| In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors
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Author:
Date:
2020-11-05
[Abstract] Glutamylation is a posttranslational modification where the amino group of a free glutamate amino acid is conjugated to the carboxyl group of a glutamate side chain within a target protein. SidJ is a Legionella kinase-like protein that has recently been identified to perform protein polyglutamylation of the Legionella SdeA Phosphoribosyl-Ubiquitin (PR-Ub) ligase to inhibit SdeA’s activity. The attachment of multiple glutamate amino acids to the catalytic glutamate residue of SdeA by SidJ inhibits SdeA’s modification of ubiquitin (Ub) and ligation activity. In this protocol, we will discuss a SidJ non-radioactive, in vitro glutamylation assay using its substrate SdeA. This will also include a second reaction to assay the inhibition of SdeA by using both modification of free Ub and ligation ...
[摘要] [摘要]谷氨酰化是翻译后修饰，其中游离谷氨酸氨基酸的氨基与目标蛋白内谷氨酸侧链的羧基缀合。SidJ是一种军团菌激酶样蛋白，最近被发现可对军团菌SdeA磷酸核糖泛素（PR- Ub ）连接酶进行蛋白多谷氨酰化，从而抑制SdeA的活性。SidJ将多个谷氨酸氨基酸附着到SdeA的催化谷氨酸残基上，从而抑制了SdeA对泛素的修饰（Ub ）和结扎活动。在此协议中，我们将讨论使用其底物SdeA的SidJ非放射性，体外谷氨酰化测定。这也将包括一个第二反应以测定抑制SdeA通过使用免费的两个修改泛素和结扎ADP-核糖基化的泛素（ADPR-泛素），以SdeA的基板Rab33b。在鉴定和公布SidJ的活性之前，尚无SdeA抑制试验。我们的小组和其他小组演示了各种方法来抑制SdeA的活性。备选方案包括使用放射性NAD测量Ub的ADP核糖基化，NAD水解以及SdeA对HA- Ub连接的蛋白质印迹分析。该方案将描述使用廉价的标准凝胶和考马斯染色对SdeA的泛素修饰和PR- Ub连接的抑制。
[背景]嗜肺军团菌是感染性细菌，其机会性感染肺泡巨噬细胞。这是通过吸入被污染的水气溶胶而发生的，引起潜在的致命性肺炎，称为军团菌病（McDade et ...
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| Radioactive Assay of in vitro Glutamylation Activity of the Legionella pneumophila Effector Protein SidJ
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Author:
Date:
2020-10-05
[Abstract] The Legionella effector protein SidJ has recently been identified to perform polyglutamylation on another Legionella effector, SdeA, ablating SdeA’s activity. SidJ is a kinase-like protein that requires the small eukaryotic protein calmodulin to perform glutamylation. Glutamylation is a relatively uncommon type of post-translational modification, where the amino group of a free glutamate amino acid is covalently linked to the γ-carboxyl group of a glutamate sidechain in a substrate protein. This protocol describes the SidJ glutamylation reaction using radioactive [U-14C] glutamate and its substrate SdeA, the separation of proteins by gel electrophoresis, preparation of gels for radioactive exposure, and relative quantification of glutamylation activity. This ...
[摘要] [摘要]在军团菌效应蛋白SidJ最近被确定为另一个执行polyglutamylation军团效应，SdeA，烧蚀SdeA的活动。SidJ是一种激酶样蛋白，需要小的真核蛋白钙调蛋白才能进行谷氨酰化。谷氨酰化是翻译后修饰的相对不常见的类型，其中游离谷氨酸氨基酸的氨基与底物蛋白质中谷氨酸侧链的γ-羧基共价连接。该协议描述了使用放射性[U- 14]的SidJ谷氨酰化反应 C]谷氨酸及其底物SdeA，通过凝胶电泳分离蛋白质，制备用于放射暴露的凝胶，以及相对定量的谷氨酰化活性。该方法可用于鉴定底物进行谷氨酰化，表征底物和由突变引起的谷氨酰胺酶活性，以及鉴定具有谷氨酰化活性的蛋白质。一些研究已经使用[ 3 H]谷氨酸（Regnard等，1998）和使用GT335抗体（Wolff等，1992）来分析谷氨酰化。但是，使用[U- 14 C] g谷氨酸盐需要更短的放射性暴露时间，而不依赖于抗体特异性。
[背景]嗜肺军团菌是感染细菌，造成军团病（麦克达德等人，1977） ，肺炎的一个潜在的致命的形式。在感染期间，Legionell ...
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| Preparation and Characterization of Ginger Lipid-derived Nanoparticles for Colon-targeted siRNA Delivery
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Author:
Date:
2020-07-20
[Abstract] Synthetic nanoparticle-based drug delivery system is widely known for its ability to increase the efficacy and specificity of loaded drugs, but it often suffers from relatively higher immunotoxicity and higher costs as compared to traditional drug formulations. Contrarily, plant-derived nanoparticles appear to be free from these limitations of synthetic nanoparticles; they are naturally occurring biocompatible vesicles that do not generate immunotoxicity and are easy to obtain. Additionally, lipids isolated from plant-derived nanoparticles have shown the capability of assembling themselves to spherical nano-sized liposomal particles. Herein, we employ lipids extracted from ginger-derived nanoparticles and load them with therapeutic siRNA (CD98-siRNA) to create CD98-siRNA/ginger-lipid ...
[摘要] [摘要 ]基于合成纳米颗粒的药物递送系统以其增加所载药物的功效和特异性的能力而广为人知，但是与传统药物制剂相比，它经常遭受相对较高的免疫毒性和较高的成本。相反，植物来源的纳米粒子似乎不受合成纳米粒子的这些限制；它们是天然存在的生物相容性囊泡，不会产生免疫毒性并且易于获得。另外，从植物来源的纳米颗粒分离的脂质已显示出将其自身组装成球形纳米尺寸脂质体颗粒的能力。在本文中，我们采用从生姜纳米颗粒中提取的脂质，并用治疗性siRNA（CD98-siRNA）加载脂质，以创建CD98-siRNA /生姜脂质纳米颗粒。CD98-siRNA /姜脂脂质纳米颗粒的表征显示它们呈球形，直径约为189.5 nm。纳米颗粒的表面ζ电势在-18.1至-18.4mV之间变化。此外，在最近的研究中，CD98-siRNA /姜脂纳米颗粒在右旋糖酐硫酸钠（DSS）诱发的结肠炎小鼠模型中显示出特定的结肠靶向能力和出色的抗炎功效。
[背景 ] 小干扰RNA（siRN As）是一种有前途的治疗剂，可以通过沉默异常上调的信使RNA（mRNA）来治疗各种疾病（Nikam and Gore ，2018）。尽管siRNA的有效性，安全有效地将siRNA递送至治疗靶标仍然是一项艰巨的任务（Tatiparti et al ...
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