信号正在成为一个新的战略来治疗恶性肿瘤和代谢小号yndromes 1,3,35-38。因此，开发以分析SCAP蛋白和N -glycosylation水平和监测其在人类细胞和患者组织贩卖一种有效的方法是重要的。

SCAP蛋白质（氨基酸540-707）在ER腔区域包含了保护，当蛋白水解胎膜与胰蛋白酶处理15两个N -glycosylation网站（N590和N641）。这个管腔片段的分子量〜30kDa的的足够小，以允许通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）15,31的SCAP的个别糖基化变体的分辨率。这里，我们提供了用于检测SCAP 氮 -glycosylation和人类细胞中的总蛋白的方法。此协议是从由布朗和戈尔茨坦实验室15和我们最近出版31出版物中描述的方法的。该协议可以在T使用他从哺乳动物细胞SCAP蛋白的研究。

Protocol

1.内源性的检测SCAP蛋白在人类细胞中细胞培养和治疗在10cm皿用Dulbecco补充有5％胎牛血清（FBS）的改良Eagle培养基（DMEM）种子〜1×10 6个 U87细胞和孵育所述细胞在37℃和5％CO 2的处理前24小时。 用磷酸盐缓冲盐水（PBS）洗一次细胞，然后切换细胞新鲜DMEM培养基有或没有葡萄糖（5毫米）12小时。 细胞膜组份和核提取物的制备洗涤细胞一次，用PB…

Representative Results

图1示出了响应于人脑胶质瘤U87细胞内源性SCAP蛋白和SREBP-1的核形式的检测通过使用免疫印迹对葡萄糖刺激。膜蛋白SCAP在“膜部分”检测的。 SREBP-1的核形式（N末端）中的“核提取物”被检测。 SCAP蛋白的水平（PDI作为ER膜蛋白内部对照）和SREBP-1的核形式由葡萄糖刺激都显着增强。这些结果表明，该协议能够检测在人类细胞中的内源性SCAP蛋白。 <p class="…

Discussion

在这项研究中，我们描述了在人细胞中膜部分的分离和用于通过使用免疫印迹制备样品用于检测SCAPÑ-glycosylation和总蛋白的一个协议。我们还提供采用GFP标记和共聚焦显微镜监测SCAP贩卖的方法。该方法是专门用于分析膜蛋白是研究SCAPñ-glycosylation和贩卖人口的重要工具。

使用各种凝集素识别不同氮肥 -glycan链，并确定ñ糖基蛋白42的方法相比?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

Keywords: SCAPN-glycosylationTraffickingHuman CellsSREBPLipid MetabolismPNGaseU87 CellsCell Membrane FractionsNuclear ExtractsSDS-PAGEWestern BlotBradford Protein Assay
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Cheng, C., Guo, J. Y., Geng, F., Wu, X., Cheng, X., Li, Q., Guo, D. Analysis of SCAP N-glycosylation and Trafficking in Human Cells. J. Vis. Exp. (117), e54709, doi:10.3791/54709 (2016).
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