A Luciferase Fusion Protein Library to Identify & Monitor Ubiquitylation Targets

用于识别的荧光素酶融合蛋白库

• 批准号: 7504000
• 负责人: WILLIAM G. KAELIN
• 金额: $ 20.52万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-15 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7504000
• 关键词: Alleles; Binding Sites; Biological Markers; Cells; Chimeric Proteins; Collection; Dioxygenases; Disease; Enzymes; Homeostasis; Human; Hydroxylation; Libraries; Link; Luciferases; Mixed Function Oxygenases; Molecular; Monitor; Open Reading Frames; Oxygen; Pathway interactions; Phosphorylation; Play; Proteins; Proteolysis; Proteome; Reporter; Resources; Role; Signal Transduction; Temperature; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes; alpha ketoglutarate; base; concept; inhibitor/antagonist; interest; novel; p27 Cell Cycle Protein; p27 Enzyme Inhibitor; response; small molecule; ubiquitin-protein ligase; vector

Regulated proteolysis plays a critical role in cellular homeostasis. Many proteins are targeted for destruction by polyubiquitylation, which flags them for proteasomal degradation. Polyubiquitylation in humans involves the sequential action of the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase. There are estimated to be hundreds of potential E3s and hence hundreds (if not thousands) of potential ubiquitylation targets in the human proteome. Fusion proteins consisting of an E3 substrate and a bioluminescent (or fluorescent) protein are often recognized by the corresponding E3 and targeted for degradation. Such reporters can then be used to monitor signals that influence the stability of the substrate moiety. For example, p27-Luciferase and HIF-luciferase fusion proteins have been used to monitor cdk2 activity and oxygen availability, respectively, in intact cells. Phosphorylation of p27 by cdk2 generates a binding site for an E3 containing Skp2 and prolyl hydroxylation of HIF, which requires oxygen and 2- oxoglutarate, generates a binding site for an E3 containing pVHL. In specific aim 1we will shuttle -12,000 open reading frames (ORFs) into an ORF-luciferase fusion vector. We will ask what % of these ORFs behave as though they are polyubiquitylated based on 1) accumulation at the restrictive temperature in ts20 cells, which harbor a temperature-sensitive E1 allele and 2) accumulation in the presence of a proteasomal inhibitor. This aim should yield a library of ORF-luciferase fusion proteins that is highly enriched for E3 substrates. This library would be a resource for the discovery of novel substrates for E3s of interest (including E3s linked to disease) and for the discovery of reporters that could be used to monitor various molecular pathways and their responses to pharmacological agents. Inspecific aim 2 and 3 we will use this library to search for novel pVHL substrates (aim 2) or novel reporters for small molecule hydroxylase inhibitors (aim 3). Reisolation of HIF in aims 2 and 3 would constitute proof of concept with respect to the potential utility of this approach.

调节的蛋白水解在细胞稳态中起着至关重要的作用。许多蛋白质成为破坏的目标 通过多泛素化，这将它们标记为蛋白酶体降解。人类的多泛素化涉及 E1 泛素激活酶、E2 泛素结合酶和 E3 的顺序作用 泛素连接酶。估计有数百个潜在的 E3，因此也有数百（如果不是数千） 人类蛋白质组中潜在的泛素化目标。由 E3 底物和 生物发光（或荧光）蛋白通常被相应的 E3 识别并靶向 降解。然后，此类报告基因可用于监测影响底物稳定性的信号 部分。例如，p27-荧光素酶和 HIF-荧光素酶融合蛋白已被用于监测 cdk2 分别是完整细胞中的活性和氧气利用率。 cdk2 磷酸化 p27 产生 含有 Skp2 的 E3 的结合位点和 HIF 的脯氨酰羟基化，需要氧气和 2- oxoglutarate，生成包含 pVHL 的 E3 的结合位点。 在具体目标 1 中，我们将-12,000 个开放阅读框 (ORF) 传送到 ORF-荧光素酶融合载体中。 我们将询问这些 ORF 中有多少百分比表现得好像它们是多泛素化的，基于 1) 的积累 ts20 细胞中的限制温度，该细胞含有温度敏感的 E1 等位基因和 2) 积累 在蛋白酶体抑制剂存在下。这个目标应该产生一个 ORF-荧光素酶融合蛋白文库 高度富集 E3 底物。这个图书馆将成为发现小说的资源 感兴趣的 E3（包括与疾病相关的 E3）的底物以及发现可以 可用于监测各种分子途径及其对药物的反应。不具体 目标 2 和 3 我们将使用该库来搜索新型 pVHL 底物（目标 2）或小型报告基因 分子羟化酶抑制剂（目标 3）。目标 2 和 3 中 HIF 的重新隔离将构成概念验证 关于这种方法的潜在效用。