核磁共振光谱法本质无序蛋白的磷酸化多元的鉴定
Summary
We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.
Abstract
Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer’s disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau’s heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.
Introduction
其中一个在21 世纪医疗保健的主要挑战是神经退行性疾病如阿尔茨海默病（AD）。头是一个微管相关蛋白，刺激微管（MT）的形成。头被均等地参与了一些神经变性疾病，所谓τ病变，其中最有名的是广告。在这些疾病中成对螺旋丝（的PHF）头自聚集，并发现修改由翻译后后修饰，如磷酸1个多残留。 Tau蛋白的磷酸化在MT稳定和功能丧失的病理表征AD神经元及其生理功能的调节都牵连。
此外，Tau蛋白，在神经元病中集成的PHF时，则不约而同地过度磷酸2。不同于含有2-3个磷酸基团正常头，在对的PHF tau蛋白含有5至9 phosphatË组3。 tau蛋白对应既能在一些网站和被称为磷酸化位点病理其他站点的磷酸化增加了化学计量。然而，重叠的AD和磷酸化的正常成人图案之间存在，尽管在4级量化的差别。具体怎么磷酸化事件的影响作用和Tau功能障碍仍是未知。我们的目标是通过翻译后修饰在分子水平上破译头监管。
深化头的分子方面的理解，我们必须解决的技术难题。首先，头在溶液中分离出来时，一个内在的无序蛋白（IDP）。这样的蛋白质缺乏在生理条件下明确定义的三维结构并需要特定的生物物理方法来研究它们的功能（S）和结构特性。头是为不断增长的国内流离失所者类的一个范例，经常发现关联疾病，如神经退行性疾病，从而增加利息，了解他们的基本功能分子参数。其次，tau蛋白磷酸表征是一个分析的挑战，随着时间最长的441氨基酸头亚型的序列80的潜在磷酸化位点。一些抗体已经开发针对头的磷酸化表位，并用于在神经元或脑组织检测病理头的。磷酸化事件可以采取由脯氨酸的激酶靶向至少有20处地点，其中大多数是在富含脯氨酸的区域内近在咫尺。定性（哪些网站？）和定量（化学计量学什么？）特性是很难甚至最新的MS技术5。
核磁共振光谱可用于研究是高度构成构象合奏的动态系统紊乱的蛋白质。高分辨率核磁共振光谱是APPLI编调查Tau蛋白的两个结构和功能。另外，该头的磷酸化信息的复杂性导致了使用NMR对磷酸化位点6的识别分子的工具和新的分析方法的发展– 8。核磁共振作为分析方法允许在全局方式识别的头磷酸化位点，在一个单一实验中的所有的单点修饰的可视化和量化磷酸并入的程度。因为虽然tau蛋白磷酸的研究在文献中比比皆是，他们大多已与抗体进行，留下了磷酸化的完整个人资料大程度的不确定性，因此个别磷酸化事件的真正影响这一点是至关重要的。重组激酶，包括PKA，糖原合酶激酶3β（GSK3β），细胞周期蛋白依赖性激酶2 /细胞周期蛋白A（CDK2 / CycA），细胞周期蛋白依赖性激酶5（CDK5）/ P25行为ivator蛋白质，细胞外信号调节的激酶2（ERK2）和微管亲合性 – 调节激酶（MARK），其显示向头磷酸化活性，可以以活性形式来制备。另外，头突变体，其允许产生与充分表征的磷酸化模式特定Tau蛋白同种型用于破译头的磷酸化的代码。然后NMR谱来表征酶改性头样品6 – 8。虽然在体外头的磷酸化是比伪磷酸更具挑战性，例如通过选定的丝氨酸/苏氨酸的突变成谷氨酸（谷氨酸）残基，该方法有其优点。实际上，无论是磷酸化的结构影响也没有相互作用参数总是可以通过谷氨酸模拟。一个例子是围绕磷酸丝氨酸202（pSer202）观察到的转基序/磷酸苏205（pThr205），这是不符合谷氨酸突变9再现。
<p class ="“jove_content”">在这里，同位素标记的牛头核磁共振调查准备将首先描述。通过ERK2磷酸化tau蛋白被修改的描述为磷酸化位点病理遗迹众多，因此代表tau蛋白的一个有趣的模式。薮中通过重组ERK2激酶磷酸化体外了详细的方案提出。 ERK2是通过磷酸化而活化有丝分裂原活化蛋白激酶/ ERK激酶（MEK）10 – 12。另外修改，同位素标记的Tau蛋白的制备中，用于翻译后修饰的识别核磁共振策略进行说明。Protocol
Representative Results
Discussion
我们已经使用NMR光谱法表征酶改性头样品。重组表达和这里描述的全长人Tau蛋白纯化可以类似地用于产生突变体头或头域。同位素是需要核磁共振光谱富集蛋白质，因此有必要重组表达。磷酸化位点的识别，需要共振分配和15 N，13 C双重标记的蛋白质。定同位素的成本，良好的产率，需要在该重组表达的步骤。葡萄糖是在M9培养基中细菌的生长，因此13 C 6 -葡萄糖?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The NMR facilities were funded by the Région Nord, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry and the University of Sciences and Technologies of Lille. We acknowledge support from the TGE RMN THC (FR-3050, France), FRABio (FR 3688, France) and Lille NMR and RPE Health and Biology core facility. Our research is supported by grants from the LabEx (Laboratory of Excellence) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer’s disease), EU ITN TASPPI and ANR BinAlz.
Materials
| pET15B recombinant T7 expression plasmid | Novagen | 69257 | Keep at -20°C |
| BL21(DE3) transformation competent E.coli bacteria | New England Biolabs | C2527I | Keep at -80°C |
| Autoclaved LB Broth, Lennox | DIFCO | 240210 | Bacterial Growth Medium |
| MEM vitamin complements 100X | Sigma | 58970C | Bacterial Growth Medium Supplement |
| 15N, 13C-ISOGRO complete medium powder | Sigma | 608297 | Bacterial Growth Medium Supplement |
| 15NH4Cl | Sigma | 299251 | Isotope |
| 13C6-Glucose | Sigma | 389374 | Isotope |
| Protease inhibitor tablets | Roche | 5056489001 | Keep at 4°C |
| 1 tablet in 1ml is 40X solution that can be kept at -20°C | |||
| DNaseI | EUROMEDEX | 1307 | Keep at -20°C |
| Homogenizer (EmulsiFlex-C3) | AVESTIN | Lysis is realized at 4°C | |
| Pierce™ Unstained Protein MW Marker | Pierce | 266109 | |
| Active human MEK1 kinase, GST Tagged | Sigma | M8822 | Keep at -80°C |
| AKTÄ Pure chromatography system | GE Healthcare | FPLC | |
| HiTrap SP Sepharose FF (5 mL column) | GE Healthcare | 17-5156-01 | Cation exchange chromatography columns |
| HiPrep 26/10 Desalting | GE Healthcare | 17-5087-01 | Protein Desalting column |
| PD MidiTrap G-25 | GE Healthcare | 28-9180-08 | Protein Desalting column |
| Tris D11, 97% D | Cortecnet | CD4035P5 | Deuterated NMR buffer |
| 5 mm Symmetrical Microtube SHIGEMI D2O ( set of 5 inner & outerpipe) | Euriso-top | BMS-005B | NMR Shigemi Tubes |
| eVol kit-electronic syringe starter kit | Cortecnet | 2910000 | Pipetting |
| Bruker 900MHz AvanceIII with a triple resonance cryogenic probehead | Bruker | NMR spectrometer for data acquisition | |
| Bruker 600MHz DMX600 with a triple resonance cryogenic probehead | Bruker | NMR spectrometer for data acquisition | |
| TopSpin 3.1 | Bruker | Acquisition and Processing software for NMR experiments | |
| Sparky 3.114 | UCSF (T. D. Goddard and D. G. Kneller) | NMR data Analysis software |
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