磷脂Scramblase活动的基于荧光的测定法
Summary
We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.
Abstract
Scramblases易位穿过膜双层磷脂双向在一个ATP无关的方式。第一scramblase被识别和生化核实被视蛋白，感光体的视紫红质的脱辅基蛋白。视紫红质是在它负责的光的感知的视网膜视杆圆盘膜局部的G蛋白偶合受体。视紫红质的scramblase活动并不取决于其配体11- 顺 -retinal， 即载脂蛋白视蛋白还积极为scramblase。虽然组成型和调节磷脂扰在细胞生理学中起重要作用，只有少数磷脂scramblases迄今已除了视蛋白鉴定。这里，我们描述视蛋白的scramblase活性的基于荧光的测定法。视蛋白被重组到磷脂酰胆碱，磷脂酰甘油和荧光NBD标记的PC（1-p的微量组成的大单层脂质体almitoyl -2- {6- [7-硝基2-1,3苯并恶-4-基）氨基]己酰基} – SN -glycero -3-磷酸胆碱）。 Scramblase活性通过测量到位于囊泡的内小叶NBD-PC分子能够访问外部小叶，他们的荧光化学由不能越过膜还原剂消除的程度来确定。我们描述的方法具有普遍的适用性，并可以用于鉴定和表征其他膜蛋白的scramblase活动。
Introduction
感光体视紫红质，一个典型的G蛋白偶联受体（参考文献1中综述例如），要被识别的第一磷脂scramblase和生物化学验证2,3。 Scramblases是磷脂转运会增加transbilayer磷脂运动的本质慢的速度在双向生理上适当水平，ATP无关的方式4-6。他们的行动的实例可于内质网和细菌胞质膜，其中需要对膜稳态和生长构加扰，以及针对各种糖基化途径5中找到。扰码是需要调节磷脂以暴露凋亡细胞的表面上的磷脂酰丝氨酸（PS），其中它充当一个“吃我” -信号为巨噬细胞7和对活化血小板提供一种促凝表面以催化生产蛋白质因子的š所需血液凝固。在感光体光盘的膜，视紫红质的扰频活动已经提出以抵消由ATP依赖，单向脂质产生的双层的两个薄膜小叶之间的磷脂失调翻转酶ABCA4 4,8,9 10-12。
尽管scramblases的生理重要性，它们的身份仍然难以捉摸直到视紫红质被报告为在感光体圆盘一个scramblase 2，TMEM16蛋白家族的成员被确定为钙离子需要在质膜PS暴露依赖性scramblases（在参考审查13），并作为脂类II scramblase所需肽合成14提出的细菌蛋白质FtsW。这些发现是基于纯化的蛋白质的脂质体中使用的方法describ重构和scramblase活性示范在所得脂蛋白体这里编。其他潜在scramblases 15-21 -的MurJ和AMJ蛋白肽聚糖的生物合成牵连，WzxE和相关蛋白牵涉于扰O-抗原的前体，MPRF蛋白需要穿过细菌胞质膜易位氨基酰化磷脂，并且已经提出Xkr8家族成员以暴露的PS凋亡细胞的表面上 – 有待生化测试。这凸显了一个强大的分析的重要性，以确定和识别scramblase活动。
这里，我们描述了纯化的视蛋白的在使用基于荧光的测定法所得到的蛋白脂质体的重建，感光体的视紫红质的脱辅基蛋白，成大单层囊泡（的LUV）和scramblase活性随后的分析。有在文献中用于异源表达和视蛋白的纯化可用几个充分描述的协议，因此，我们将不描述它在这个协议;我们使用这产生FLAG标签，热稳定视蛋白在约100纳克在戈伦等 3所述的协议/微升的0.1％（重量/体积）十二烷基麦芽糖（DDM）。
重构是通过使得它们溶胀但不溶于治疗与足够洗涤剂的LUV实现。在这些条件下，膜蛋白 – 蛋白质 – 去污剂胶束的形式提供 – 将整合入脂质体和成为重建成在洗涤剂除去脂质体膜，导致脂蛋白体。重构视蛋白（作为纯化蛋白得到的0.1％（重量/体积），DDM），的LUV由（1- POPC（1-棕榈酰-2-油酰- SN -glycero -3-磷酸胆碱）和POPG的混合物制备棕榈酰-2-油酰- SN -glycero -3- [磷酸外消旋- （1-甘油）]）和添加视蛋白和NBD-PC之前，用DDM饱和。洗涤剂然后通过用聚苯乙烯珠粒的样品中除去。
<p c小姑娘="“jove_content”">的基于荧光的测定法的基本原理示于图1B。的LUV对称用NBD-PC或其他NBD标记的荧光磷脂记者（ 图1A）的微量重构。上加入连二亚硫酸盐，膜不通透性二价阴离子，在的LUV的外部小叶NBD-PC分子呈现非荧光作为硝基组NBD的被还原成非荧光氨基 – 基团。既不NBD-PC分子也不连二是能够在时间尺度的实验（<10分钟）的遍历膜，这导致荧光信号的减少50％。然而，如果脂质体与scramblase重构，在内小叶NBD-PC分子可以迅速加扰向外部在那里它们被降低。这导致荧光的在理想的情况下（ 图1C）的总损耗。ES / ftp_upload / 54635 / 54635fig1.jpg“/>
图1：scramblase活性测定的示意图的测定使用荧光NBD标记的报告脂质; NBD-PC如图（A）。大单层囊泡重构与NBD-PC的痕量。重组产生对称囊泡，以在外部和内部小叶均匀分布NBD-PC。连二亚硫酸盐（S 2 O 4 2-）化学还原硝基组NBD到一个非荧光氨基-基团。用连二亚硫酸盐（B，上图）无蛋白质的脂质体处理引起，因为只有在该外部小叶的NBD-PC分子荧光的减少50％的降低：连二亚硫酸盐被带负电荷的，并且不能穿过膜与NBD-PC分子反应在内部小叶。含有视蛋白，脂蛋白（B，下），连二亚的治疗即 scramblase活性脂蛋白，导致的F 100％的损失luorescence如视蛋白促进内层和外层小叶之间的NBD-PC的移动。 （C）表示对治疗无蛋白质的脂质体和含有视蛋白-蛋白脂质体与连二亚得到理想化荧光痕迹。荧光损失率是在两种情况下，指示由连二亚化学还原NBD的是限速，并且加扰出现在比化学反应的速率等于或大于一个速率是相同的。从实际实验中获得痕迹如图3所示。 请点击此处查看该图的放大版本。
我们描述的方法可以被用来重建和测定等纯化的蛋白质，以及获得，例如，通过用洗涤剂22提取微粒的膜蛋白的混合物。
Protocol
Representative Results
Discussion
该scramblase活性测定，使我们原来确定的视蛋白具有磷脂scramblase活动2。该测定法还允许我们通过测试特异性表征视蛋白的scramblase活性（我们采用了各种NBD标记的报告脂质如NBD-磷脂酰乙醇胺，如图所示为NBD-PC在图1A中，或在上的酰基链与NBD标记的首基，NBD-鞘磷脂或NBD-磷脂2），囊泡脂质组合物（的效应， 例如 ，在不同量的作为囊泡组分胆固醇），和是否视蛋白?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This study was supported by the Velux Stiftung (A.K.M.), NIH grant EY024207 (A.K.M.) and the Austrian Science Fund (FWF) project J3686 (B.P.).
Materials
| 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine | Avanti Polar Lipids | 850457C | POPC |
| 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt) | Avanti Polar Lipids | 840457C | POPG |
| 1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-an-glycero-3-phosphocholine | Avanti Polar Lipids | 810130C | NBD-PC |
| 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid | VWR Scientific | EM-5330 | HEPES |
| NaCl | Sigma | S7653-1KG | NaCl |
| Dodecyl-β-d-maltoside | Anatrace | D310 5 GM | DDM |
| Fluorimeter cuvettes | sigma | C0918-100EA | cuvettes |
| Spectrofluorometer | Photon Technology International, Inc. | fluorimeter | |
| Sodium hydrosulfite technical grade, 85% | Sigma | 157953-5G | dithionite |
| GraphPad Prism 5 software | Prism | ||
| Tris Base | VWR | JTX171-3 | Tris |
| LIPEX 10 mL extruder | Northern Lipids, Inc. | Extruder | |
| Whatman, Drain disc, PE, 25 mm | Sigma | 28156-243 | Disc support |
| Whatman Nuclepore Track-Etched Membranes, 0.4 µm, 25 mm diameter | Sigma | WHA110607 | 400 nm membrane |
| Whatman Nucleopore Track-Etched Membranes, 0.2 µm, 25 mm diameter | Sigma | WHA110606 | 200 nm membrane |
| sodium phosphate | Sigma | S3264-500G | |
| VWR Culture Tubes, Disposable, Borosilicate Glass, 13×100 mm | VWR Scientific | 47729-572 | glass tubes |
| Perchloric acid | Sigma | 30755-500ML | |
| Ammonium Molybdate Tetrahydrate | Sigma | A-7302 | ammonium molybdate |
| (+)-Sodium L-ascorbate | Sigma | A7631-25G | sodium ascorbate |
| Bio-Beads SM2 adsorbents | Bio Rad | 1523920 | polystyrene beads |
| 2.0 mL Microtubes clear | VWR Scientific | 10011-742 | Reconstitution tubes |
| Reconstitution glass tube | VWR Scientific | 53283-800 | Reconstitution glass tubes |
| Zetasizer | Malvern | DLS | |
Riferimenti
- Ernst, O. P., et al. Microbial and animal rhodopsins: structures, functions, and molecular mechanisms. Chem. Rev. 114, 126-163 (2014).
- Menon, I., et al. Opsin is a phospholipid flippase. Curr. Biol. CB. 21, 149-153 (2011).
- Goren, M. A., et al. Constitutive phospholipid scramblase activity of a G protein-coupled receptor. Nat. Commun. 5, 5115 (2014).
- Ernst, O. P., Menon, A. K. Phospholipid scrambling by rhodopsin. Photochem. Photobiol. Sci. Off. J. Eur. Photochem. Assoc. Eur. Soc. Photobiol. , (2015).
- Sanyal, S., Menon, A. K. Flipping lipids: why an’ what’s the reason for?. ACS Chem. Biol. 4, 895-909 (2009).
- Bevers, E. M., Williamson, P. L. Phospholipid scramblase: an update. FEBS Lett. 584, 2724-2730 (2010).
- Leventis, P. A., Grinstein, S. The distribution and function of phosphatidylserine in cellular membranes. Annu. Rev. Biophys. 39, 407-427 (2010).
- Quazi, F., Lenevich, S., Molday, R. S. ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer. Nat. Commun. 3, 925 (2012).
- Quazi, F., Molday, R. S. ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal. Proc. Natl. Acad. Sci. U. S. A. 111, 5024-5029 (2014).
- Ben-Shabat, S., et al. Formation of a nonaoxirane from A2E, a lipofuscin fluorophore related to macular degeneration, and evidence of singlet oxygen involvement. Angew. Chem. Int. Ed Engl. 41, 814-817 (2002).
- Sparrow, J. R., Wu, Y., Kim, C. Y., Zhou, J. Phospholipid meets all-trans-retinal: the making of RPE bisretinoids. J. Lipid Res. 51 (2010), 247-261 (2010).
- Schütt, F., Davies, S., Kopitz, J., Holz, F. G., Boulton, M. E. Photodamage to human RPE cells by A2-E, a retinoid component of lipofuscin. Invest. Ophthalmol. Vis. Sci. 41, 2303-2308 (2000).
- Picollo, A., Malvezzi, M., Accardi, A. TMEM16 proteins: unknown structure and confusing functions. J. Mol. Biol. 427, 94-105 (2015).
- Mohammadi, T., et al. Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane. EMBO J. 30, 1425-1432 (2011).
- Meeske, A. J., et al. MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis. Proc. Natl. Acad. Sci. 112, 6437-6442 (2015).
- Ruiz, N. Lipid Flippases for Bacterial Peptidoglycan Biosynthesis. Lipid Insights. 8, 21-31 (2015).
- Rick, P. D., et al. Evidence that the wzxE gene of Escherichia coli K-12 encodes a protein involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen. J. Biol. Chem. 278, 16534-16542 (2003).
- Suzuki, J., Imanishi, E., Nagata, S. Exposure of phosphatidylserine by Xk-related protein family members during apoptosis. J. Biol. Chem. 289, 30257-30267 (2014).
- Suzuki, J., Nagata, S. Phospholipid scrambling on the plasma membrane. Methods Enzymol. 544, 381-393 (2014).
- Alaimo, C., et al. Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides. EMBO J. 25, 967-976 (2006).
- Ernst, C. M., Peschel, A. Broad-spectrum antimicrobial peptide restistance by MprF-mediated aminoacylation and flipping of phospholipids. Mol. Microbial. 80 (2), 290-299 (2011).
- Vehring, S., et al. Flip-flop of fluorescently labeled phospholipids in proteoliposomes reconstituted with Saccharomyces cerevisiae microsomal proteins. Eukaryot. Cell. 6, 1625-1634 (2007).
- Rouser, G., et al. Two dimensional then [sic] layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 5, 494-496 (1970).
- Rigaud, J. -. L., Lévy, D. Reconstitution of membrane proteins into liposomes. Methods Enzymol. 372, 65-86 (2003).
- Geertsma, E. R., Nik Mahmood, N. A. B., Schuurman-Wolters, G. K., Poolman, B. Membrane reconstitution of ABC transporters and assays of translocator function. Nat. Protoc. 3, 256-266 (2008).
- Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C., Reynolds, J. A. Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside. Biochimica. 20, 833-840 (1981).
- Malvezzi, M., et al. Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel. Nat. Commun. 4, 2367 (2013).
- Eckford, P. D. W., Sharom, F. J. The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids. Biochem. J. 389, 517-526 (2005).
- Marek, M., Günther-Pomorski, T. Assay of Flippase Activity in Proteoliposomes Using Fluorescent Lipid Derivatives. Methods Mol. Biol. 1377, 181-191 (2016).
- Coleman, J. A., Kwok, M. C. M., Molday, R. S. Localization purification, and functional reconstitution of the P4-ATPase Atp8a2, a phosphatidylserine flippase in photoreceptor disc membranes. J. Biol. Chem. 284, 32670-32679 (2009).
- Coleman, J. A., Quazi, F., Molday, R. S. Mammalian P4-ATPases and ABC transporters and their role in phospholipid transport. Biochim. Biophys. Acta. 1831, 555-574 (2013).
- Romsicki, Y., Sharom, F. J. Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter. Biochimica. 40, 6937-6947 (2001).
- Zhou, X., Graham, T. R. Reconstitution of phospholipid translocase activity with purified Drs2p, a type-IV P-type ATPase from budding yeast. Proc. Natl. Acad. Sci. U. S. A. 106, 16586-16591 (2009).
