Name: assaying proteasomal degradation in a cell-free system in plants
Uploaded: 2014-03-26T17:45:00
Description: Watch this Scientific Journal Video about Assaying Proteasomal Degradation in a Cell-free System in Plants at JoVE.com

这项工作导致本出版物已收到的资金从居里夫人COFUND节目“U-移动”，由马拉加大学和欧盟第七框架计划（FP7/2007-2013）根据GA编号246550共同出资。在我们实验室工作的支持，赠款从美国国立卫生研究院，美国农业部/ NIFA，美国国家科学基金会，BARD和BSF到VC

1。蛋白表达<ol><li> 表达系统的选择 选择系统，即向量和向量配送方式，最适合在特定的模式生物/细胞目的蛋白的表达。注意，我们的测定需要在容易可检测的量，这是最好的大批细胞的瞬时转化取得的测试蛋白质的表达。在植物中，例如，这是一个使用二进制质粒作为表达载体和农杆菌作为输送系统的最佳实现。 </li><li> 二元表达载体的构建 克隆感兴趣的蛋...

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该测定依赖于植物组织中的测试蛋白质的表达，因此，潜在的蛋白酶体降解过程显然已经发生的活体组织内。然而，我们测定蛋白质不稳定，仅在提取物，用时间为零的样品作为初始参考点。因此，我们将其定义为不含细胞的测定法。 对于该测定法的成功的一个重要的方面是正确的选择从其中被测试的蛋白质（s）将制备的表达载体的构建。除可对供试蛋白特异性抗体，它应?...

在泛素蛋白酶体途径正在成为一个普遍的机制，不同的生物反应，包括转录调控，细胞周期进程和信号转导，受体下调或胞吞作用，其中包括处理1-4的控制权。在此途径中，靶蛋白被标记由泛素，它们通过硫酯键的第一个连接到泛素激活酶E1，然后易位到的泛素缀合的酶E2的半胱氨酸氨基酸残基的残基;最后，E2具有泛素连接酶E3的交互，导致蛋白质底物的泛素化。最终，该多聚泛素蛋白被26S蛋白酶体识别并降解。在这种机制下，在E3酶指定的基板，并作为泛素蛋白酶体系统（UPS）的关键调控元件。 E3连接酶可以独立地采取行动，如RING结构域连接酶，或作为一个多亚基SCF（S部分kp1/Cullin/F-box蛋白）复合物，如F-盒结构域连接酶。 SCF-介导的蛋白酶体降解途径中涉及转录，细胞周期，信号转导5-10和许多其他主要的细胞功能的调节。 除了在细胞过程的调控这些关键角色，UPS采取中心舞台在许多植物 - 病原体相互作用。例如，越来越多的证据表明，一些植物病原体，包括根癌农杆菌，依赖于宿主的UPS，以促进感染过程11。土壤杆菌引发的植物，其中表示它的天然宿主的肿瘤生长，并且它也可以改变一个范围广泛的其他真核生物中，从真菌1,2对人类细胞12,13。在其感染时，根癌农杆菌出口的DNA元件（T-DNA）和几个毒力（虚象）的蛋白质进入宿主细胞12-13。这些蛋白质之一是virF等，找到的第一个F-box蛋白通过原核基因组14进行编码。由于SCF泛素连接酶复合物，virF等，其功能主机同源VBF 15的一部分， 通过 UPS介导的蛋白质降解，这大概是有利于入侵细菌的T-DNA的脱壳从与之配套的细菌和宿主蛋白，性virE2利于农杆菌感染和VIP1，分别为16,17。有趣的是，许多F-box蛋白，包括virF等，是由于自身蛋白酶解，这是由自身泛素化活性的18,19或由其他E3连接酶的量的F-box蛋白可以用作基板20-23介导的内在的不稳定。 当研究F-box蛋白，其它泛素连接酶，和/或它们的底物的生化活性，这将是非常有用的，采用一个简单而可靠的测定蛋白酶体降解。在这里，我们描述了一个这样的协议，用于分析在细胞蛋白质的稳定性- 免费的系统。在该测定中，UPS基板的稳定性在蛋白酶体降解途径中，诸如F-box蛋白，在无细胞系统的主要部件之一的存在或不存在进行了分析。一般来说，我们表示测试蛋白（S）在植物组织中，从这些组织准备的无细胞提取物和监测免疫印迹感兴趣的蛋白（S）的款项。蛋白降解的UPS依赖性机制被证明通过包含特定的蛋白酶体抑制剂和/或使用的一个SCF成分，卡林的显性负形式的共表达。而我们使用拟南芥VIP1 17蛋白的蛋白酶体降解的F-box蛋白VBF 15示出该试验中，它可被用来考察的其它任何蛋白酶底物的稳定性。

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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Protein assay kit</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">500-0001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Proteinase inhibitor cocktail&#160;</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mini-Protean system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">165-8000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Semi-dry western blotting SD electrotransfer system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">170-3940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Affinity Purified Rabbit Anti-Ha</td>
			<td style="width:129px;">icllab</td>
			<td>RHGT-45A-Z</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Goat anti-Rabbit IgG Peroxidase Conjugate</td>
			<td style="width:129px;">Thermo Scientific</td>
			<td style="width:119px;">31460</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">BioTrace, NT nitrocellulose transfer membrane</td>
			<td style="width:129px;">Pall Corportation</td>
			<td style="width:119px;">27377-000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Immobilon western chemiluminescent HRP substrate</td>
			<td style="width:129px;">EMD Millipore</td>
			<td style="width:119px;">WBKL S0 050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MG132</td>
			<td style="width:129px;">EMD Millipore</td>
			<td>474790-1MG</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:247px;">Lactacystin</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td>L6785</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Thermo Scientific Pierce Fast Western Blot Kit, ECL Substrate</td>
			<td style="width:129px;">Pierce</td>
			<td style="width:119px;">35055</td>
			<td></td>
		</tr>
	</tbody>
</table>

assaying proteasomal degradation in a cell-free system in plants

泛素 - 蛋白酶体途径降解蛋白质已经成为了一个广泛的细胞功能的频谱调控几乎所有真核生物中最重要的机制之一。具体而言，在植物中，泛素蛋白酶体系统（UPS），调节蛋白质降解和显著到的广​​泛的过程，包括免疫应答，开发和程序性细胞死亡的发展做出贡献。此外，越来越多的证据表明，许多植物病原体，如农杆菌 ，利用主机的UPS进行有效感染，强调的UPS在植物-病原体相互作用的重要性。 UPS的底物特异性是由E3泛素连接酶，其作用在音乐会与E1和E2连接酶识别和标记通过附加到它们的泛素分子链去往降解特定蛋白质分子来实现。一类的E3连接酶的是SCF（SKP1 / C阿林/ F-box蛋白）复合物，其特异性识别UPS的底物，并通过它的F-box蛋白组分瞄准他们对泛素化。调查的UPS中的感兴趣的生物过程中的潜在作用，设计一种简单，可靠的测定为UPS介导的蛋白质降解是重要的。在这里，我们描述了使用植物无细胞系统中的一个这样的测定。此法可以适用于不同的细胞过程调节的蛋白质降解的作用的研究，特别注重对F-box蛋白底物的相互作用。

图1中 ，用于从Zaltsman 等人 17，示出了用于检测蛋白酶体降解的无细胞系统代表实验。具体来说，我们通过 SCF VBF通路在全展示出一种植物防御相关蛋白VIP1由VBF F-box蛋白的不稳定本塞姆氏 。拟南芥VBF和HA标记VIP1（HA-VIP1）的蛋白质瞬时共表达，以及表达叶提取物中的HA-VIP1含量蛋白免疫印迹分析。这一分析表明，VIP1金额减少至一个显著程度，...

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Assaying Proteasomal Degradation in a Cell-free System in Plants

测定蛋白酶体降解的植物无细胞系统

有针对性的蛋白质降解是一个重大的监管机制，细胞的功能。它发生通过一个保守的泛素-蛋白酶体通路，其高度多聚泛素链的靶蛋白，然后作为分子的“标签”为26S蛋白酶体。在这里，我们描述了用于蛋白质的蛋白酶体降解的简单和可靠的无细胞测定法。

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再水化冻干水凝胶中的无细胞蛋白质合成