| Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate
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Author:
Date:
2016-10-05
[Abstract] This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin chains as alternative substrates. Although there are commercially available antibodies, we have not tested their performance in this assay since, but used validated antibodies from our laboratory. An alternative antibody-independent method, the use of phos-tag gels to detect pS65-ubiquitin and pS65-Parkin, is described in addition.
[摘要] 该协议描述了通过激酶PINK1使用重组蛋白的泛素和帕金蛋白的体外磷酸化。两种底物，泛素和帕金蛋白，在保守的丝氨酸65残基（pS65-泛素和pS65-帕金蛋白）上磷酸化。该方案还包括使用单体和K48和K63连接的聚泛素链作为替代底物。虽然有市售的抗体，我们没有测试他们在这个测定中的性能，因为，但使用我们实验室的验证抗体。另外描述了另一种抗体非依赖性方法，使用phos-标记凝胶检测pS65-泛素和pS65-帕金。
[背景] 在细胞中， PINK1是稳定和激活的线粒体膜去极化和其他形式的应力，导致线粒体损伤。活化的PINK1磷酸化泛素，其作为线粒体表面上胞质E3泛素连接酶Parkin的受体。 Parkin对PINK1的磷酸化是Parkin对线粒体底物的完全活性所必需的。活性pS65-Parkin的存在在前馈机制中扩增了作为线粒体标记的线粒体上的pS65-泛素的量。最终，受损的线粒体被自噬噬菌体衔接子识别，并将被蛋白酶体和自噬（mitophagy）降解。这种关键的线粒体质量控制通路促进线粒体的周转，并防止可导致细胞变性的功能障碍线粒体的积累。 PINK1或Parkin中的功能缺失突变与早发性帕金森病相关。
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| ACE-score-based Analysis of Temporal miRNA Targetomes During Human Cytomegalovirus Infection Using AGO-CLIP-seq
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Author:
Date:
2016-04-20
[Abstract] Although temporal regulation of gene expression during the course of infection is known to be critical for determining the outcome of host-virus interactions, systematic temporal analysis of the miRNA targetomes during productive viral infection has been technically challenging due to the large range of miRNA-mRNA cross-talks at the host-virus interface. High-confidence quantifying models of the suppression efficacy in targeting sites by integrating bioinformatics with Argonaute-crosslinking and immunoprecipitation followed by high-throughput sequencing (AGO-CLIP-seq) (Chi et al., 2009) data have been poorly developed. To accurately identify miRNA target sites and calculate the targeting efficacy of miRNA-target interactions, we developed a new bioinformatic quantitation method, ...
[摘要] 尽管已知在感染过程中基因表达的时间调节对于确定宿主 - 病毒相互作用的结果是至关重要的，但是在生产性病毒感染期间对miRNA targetomes的系统时间分析在技术上是具有挑战性的，因为大范围的miRNA- mRNA在主机 - 病毒接口交叉对话。数据通过将生物信息学与Argonaute-交联和免疫沉淀接着高通量测序（AGO-CLIP-seq）数据（Chi等人，2009）数据结合，已经不发达。为了准确地鉴定miRNA靶位点并计算miRNA-靶相互作用的靶向效果，我们开发了新的生物信息学定量方法，即AGO-CLIP-seq富集（ACE） - 评分算法（Kim等， 2015）。在我们的AGO-CLIP-seq分析中包括未感染的对照可以显着提高病毒或人miRNA的真实靶位点识别的准确性，并且在我们的ACE评分方法中提取生产性人巨细胞病毒（HCMV）感染期间的生理学显着变化。因此，我们建议我们新的基于ACE评分的方法可以应用于各种miRNA targetome研究，这将在其他类型的时间背景下进行，如发展阶段，细胞因子或病原体的免疫刺激和其他病毒。
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| In vitro Deneddylation Assay
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Author:
Date:
2016-03-20
[Abstract] Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated by cycles of neddylation (CulA-N8, ~98 kDa) and deneddylation (CulA ~89 kDa). The COP9 signalosome (CSN) and Deneddylase A (DenA) are capable of cleaving the isopeptide bond between Nedd8 and CullinA. In contrast to the single protein DenA, CSN is an eight subunit multiprotein complex. Protein crude extracts of different Aspergillus nidulans csn deletion strains were mixed with recombinant CSN subunits expressed and purified from Escherichia coli (E. coli). Western ...
[摘要] Nedd8是共价连接到作为cullin-RING连接酶（CRL）的一部分的cullin蛋白的保守赖氨酸残基的小的遍在蛋白样蛋白（9kDa）。 CRL是对泛素 - 蛋白酶体途径（UPP）中的蛋白质泛素化重要的主要E3连接酶。 CRL的活性通过脱甲基化（CulA-N8，〜98kDa）和脱甲基化（CulA〜89kDa）的循环调节。 COP9信号体（CSN）和丁二酸酶A（DenA）能够切割Nedd8和CullinA之间的异肽键。与单一蛋白DenA相反，CSN是八亚基多蛋白复合物。将不同的构巢曲霉csn 缺失菌株的蛋白质粗提物与从大肠杆菌（大肠杆菌）表达和纯化的重组CSN亚基混合。使用抗CulA或抗Nedd8抗体的Western杂交实验可以显示Nedd化化合物与Denedd化CulA的比率。使用deneddylation测定，我们可以显示CsnE是在体外连接7-亚基预组装的CSN的最后一个亚基，然后CSN可以通过金属蛋白酶亚基CsnE执行cullin deneddylation。该测定法是一种快速且非昂贵的方法，其显现了用于脱蛋白的蛋白质的酶活性。它还可用于测试除去来自构巢曲霉（构巢曲霉）或其他生物体中的底物的翻译后修饰的其它藻肽的活性。
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