笔者想感谢安捷伦为他们提供技术援助。凯特·马卡姆和拉斐尔·帕洛米诺都记在合成的初始帮助纯化的Aβ肽和Hsiau伟李博士在准备手稿图1感谢他的帮助。

1.Aβ40或Aβ42肽的制备型HPLC纯化<ol><li> 准备以下缓冲区的HPLC纯化。 <ol><li>加入1.3毫升NH 4 OH（28％溶液）到1000毫升超纯水制备缓冲液A（20毫米NH 4 OH）。 </li><li>通过加入1.3毫升的 NH 4 OH（28％溶液）的800毫升的HPLC级乙腈和200毫升的超纯水的溶液中制备的缓冲液B（80％乙腈的20mM NH 4 OH）。 </li><li>通过加入100微升的NH 4 OH（28％溶液）至100毫升的超纯水制备样品溶解缓冲液（0.1％的NH 4 OH）。 </li></ol></li><li> 设置为在图1所示的高效液相色谱仪。 <ol><li>适合的溶剂瓶是包含缓冲A和缓冲B到HPLC泵的使用聚合物管道入口。附加聚合物管与单件式接头的HPLC泵。确保聚合物管每个缓冲器被装配到仪器的正确入口阀。契合脱气HPLC泵。 </li><li>耦合HPLC泵与300埃为8μm25毫米х300毫米制备柱的入口使用聚合物管（ 图1B，最左边列中，见材料列表）。 <ol><li>附加聚合物管连接到制备柱上用一单件手指紧接头。确保聚合物列以正确的方式定向。 注:制备柱的固定相由聚（苯乙烯 - 二乙烯基苯）的颗粒。该聚合物列的正确方向上标有与单个方向箭头的外壳。 </li></ol></li><li>使用聚合物管列的出口连接到双波长检测器和波长检测器设定至214纳米和280纳米。 <ol><li>在的设置仪器方法选项改变检测波长参数改变波长内置型HPLC软件。 </li></ol></li><li>附加聚合物管波长检测器的入口有一个单件的手指紧身。附加聚合物管连接到波长检测器的输出阀。与单件式手指紧身附加聚合物管连接到HPLC检测器的出口阀。这将是样品收集管。 注:缺乏对Aβ肽的强烈的发色团的决定了214nm处被用作初级紫外线（UV）波长为峰集合。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/55482/55482fig1.jpg" /> 图1:用于淀粉样蛋白β肽的纯化的HPLC仪器的实验装置。 （A）中的季HPLC泵装有一个脱气器和可变波长检测器设定至214纳米和280纳米;用于淀粉样蛋白β肽的纯化（B）的HPLC柱，从从左到右，25×300毫米2制备柱，7.5×300毫米2半制备柱和4.6×250毫米2分析柱; （C）手动进样器与用于HPLC分析20微升不锈钢注射循环; （D）的手动喷射器与用于制备和半制备纯化10mL的不锈钢注射环。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55482/55482fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="3"><li> 编程性HPLC软件以运行该纯化方法如表1所示，通过改变溶剂时间表参数（在内置到HPLC软件的安装仪器方法选项）输入的纯化方法。通过点击HPLC软件的"上"按钮打开HPLC泵。 注:该泵将开始提供缓冲液A开始比，并通过预备缓冲液Barative柱和HPLC仪器。 <ol><li>离开系统30分钟以充分平衡。 </li></ol></li></ol><table fo:keep-together.within-page="1"><tbody><tr><td> 次/分 </td><td> 缓冲液A 一的％ </td><td> 缓冲液B 的B％ </td><td> 流量d /毫升-1分 </td></tr><tr><td> 0 </td><td> 80 </td><td> 20 </td><td> 6 </td></tr><tr><td> 45 </td><td> 75.5 </td><td> 24.5 </td><td> 6 </td></tr><tr><td> 45.01 </td><td> 80 </td><td> 20 </td><td> 6 </td></tr><tr><td> 52.01 </td><td> 80 </td><td> 20 </td><td> 6 </td></tr><tr><td> 52.02 </td><td> 73 </td><td> 27 </td><td> 6 </td></tr><tr><td> 85 </td><td> 73 </td><td> 27 </td><td> 6 </td></tr><tr><td> 92 </td><td>五</td><td> 95℃ </td><td> 6 </td></tr></tbody></table> &lt;强&gt;表1:时间表使用25×300毫米聚合物列中的Aβ42和Aβ40肽的纯化。 一个缓冲A - H 2 O运用20毫米NH 4 OH; B 80％乙腈/ 20％H 2 O 20毫米NH 4 OH; Ç冉15分钟以洗涤之前样品的下一注射柱; d在为了运行上的HPLC仪器6毫升/分钟的流速，压力限制需要被降低到200巴。 <ol start="4"><li> 该Aβ肽样品的分离纯化 注意:通过自动固相肽合成而获得粗肽。 10 <ol><li>溶解3毫克粗的Aβ肽在4毫升的样品溶解缓冲液中。超声处理样品30-60■在室温下，并在40千赫兹以促进溶解的频率。 </li><li>注入整个样品上用装有16号5毫升塑料注射器HPLC柱不锈钢针。如在子步骤1.3中概述运行的纯化方法。 注:该系统使样品注入到通过使用安装有10毫升不锈钢注射环（ 图1D）的手动喷射器来完成。所需的Aβ肽将洗脱分钟72和74之间作为一个尖锐分辨峰（ 图2C）。 </li><li>收集样品到50mL锥形离心管中。确认Aβ峰通过收集洗脱液直接注入质谱的身份。 11商店洗脱液为在-20℃达12小时。 注:长于12小时没有到期的肽的氧化宜电位期间将溶液的贮藏。 </li><li>隔离通过快速纯化肽冷冻收集的等分试样/液氮冷冻干燥的Aβ肽的等分试样。通过冷冻干燥该样品在-60℃，PRES的温度下进行冷冻干燥确保20毫托，为期24小时的。 </li><li>运行分析型HPLC协议如下所述来确定Aβ肽的纯度。商店肽在-20℃，为期6个月的冷冻干燥的形式。 </li></ol></li></ol> 2.纯化的Aβ蛋白的分析性HPLC分析<ol><li>准备HPLC缓冲区在小节1.1概述。上述协议。 </li><li>设置的分析性HPLC按步骤1.2.1并且与4.6×250毫米分析柱（ 图1B，最右列），并用20微升不锈钢注入环的手动进图1所示（ 图1C）装配到仪器。 </li><li>编程性HPLC软件以运行该分析方法如表2所示下列类似于在步骤1.3的指令。 </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td> 时间 /分</td><td> 缓冲液A 一的％ </td><td> 缓冲液B 的B％ </td><td> 流量/毫升-1分 </td></tr><tr><td> 0 </td><td> 95 </td><td>五</td><td> 1 </td></tr><tr><td>三十</td><td> 50 </td><td> 50 </td><td> 1 </td></tr></tbody></table> 表2:时间表的Aβ肽的HPLC纯度分析。 一个缓冲A - H 2 O运用20毫米NH 4 OH; B 80％乙腈/ 20％H 2 O 20毫米NH 4 OH。 <ol start="4"><li> 该Aβ肽的纯度分析 <ol><li>通过在样品缓冲液中的肽的溶解制备纯化的肽的1毫克/毫升溶液。 注:缓冲区配方可在小节1.1中找到。蛋白质浓度通过在280nm（A 280毫微米 ）测定蛋白质的吸收来确定。 12 m个<html>用于确定浓度OLAR消光系数（ε）为ε= 1,490分米3摩尔-1 -1。 13 </li><li>注入1毫克/毫升（222μM）溶液到HPLC柱20微升并运行的分析方法，该方法是设置在步骤2.3。 注:不用于分析剩余的溶液可以在液氮快速冷冻和冷冻干燥以回收的Aβ肽。冻干细节可以在子1.4.4节中找到。 Aβ的肽将洗脱从16和18分钟（ 图2D）之间的分析柱。使用伴随HPLC仪器来确定Aβ肽的纯度内置整合分析软件。纯度通过每个单独的峰的积分在光谱并计算肽峰面积百分比来确定。通常情况下，"95％的纯化应来确定。 </li></ol></li></ol><img alt="&lt;/ HTML">"图2"SRC ="/文件/ ftp_upload / 55482 / 55482fig2.jpg"/&gt; 图2:Aβ42的代表性HPLC曲线。 （A）的传统的 C 4的二氧化硅纯化，条件为:缓冲液A:H 2 O与0.1％三氟乙酸（TFA），缓冲液B:MeCN中（乙腈），0.1％TFA，梯度:20到27％缓冲液B，经40分钟，随后通过等度27％缓冲液B; （B）使用25×300毫米2聚合物列，条件制备纯化:缓冲液A:H 2 O 20毫米NH 4 OH，缓冲液B:80％乙腈/ 20％H 2 O 20毫米NH 4 OH，渐变:70分钟; 20到27％缓冲液B，接着等度27％缓冲液B; （ 三 ）优化使用25×300毫米2聚合物制备色谱柱净化，条件在位于小节协议描述文本1.3表1所示; （D）分析用HPLC使用4.6×250毫米2聚合物柱，CONDitions:缓冲液A:H 2 O 20毫米NH 4 OH，缓冲液B:80％乙腈/ 20％H 2 O超过30分钟20毫米NH 4 OH，梯度5至50％缓冲液B。对于部分A，B和C对应Aβ42峰用星号标记。标记Aβ42峰值在C部分揭示了&gt; 95％的纯度所示部分D质谱的集合被用来确定Aβ42峰的身份。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55482/55482fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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所述Aβ肽的HPLC纯化是高度依赖于两者在纯化中使用的固定相和选择，以洗脱所述肽的流动相的选择。肽和高倾向聚集低等电点呈现疏水蛋白质的分离（C4或加上酸性移动洗脱液C8固定相）挑战传统色谱条件下，与Aβ肽洗​​脱作为延长的宽，非分辨峰（ 图2A）。 为了避免这个问题，PLRP-S固定相，在高pH下是化学稳定的被发现是有效的Aβ肽（ 图2B和<stron...

阿尔茨海默氏症是一种神经退行性疾病的全球影响超过35万人。 1在疾病的发作和发展强烈牵连，是高度易聚集，疏水肽β淀粉样蛋白（Aβ）。 2Aβ从长度36至43个氨基酸的范围内，但是，它被认为是42-氨基酸变体，淀粉样蛋白β42（Aβ42），是蛋白质的最毒的形式。 3这是由于在大多数情况下，以Aβ42的容易地形成被认为是特别神经毒性实体扩散，低聚物质的能力。 4为了进一步提供了对Aβ肽的了解，有必要定期地获得高纯度的材料。微量杂质的存在已经显示出显着改变肽的聚集倾向的属性。 五 ŧraditionally，高效液相色谱（HPLC）的疏水性肽，例如Aβ的分离已通过使用C 4或C 8的二氧化硅基固定相和酸性流动相的组合完成的。 6然而，这样的条件可以提出一个挑战的肽的纯化。所述Aβ肽的低等电点（pI约5.5）7意味着在酸性条件下，肽聚集增加，并且作为结果宽，非分辨的HPLC峰是常常难以分离产生（ 图2A）。此外，这样的宽峰常常含有可能影响所述肽的聚集的空间，通常需要后续的轮次的纯化，其可以极大地影响肽的产生量的杂质。 的聚（苯乙烯/二乙烯基苯）固定相，PLRP-S，表示PURIF的替代手段英疏水性肽。固定相已在许多不同的蛋白质和信使核糖核酸（mRNA）的的纯化被使用。 8，9 PLRP-S固定相需要用于反相分离没有额外的烷基配体，而且更重要的是化学稳定在高pH导致肽的解聚。 7在此，我们报告说，得到高纯度的β淀粉样蛋白42（Aβ42）和β淀粉样蛋白40（Aβ40）肽量身定制的HPLC纯化协议。

<table><tbody><tr><td>Agilent 1260 Infinity II quarternary pump</td><td>Agilent</td><td>G7111B</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-pumps-vacuum-degassers/1260-infinity-ii-quaternary-pump</td></tr><tr><td>Agilent 1260 Infinity II Dual variable wavelength detector</td><td>Agilent</td><td>G7114A</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-detectors/1260-infinity-ii-variable-wavelength-detector</td></tr><tr><td>Agilent 1260 Infinity II Manual Injector fitted with 10 mL stainless steel sample loop</td><td>Agilent</td><td>0101-1232</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td></tr><tr><td>Agilent 1260 Infinity II Manual Injector fitted with 20 &amp;micro;L stainless steel sample loop</td><td>Agilent</td><td>G1328C</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td></tr><tr><td>Ring Stand Mounting Bracket</td><td>Agilent</td><td>1400-3166</td><td></td></tr><tr><td>Agilent PLRP-S 300 &amp;Aring; 5&amp;nbsp;&amp;micro;m 4.6 x 250 mm (Analytical)</td><td>Agilent</td><td>PL1512-5501</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features</td></tr><tr><td>A&amp;beta;42 or A&amp;beta;40 peptide</td><td></td><td></td><td>Synthesized in-house using a CEM liberty automated peptide synthesizer.</td></tr><tr><td>Ammonium Hydroxide (NH&lt;sub&gt;4&lt;/sub&gt;OH, 28% solution)</td><td>Fisher Scientific</td><td>A669-500</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific</td><td>A998-4</td><td></td></tr><tr><td>HPLC grade water</td><td>Fisher Scientific</td><td>W5-4</td><td></td></tr><tr><td>Falcon 50 mL conical centrifuge tube</td><td>Fisher Scientific</td><td>14-954-49A</td><td></td></tr><tr><td>Supelco PEEK Fitting One-piece fingertight, pkg of 5 ea</td><td>Sigma-Aldrich</td><td>Z227250</td><td></td></tr><tr><td>Normject 5&amp;nbsp;cc sterile syringe</td><td>Fisher Scientific</td><td>1481729</td><td></td></tr><tr><td>16 Gauge SS Needle</td><td>Rheodyne</td><td>3725-086</td><td></td></tr></tbody></table>

a tailored hplc purification protocol that yields high-purity amyloid beta 42 and amyloid beta 40 peptides, capable of oligomer formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

所述Aβ42肽的使用和PLRP-S固定相的组合的高pH的流动相在用于Aβ肽的尖锐，解析峰的形成在保留时间72和74分钟（ 图2C）之间的结果的纯化。峰的身份的确认是通过收集洗脱液直接注射质谱完成。洗脱液可以存储在长达12小时的溶液-20℃。存储的时间较长，可能会导致该蛋白质的氧化。以分离纯化的肽，洗脱剂是闪光灯在液氮中并冻干冷冻。该纯化的肽的分析型H...

Watch this Scientific Journal Video about A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation at JoVE.com

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

在此，我们报告说，得到高纯度的β淀粉样蛋白42（Aβ42）和β淀粉样蛋白40（Aβ40）肽，能够形成低聚物量身定制的HPLC纯化协议。 β淀粉样蛋白在阿尔茨海默氏症牵连高度易聚集，疏水肽。该肽的淀粉样蛋白性质使得其净化是一个挑战。

一个定制HPLC纯化协议，产量高纯度淀粉样蛋白β42和贝塔淀粉样蛋白肽40，有能力寡聚物形成的

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

a-tailored-hplc-purification-protocol-that-yields-high-purity-amyloid

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

11.8K 次观看.  University of California, Santa Cruz. 在此，我们报告说，得到高纯度的β淀粉样蛋白42（Aβ42）和β淀粉样蛋白40（Aβ40）肽，能够形成低聚物量身定制的HPLC纯化协议。 β淀粉样蛋白在阿尔茨海默氏症牵连高度易聚集，疏水肽。该肽的淀粉样蛋白性质使得其净化是一个挑战。 

视频: A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

低聚β-淀粉样蛋白的制备 1-42和突触可塑性损伤诱导海马脑片

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

科研

神经科学

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

淀粉样β-蛋白，阿尔茨海默氏病的病原体适配的选择

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

用斑点印迹分析 A11-positive β淀粉样寡聚物的制备与评价

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

生物化学

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

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 ...

医学

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

自组装多胺基多肽 Amphiphiles (PPAs) 及相关生物材料的简易合成协议

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

化学

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

12-Dithiolane 改性自组装肽的合成与表征

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy and transmission electron microscopy (TEM). The observed pH-dependent shift of the SPR band of A&#946;1-40-coated 20 nm gold particles was correlated with alternation of aggregation and disaggregation observed in the TEM images. A pH of ~4&#160;induced an unfolded conformation, and a pH of ~10&#160;induced a folded conformation of A&#946;1-40 on the gold surface. This reversible aggregation process was observed by Raman imaging as the pH was gradually changed from pH 4 to pH 10. We observed a pH-dependent morphology change in A&#946;1-40 on the gold surface, where clear aggregates were observed at pH 4. However, we observed very subtle differences in the surface-enhanced Raman spectroscopy (SERS) spectrum between pH 4 and pH 10 conditions, with the most striking difference being spectral density in the region of 250 cm-1 and 1750 cm-1. Specifically, the mode analysis of the reversible aggregation indicated that the aggregates were formed by the unfolded conformation of A&#946;1-40 which involved the benzene ring section of Tyrosine and Phenylalanine. Conversely, the disassembly of the aggregates was associated with conformational changes in protein folding of A&#946;1-40 involving Histidine, Glutamine, Methionine, and Aspartic acid.

Here, we present a systematic method describing the quasi-reversible self-assembly of the amyloid beta 1-40 (A&#946;1-40)-coated 20 nm gold aggregates. The nano-size-dependent quasi-reversible networking between peptides was correlated with specific amino acids or sections of the A&#946;1-40 monomer.

β 淀粉样蛋白 1-40 包被的金胶体的 pH 依赖性可逆自组装的表征

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

生物学

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

一个定制HPLC纯化协议，产量高纯度淀粉样蛋白β42和贝塔淀粉样蛋白肽40，有能力寡聚物形成的

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低聚β-淀粉样蛋白的制备_1-42和突触可塑性损伤诱导海马脑片

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