| In vitro Ubiquitin Dimer Formation Assay
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Author:
Date:
2017-01-05
[Abstract] The process of protein ubiquitination typically consists of three sequential steps to add an ubiquitin (Ub) or Ub chain to a substrate protein, requiring three different enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Most E2s possess the classical E2 activity in forming E2-Ub complex through a thioester linkage, in presence of an E1 and Ub. Additionally, some E2s have the ability of catalyzing the formation of free Ub dimer. Such activity indicates an important role of these E2s in ubiquitination pathway. Thus, we developed an in vitro Ub dimer formation assay to determine the activity of certain E2s. Moreover, by using Ub mutants, in which different lysine residues are mutated, the specific linkage of dimer can ...
[摘要] 蛋白质泛素化的过程通常包括三个顺序步骤：向底物蛋白添加泛素（Ub）或Ub链，需要三种不同的酶，泛素活化酶（E1），泛素缀合酶（E2）和泛素蛋白连接酶E3）。大多数E2在E1和Ub的存在下具有通过硫酯键形成E2-Ub复合物的经典E2活性。另外，一些E2具有催化游离Ub二聚体形成的能力。这种活性表明这些E2在泛素化途径中的重要作用。因此，我们开发了体外的Ub二聚体形成测定来确定某些E2的活性。此外，通过使用不同赖氨酸残基突变的Ub突变体，也可以确定二聚体的特异性连接。
背景 E2共轭启动试验（不添加E3和底物）的现有方案旨在检测硫酯键（E2-S-Ub）。我们的方法着重于催化游离Ub二聚体形成（Ub-Ub）的E2活性。提供了一种检测不同物种E2重要生化特征的便捷方式。此外，二聚体的特异性连接可以通过使用不同的Ub突变体来确定。
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| Conjugation of Duplexed siRNN Oligonucleotides with DD-HyNic Peptides for Cellular Delivery of RNAi Triggers
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Author:
Date:
2016-04-05
[Abstract] Despite the great promise that short interfering RNA (siRNA) induced RNAi responses hold as a therapeutic modality, due to their size (~15 kDa) and high negative charge (Bumcrot et al., 2006), siRNAs have no bioavailability and require a delivery agent to enter cells (Figure 1). TAT peptide transduction domain (PTD) has been developed as an agent that mediates cellular delivery of macromolecular therapeutics that otherwise lack bioavailability, making it a tantalizing candidate for siRNA delivery (Farkhani et al., 2014). Unfortunately, when conjugated to TAT PTD, the presence of 40 negative phosphodiester backbone charges on siRNA neutralizes the cationic PTD resulting in aggregation and poor cellular delivery (Meade and Dowdy, 2007). In light of this, we synthesized a ...
[摘要] 尽管由于它们的大小（〜15kDa）和高负电荷（Bumcrot等人，2006），短干扰RNA（siRNA）诱导的RNAi反应保持作为治疗模式的巨大希望，siRNA没有生物利用度，需要递送剂进入细胞（图1）。 TAT肽转导结构域（PTD）已经被开发为介导大分子治疗剂的细胞递送的药剂，否则其缺乏生物利用度，使其成为siRNA递送的诱人候选物（Farkhani等人，2014）。不幸的是，当缀合到TAT PTD时，siRNA上40个负磷酸二酯主链电荷的存在中和了导致聚集和差的细胞递送的阳离子PTD（Meade和Dowdy，2007）。鉴于此，我们合成了称为siRibo核中性的中性RNAi触发物，用于与TAT PTD缀合（Meade等人，2014）。简言之，带负电荷的磷酸二酯主链通过具有生物可逆磷酸三酯保护基团的合成来中和，其通过细胞质限制性硫酯酶的作用特异性地转化为细胞内的带电磷酸二酯键，产生可诱导RNAi应答的野生型siRNA。在这里我们描述了与TAT PTD递送结构域（DD）HyNic肽的siRNN寡核苷酸的缀合和细胞递送。
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| Chromatin Immunoprecipitation (ChIP), Streptavidin and ATP-agarose Mediated Pull-down Analyses
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Author:
Date:
2013-09-20
[Abstract] Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) induces expression of both viral and cellular genes in virus infected B cells by mimicking activated Notch receptors (Notch-IC) that mediate transcription activation through binding to the repressing domain of the recombining binding protein suppressor of hairless (RBP-Jκ). In general, chromatin immunoprecipitation (ChIP) assays, electrophoresis mobility shift assays (EMSA), streptavidin-agarose mediated DNA pull-down assays, together with cell-based transcription reporter assays were conducted to verify whether the query protein is involved in EBNA2-dependent transcription. The ATP-bound state of nuclear chaperone nucleophosmin (NPM1) has been implicated in pleiotropic biological processes. An ATP-agarose-mediated pull-down protocol was ...
[摘要] EB病毒（EBV）核抗原2（EBNA2）通过模拟激活的Notch受体（Notch-IC）诱导病毒感染的B细胞中病毒和细胞基因的表达，所述Notch受体通过结合重组结合的抑制结构域介导转录激活无毛蛋白抑制剂（RBP-Jκ）。通常，进行染色质免疫沉淀（ChIP）测定，电泳迁移率变动测定（EMSA），链霉亲和素 - 琼脂糖介导的DNA下拉测定以及基于细胞的转录报道基因测定，以验证查询蛋白是否参与EBNA2依赖性转录。核伴侣核蛋白（NPM1）的ATP结合状态已涉及多效生物过程。开发ATP-琼脂糖介导的下拉方案以监测由ATP结合的NPM1诱导的引发前复合物的形成。根据EBNA2和Notch-IC已经显示出在B细胞系中靶基因的活化方面是部分可互换的，可以想象EBNA2是活化的Notch IC的生物学等价物。
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