Name: peptide-based identification of functional motifs and their binding partners
Uploaded: 2013-06-30T17:00:00
Description: Watch this Scientific Journal Video about Peptide-based Identification of Functional Motifs and their Binding Partners at JoVE.com

这项工作是由NIH / NIGMS / MBRS（格兰特58268），美国国立卫生研究院/（格兰特G12-RR03034）NCRR / RCMI的的资金补助GRA.VAC08.W，乔治亚研究联盟，NIH / NIAID / NRSA的的补助F31AI091484，埃默里CFAR授予P30支持A1050409。进行这项调查是在设施建造与研究设施改善格兰特＃C06 RR18386支持NIH / NCRR。 Jurkat细胞，20组HIV-1 Nef的肽，以及来自美国国立卫生研究院艾滋病研究和参考试剂计划，（在Rockville，MD）获得兔抗HIV-1 Nef的抗血清。

I.使用短肽在生物分析一.1。测绘使用肽的生物功能图案<ol><li>治疗细胞与NEF扫描的肽<ol><li>促使扫描感兴趣区域的一组肽。对于我们的实验中，20聚体肽，带有10个氨基酸重叠，跨越整个HIV-1 Nef蛋白（205个氨基酸），得到，艾滋病试剂从程序。单独测定的肽，如下所示： </li><li>加入10毫微克/毫升的细胞培养液中的肽。 </li><li>添加10ml的培养基中每板Jurkat细胞培养。 </li><li>培养24...

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理解Nef的能力去操纵的exosomal贩运途径将是有益的工程新型抑制剂的exNef分泌机制。应减少抑制exNef分泌的CD4 + T细胞耗竭和中央情报局驱动艾滋病毒/艾滋病的发病机制。为了实现这一目标，我们制定了一些新颖的试剂和方法，让我们来分析的的遗传学exNef分泌，并开始确定所涉及的细胞蛋白质。这种方法也导致了发展的第一个直接针对exNef分泌，使SMRwt的肽抑制剂。 我们工作的...

随着抗逆转录病毒疗法的问世，在西方世界的艾滋病疫情已经放缓，但不会削减，和艾滋病毒的蔓延仍然是一个主要的健康负担，在世界各地。除了轻微有效RV144泰国试用，艾滋病毒疫苗迄今未能保护免受感染。因此，成额外的，潜在的治疗靶点的研究仍是必要的。 随着CD4 + T细胞耗竭，持续性全身免疫激活是一个标志性的艾滋病毒感染。这种慢性免疫激活（CIA）导致增加细胞的新陈代谢，激活和分化的淋巴细胞亚群，细胞的疲惫和衰老，并杀害的T细胞和B细胞通过激活诱导的细胞死亡（AICD）1,2,3，它是公认为疾病进展4,5,6,7,8,9,10,11,12最强的预测之一。然而，机制中央情报局（CIA）和CD4在HIV感染的T细胞耗竭，仍然完全阐明。 从我们的实验室和其他证据，导致疾病进展到一个模型（ 图1），其中HIV蛋白Nef的（负调控因子）诱导其分泌外来HIV-1感染细胞13,14。在这些含Nef的的外来（exNef）诱导细胞凋亡中的细胞谱系数，包括未感染的CD4 + T细胞15,16。另外，在单核细胞/巨噬细胞exNef改变基因表达模式， 如细 ​​胞因子的表达，出现不定期的免疫激活诱导的状态。该机构的证据表明在中央情报局（CIA）和CD4 + T细胞耗竭exNef了重要作用。 理解Nef的能力去操纵的exosomal贩运途径将是有益的工程新型抑制剂的exNef分泌机制。应减少抑制exNef分泌的CD4 + T细胞耗竭中情局驱动艾滋病毒/艾滋病的发病机制。 为了收集证据，导致我们的模型为艾滋病毒/艾滋病疾病的进展和后续的数据，建到这个模型，我们开发了一些新颖的试剂和方法，使我们能够分析遗传学的exNef分泌，并开始确定细胞的蛋白质参与。在最初的工作中，我们发现，Nef蛋白诱导旁观者细胞凋亡，并释放到细胞外从奈夫转染和HIV感染的细胞15。肽SDF-1α（基质细胞衍生因子-1，与替代拼接阿尔法），来自先前的研究显示，保留绑定和信令活动分子全长17。我们推测的Nef肽可能保留一些细胞凋亡活性全蛋白，，这些肽必然会包含NEF凋亡域。要识别这些肽，因此奈夫凋亡域（S），我们获得了一组20例HIV-1 Nef的扫描肽从美国国立卫生研究院艾滋病研究和参考试剂计划。在这些20个氨基酸的肽，每一个重叠的10个氨基酸，它的邻居，被命名为他们的最后的氨基酸， 即 N20跨度Nef的氨基酸1-20，N30的跨度的Nef的氨基酸11-30，等16的数量。我们发现，将T细胞暴露以重叠的两个不同的10个氨基酸的结构域的特异性肽全长的Nef蛋白在这些细胞中诱导细胞凋亡的细胞外。随后的分析显示这些Nef的衍生细胞凋亡肽的物理交互的趋化因子受体CXCR4的T细胞的表面上，结合动力学，使这些肽的竞争性抑制CXCR4和其天然配体SDF-1α之间的结合的能力。最后，NEF源性凋亡肽与CXCR4的相互作用被发现在这些T细胞，导致细胞凋亡诱导应激反应。这方面的证据使我们能够QuIC的地图NEF千光年的功能域;一个过程，会花费更长的使用标准DNA诱变技术，如丙氨酸扫描诱变。它还表明，这些短Nef的衍生肽保留了​​细胞凋亡的生物学功能域的全长蛋白。 经鉴定外奈夫的作用， 即 T细胞凋亡，我们寻求更好的理解奈夫如何从细胞分泌。我们使用了一系列突变的Nef的构造，映射Nef蛋白高度保守的基序的N-末端区域的同时感染HIV Nef的，和：相当于猕猴SIV 的mac（猴免疫缺陷病毒）Nef的，这是至关重要的为分泌exNef 13。这些图案之一，分泌修改地区（SMR 66VGFPV70），是特别重要的，因为任何其五个氨基酸要么大大减少或取消exNef，分泌丙氨酸更换。当传递到细胞通过Active Motif公司的C结果发现，包含连接到一个FLAG肽序列（SMRwt）的SMR的肽hariot蛋白交货试剂从Nef的转染和HIV感染的细胞18抑制exNef分泌。根据我们以往的经验与肽上，我们决定使用这种肽，阐明分子和机制的Nef的SMR的作用在exNef分泌相关。 ，我们使用肽作为我们的“诱饵蛋白”SMRwt，共同免疫沉淀的细胞的结合配偶体的SMR来自未感染的T细胞的溶胞产物18。 FLAG肽序列提供了一个方便的把手，使用抗FLAG亲和树脂捕捉SMRwt的肽。我们以前发现，一个单一的缬氨酸，丙氨酸突变在SMRwt肽废除其抑制分泌exNef的足够确定了方便，具有高度特异性的控制的肽（SMRmut），我们用来排除联合免疫沉淀的蛋白质，不特定的SMR。使用有s的肽pecific领域的利益，而不是全长蛋白允许，我们绕过筛选几十种细胞因子结合到其他领域奈夫19。 一旦我们确定了的SMR结合合作伙伴，一个合乎逻辑的下一步是要表明细胞结合合作伙伴是非常重要的生物学功能18。的标准程序来完成，这是使用目标蛋白特异性的miRNA或siRNA，并随后测定效果的生物学功能拦截蛋白水平。我们进行的miRNA的拦截，从而抑制靶mRNA的翻译，降低生产该目标，在这种情况下，SMR-结合蛋白。这种减少的目标蛋白是一种间接的影响，并可能有一个延迟效应对目标蛋白的生物学功能发挥了作用。因此，我们还采用了不常见的抗体，以确定是否直接破坏的活性的抑制技术目标蛋白的生物学功能降低或消除了。在此过程中，对目标蛋白的抗体的转染进入细胞内使用战车试剂的，并直接与目标蛋白的任一螯合剂从它的网站的功能，或阻断其相关的结合结构域。抑制靶蛋白通过此程序直接破坏其功能，可以互补RNA拦截程序，进一步确认的目标蛋白的生物学功能的重要性。 虽然战车试剂在进入细胞提供的肽和蛋白质是有效的，这个过程是耗时的，并限制类型的实验， 例如，长期或反复暴露在体内动物研究中可以执行的。因此，我们增加了一个细胞穿透肽（CPP）序列的SMRwt肽（SMRwt CPP）18生成肽，可以采取细胞被动从培养基。这个版本是有效的，因为前者在抑制exNef分泌。 从这些发表的实验证据表明含有特定的功能图案，小肽拮抗的全长蛋白的功能通过竞争性抑制，并分离的蛋白质结合这些图案的能力。人们期望，这些技术在许多实验的协议应该是有用的。他们也应该是有效的工程新型肽抑制剂的许多细胞过程中的功能，可以进一步增强联动CPP序列。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent </td>
 <td>Company </td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>20mer peptide set with 10 amino acid overlap</td>
 <td>NIH AIDS Research and Reference Reagent Program</td>
 <td>4641</td>
 <td></td>
 </tr>
 <tr>
 <td>TUNEL Assay</td>
 <td>Roche</td>
 <td>11 684 809 910</td>
 <td></td>
 </tr>
 <tr>
 <td>Chariot Protein Delivery Reagent</td>
 <td>Active Motif</td>
 <td>30100</td>
 <td></td>
 </tr>
 <tr>
 <td>Tecan GENEios fluorimeter</td>
 <td> (Tecan Group, Switzerland)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>96-well black microtiter plate</td>
 <td>Corning</td>
 <td>3792</td>
 <td></td>
 </tr>
 <tr>
 <td>anti-FLAG M2 Affinity Gel</td>
 <td>Sigma</td>
 <td>A2220</td>
 <td></td>
 </tr>
 <tr>
 <td>Dynabeads Protein G magnetic beads </td>
 <td>Invitrogen</td>
 <td>100.03D</td>
 <td></td>
 </tr>
 <tr>
 <td>MagnaSphere Technology Magnetic Separation Stand (two position)</td>
 <td>Promega Corp., Madison, WI</td>
 <td>Z5332</td>
 <td></td>
 </tr>
 <tr>
 <td>C-18 ZipTip </td>
 <td>Millipore</td>
 <td>ZTC18S096</td>
 <td>C18 Resin (0.6 &mu;l or 0.2 &mu;l bed volumes). Oligonucleotides or small (&lt;50 kDa) proteins/ peptides in aqueous solution</td>
 </tr>
 <tr>
 <td>MALDI TOF/TOF</td>
 <td>Bruker Daltonics ultraflex III TOF/TOF</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

peptide-based identification of functional motifs and their binding partners

全长蛋白的基序发现，在我们的例子中的HIV-1 Nef的是来自于特定的短肽，不仅保留其生物学功能，但也可以竞争性抑制的全长蛋白的功能。 20 Nef的扫描肽，20个氨基酸的长度与每个重叠10个氨基酸，它的邻居，一组被用来确定负责其诱导细胞凋亡的Nef的图案。包含这些细胞凋亡的基序的肽的诱导细胞凋亡的水平相媲美Nef蛋白的全长。第二肽，来自分泌修改地区（SMR）的Nef保留奈夫的分泌的外来（exNef）细胞的蛋白质参与互动的能力。作为“诱饵”蛋白此SMRwt肽免疫共沉淀实验中分离的细胞蛋白特异性结合的Nef的SMR动机。蛋白转染和抗体抑制使用的物理破坏的相互作用的赌注测定碱之间mortalin，隔离SMR-结合蛋白之一的，而且效果与的基于荧光exNef分泌测定。该SMRwt肽的能力胜过全长Nef的细胞蛋白质结合的SMR动机，使第一抑制剂分泌exNef的。因此，利用这里描述的技术，利用图案全长蛋白中发现的来自于特定的短肽的独特性能，可加速蛋白质中的功能性基序的识别和致病性的功能的肽类抑制剂的发展。

使用肽的生物功能图案映射。两个区域被确定在NEF蛋白诱导细胞凋亡。观察细胞凋亡肽驱动（ 图2）开始用肽N50（AA30-50），峰值在N60（AA40-60）和7610（AA50-70），并减少在肽N100（AA80-100）的背景水平。的主要母题1（M1）峰值，围绕AA50-60，诱导80％全长Nef蛋白的细胞凋亡水平。第二个较小的跨越肽N180（aa160-180）和N190（aa170-190）观察凋亡峰。这一个小小的母题2（M2）的高峰期...

Watch this Scientific Journal Video about Peptide-based Identification of Functional Motifs and their Binding Partners at JoVE.com

Peptide-based Identification of Functional Motifs and their Binding Partners

解剖机制的分泌HIV-1 Nef的外来技术进行了阐述。来自NEF和蛋白转染的特殊短肽被利用来确定Nef的分泌修改区域的结构，功能和有约束力的合作伙伴。这些程序在许多机理研究具有普遍意义。

肽为基础的识别功能图案和具有约束力的合作伙伴

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

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