Name: purification and aggregation of the amyloid precursor protein intracellular domain
Uploaded: 2012-08-28T12:00:00
Description: Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

作者要感谢郑晖博士（美国贝勒医学院）的APP基因。这个工作是由美国国立卫生研究院资助R21AG031948（DB，JMB），F30AG030878（ESS），R01DK073394（AFO），约翰·西利纪念基金会生物医学研究基金（AFO），并在Jean C.和威廉·D·威利斯神经研究基金（ESS）。 ，JMB是一个学者的翻译研究学者项目和大学的德克萨斯州医学科克劳德·E.辣椒年长美国人独立中心的成员（由美国国立卫生研究院资助UL1RR029876和P30-AG-024832，分别支持）。

<html> 1。表达的重组APP胞内结构域（AICD） <ol><li>变换E. coli菌株BL21与人类AICD（残基649-695的APP，神经元亚型编号）克隆到载体pGEX-4T-1（GE Healthcare公司）。该载体将快递AICD作为谷胱甘肽-S-转移酶（GST）的融合蛋白的C-末端基团。该载体也编码凝血酶裂解序列的GST部分以有利于去除。 AICD的克隆到pGEX-4T-1的详细信息可以在我们以前出版4。 </li><li>从单菌落，接种用氨苄青霉素（100微克/?...

<ol>
	<li>De Strooper, B., Vassar, R., Golde, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretases:+enzymes+with+therapeutic+potential+in+Alzheimer+disease.">The secretases: enzymes with therapeutic potential in Alzheimer disease.</a> Nature reviews. Neurology. 6, 99-107 (2010).</li><li>Chang, K. A., Suh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+roles+of+amyloid+intracellular+domain+of+amyloid+precursor+protein.">Possible roles of amyloid intracellular domain of amyloid precursor protein.</a> BMB reports. 43, 656-663 (2010).</li><li>McLoughlin, D. M., Miller, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FE65+proteins+and+Alzheimer's+disease.">The FE65 proteins and Alzheimer's disease.</a> J. Neurosci. Res. 86, 744-754 (2008).</li><li>Stieren, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquilin-1+is+a+molecular+chaperone+for+the+amyloid+precursor+protein.">Ubiquilin-1 is a molecular chaperone for the amyloid precursor protein.</a> J. Biol. Chem. 286, 35689-35698 (2011).</li><li>Ramelot, T. A., Nicholson, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation-induced+structural+changes+in+the+amyloid+precursor+protein+cytoplasmic+tail+detected+by+NMR.">Phosphorylation-induced structural changes in the amyloid precursor protein cytoplasmic tail detected by NMR.</a> J. Mol. Biol. 307, 871-884 (2001).</li><li>Ramelot, T. A., Gentile, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+structure+of+the+amyloid+precursor+protein+cytoplasmic+tail+indicates+preordering+of+structure+for+binding+to+cytosolic+factors.">Transient structure of the amyloid precursor protein cytoplasmic tail indicates preordering of structure for binding to cytosolic factors.</a> Biochem. 39, 2714-2725 (2000).</li><li>Radzimanowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+intracellular+domain+of+the+amyloid+precursor+protein+in+complex+with+Fe65-PTB2.">Structure of the intracellular domain of the amyloid precursor protein in complex with Fe65-PTB2.</a> EMBO Rep. 9, 1134-1140 (2008).</li><li>Ohkawara, T., Nagase, H., Koh, C. S., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+precursor+protein+intracellular+domain+alters+gene+expression+and+induces+neuron-specific+apoptosis.">The amyloid precursor protein intracellular domain alters gene expression and induces neuron-specific apoptosis.</a> Gene. 475, 1-9 (2011).</li><li>von Rotz, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+APP+intracellular+domain+forms+nuclear+multiprotein+complexes+and+regulates+the+transcription+of+its+own+precursor.">The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor.</a> J. Cell Sci. 117, 4435-4448 (2004).</li><li>Hamid, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+intracellular+domain+modulates+cellular+calcium+homeostasis+and+ATP+content.">Amyloid precursor protein intracellular domain modulates cellular calcium homeostasis and ATP content.</a> J. Neurochem. 102, 1264-1275 (2007).</li><li>Ghosal, K., Stathopoulos, A., Pimplikar, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+intracellular+domain+impairs+adult+neurogenesis+in+transgenic+mice+by+inducing+neuroinflammation.">APP intracellular domain impairs adult neurogenesis in transgenic mice by inducing neuroinflammation.</a> PLoS ONE. 5, e11866 (2010).</li><li>Pimplikar, S. W., Suryanarayana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+APP+intracellular+domain+in+brain+tissue.">Detection of APP intracellular domain in brain tissue.</a> Met. Molecul. Biol. 670, 85-91 (2011).</li><li>Buchner, J., Grallert, H., Jakob, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+chaperone+function+using+citrate+synthase+as+nonnative+substrate+protein.">Analysis of chaperone function using citrate synthase as nonnative substrate protein.</a> Met. Enzymol. 290, 323-338 (1998).</li><li>Hansma, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+atomic+force+microscopy+of+DNA.">Recent advances in atomic force microscopy of DNA.</a> Scanning. 15, 296-299 (1993).</li><li>Valbuena, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quasi-simultaneous+imaging/pulling+analysis+of+single+polyprotein+molecules+by+atomic+force+microscopy.">Quasi-simultaneous imaging/pulling analysis of single polyprotein molecules by atomic force microscopy.</a> Rev. Sci. Instrum. 78, 113707 (2007).</li><li>Radzimanowski, J., Beyreuther, K., Sinning, I., Wild, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overproduction,+purification,+crystallization+and+preliminary+X-ray+analysis+of+human+Fe65-PTB2+in+complex+with+the+amyloid+precursor+protein+intracellular+domain.">Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain.</a> Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 64, 409-412 (2008).</li><li>Chen, T. Y., Liu, P. H., Ruan, C. T., Chiu, L., Kung, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+domain+of+amyloid+precursor+protein+interacts+with+flotillin-1,+a+lipid+raft+protein.">The intracellular domain of amyloid precursor protein interacts with flotillin-1, a lipid raft protein.</a> Biochem. Biophys. Res. Commun. 342, 266-272 (2006).</li><li>Kim, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Notch1+signaling+by+the+APP+intracellular+domain+facilitates+degradation+of+the+Notch1+intracellular+domain+and+RBP-Jk.">Regulation of Notch1 signaling by the APP intracellular domain facilitates degradation of the Notch1 intracellular domain and RBP-Jk.</a> J. Cell Sci. 124, 1831-1843 (2011).</li><li>Lazarov, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport,+amyloid+precursor+protein,+kinesin-1,+and+the+processing+apparatus:+revisited.">Axonal transport, amyloid precursor protein, kinesin-1, and the processing apparatus: revisited.</a> J. Neurosci. 25, 2386-2395 (2005).</li><li>Kakuda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equimolar+production+of+amyloid+beta-protein+and+amyloid+precursor+protein+intracellular+domain+from+beta-carboxyl-terminal+fragment+by+gamma-secretase.">Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from beta-carboxyl-terminal fragment by gamma-secretase.</a> J Biol. Chem. 281, 14776-14786 (2006).</li><li>Gosal, W. S., Myers, S. L., Radford, S. E., Thomson, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+under+the+atomic+force+microscope.">Amyloid under the atomic force microscope.</a> Protein Pept. Lett. 13, 261-270 (2006).</li></ol>

在这个协议中，我们提出的结构，生物物理和生物化学分析，获得高纯度的AICD的程序。此过程不需要复杂的色谱仪，因此大多数实验室访问。其他团体纯化的AICD 5-7,16，包括GST-AICD 17日至19日 ，生化/结构的分析。到先前的协议的缺点包括AICD 16的溶解性差，小于理想纯度17，和尺寸排阻16的要求，或反相色谱法6,20。因此，这里所描述的协议，极大地?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> PGEX-4T-1 </td><td> GE医疗集团</td><td> 28-9545-49 </td><td></td></tr><tr><td>凝血酶</td><td> GE医疗集团</td><td> 27-0846-01 </td><td></td></tr><tr><td>氨苄青霉素</td><td> Fisher Scientific则</td><td> BP1760 </td><td></td></tr><tr><td> Bradford蛋白测定试剂</td><td> Bio-Rad公司</td><td> 500-0002 </td><td></td></tr><tr><td>考马斯亮蓝</td><td> Bio-Rad公司</td><td> 161-0786 </td><td></td></tr><tr><td> IPTG（异丙基-β-D硫代半乳糖苷） </td><td> Sigma-Aldrich公司</td><td> I6758 </td><td></td></tr><tr><td>谷胱甘肽 - 琼脂糖</td><td> Sigma-Aldrich公司</td><td> G4510 </td><td></td></tr><tR&gt; <td>对氨基苯甲脒琼脂糖</td><td> Sigma-Aldrich公司</td><td> A7155 </td><td></td></tr><tr><td>完整的蛋白酶抑制剂混合物</td><td>罗氏公司</td><td> 11836170001 </td><td></td></tr><tr><td>幻灯片-A仪透析磁带的</td><td> Thermo Scientific的</td><td> 66380 </td><td></td></tr><tr><td>层析柱</td><td>长荣科学</td><td> 208-3367-050 </td><td></td></tr><tr><td>乳化剂</td><td> Avestin公司</td><td> EmulsiFlex-C3 </td><td>强烈推荐</td></tr><tr><td>的Eppendorf恒温</td><td> Eppendorf公司</td><td> 022670107 </td><td></td></tr><tr><td>云母磁盘</td><td>特德·佩拉</td><td> 50-12 </td><td></td></tr><tr><td>原子力显微镜的悬臂</td><td>布鲁克</td><td> MSNL-10 </td><td></td></tr><tr><td> WSxM软件</td><td> NANOTEC </td><td> N / A </td><td>神父EE下载</td></tr></tbody></table>

purification and aggregation of the amyloid precursor protein intracellular domain

淀粉样前体蛋白（APP）是与阿尔茨海默氏病（AD）的发病机制相关联的I型跨膜蛋白。 APP是其特征在于由一个大的胞外结构域和一个短的胞质域称为APP胞内结构域（AICD）。在成熟过程中通过分泌途径，APP可以由蛋白酶被称为α，β，和γ-分泌1裂解。顺序的蛋白水解切割APP的β和γ-分泌导致一个小的蛋白水解肽，称为Aβ是淀粉样蛋白形成和老年斑的核心成分的生产。 AICD是从膜中解放出来后分泌处理，并通过FE65和TIP60的相互作用，可转位到细胞核内参与多个靶基因的转录调节2,3。蛋白质 - 蛋白质相互作用的AICD可能会影响贩运，加工，细胞功能的全息APP和C-termin人的片段。最近，我们已经表明，AICD可以聚集在体外 ，而这个过程被抑制的AD-牵连分子伴侣ubiquilin的，1 4。与这些结果一致，AICD暴露出疏水区，并有内在的紊乱体外 5,6，但它获得稳定的二级结构时，势必FE65 7。我们已经提出，ubiquilin-1防止AICD不适当间和分子内相互作用，防止聚集在体外和在完整细胞4。虽然大多数研究专注于APP在AD的发病机制中的作用，AICD在这个过程中的作用目前尚不清楚的。已被证明的表达AICD诱导细胞凋亡8，调制信号通路9，调节钙信号10。 AICD和FE65过度表达的转基因小鼠模型中诱导阿尔茨海默氏症样病变11日 ，并于最近AICD中发现了胸罩在裂解液免疫印迹当使用合适的抗原修复技术12。为了方便的AICD的结构，生化和生物物理研究，我们开发了一个程序，以重组产生大量的高纯度AICD蛋白。我们进一步对诱导在体外热聚集，的​​AICD并分析利用原子力显微镜的描述方法。描述的方法是有用的生物化学，生物物理与结构表征，AICD的影响AICD聚集的分子伴侣。

Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

APP胞内结构域（AICD）用于大规模纯化的方法，进行说明。我们也将介绍的方法来诱导<em在体外</emAICD聚合和原子力显微镜的可视化。所描述的方法是有用的生化/结构表征AICD和聚集在其分子伴侣的影响。

淀粉样前体蛋白胞内结构域的分离纯化和聚集

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Research

JoVE Journal

Medicine

Performing and Processing FNA of Anterior Fat Pad for Amyloid

表演和加工为淀粉样蛋白前脂肪垫活检

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

科研

医学

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

MACS纯化光感受器前体细胞的亚视移植到成年小鼠视网膜中

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

在实验颈脊髓损伤协同使用神经前体细胞，并自组装肽的

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

自发交替评估，淀粉样蛋白β和脑神经病理学转基因小鼠模型中的新物体识别和肢体紧扣

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

比浊法对人洗涤血小板：所述Pannexin1抑制剂的影响亮蓝FCF对胶原诱导的聚集

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

肌网钙储备与细胞内舒张钙在心室肌细胞中的分离评价

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Isolation and Purification of Murine Cardiac Pericytes

鼠心围细胞的分离与纯化

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

原位小鼠肝脏灌注模型，用于研究对底物的代谢反应

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

作者聚焦：研究肝脏疾病的肝脏适应和糖尿病前期进展 

使用灌注小鼠肝脏模型量化肝葡萄糖生成、尿素生成和脂肪分解

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused ...