A Luciferase Fusion Protein Library to Identify & Monitor Ubiquitylation Targets

描述（由申请人提供）：调节蛋白水解在细胞稳态中起关键作用。许多蛋白质是多泛素化破坏的目标，这标志着它们的蛋白酶体降解。人类多泛素化涉及E1泛素激活酶、E2泛素结合酶和E3泛素连接酶的顺序作用。据估计，人类蛋白质组中有数百种潜在的e3，因此有数百种（如果不是数千种的话）潜在的泛素化靶点。融合蛋白由E3底物和生物发光（或荧光）蛋白组成，通常被相应的E3识别并靶向降解。这样的报告器可以用来监测影响衬底部分稳定性的信号。例如，p27-荧光素酶和hif -荧光素酶融合蛋白已分别用于监测完整细胞中的cdk2活性和氧可用性。cdk2磷酸化p27产生含有Skp2的E3的结合位点，HIF的脯氨酰羟基化（需要氧和2-氧葡萄糖酸酯）产生含有pVHL的E3的结合位点。在具体目标1中，我们将把-12,000个开放阅读帧（orf）穿梭到orf -荧光素酶融合载体中。我们将询问这些orf中有多少%表现得像多泛素化，这是基于：1)在ts20细胞的限制性温度下的积累，ts20细胞含有温度敏感的E1等位基因；2)在蛋白酶体抑制剂存在下的积累。这一目标应该产生一个orf -荧光素酶融合蛋白库，该蛋白库对E3底物高度富集。该文库将成为发现感兴趣的E3s（包括与疾病相关的E3s）的新底物和发现可用于监测各种分子途径及其对药理学药物反应的报告因子的资源。在特定的目标2和3中，我们将使用该文库搜索新的pVHL底物（目标2）或小分子羟化酶抑制剂的新报告（目标3）。在目标2和3中重新隔离HIF将证明这一方法的潜在效用。

DESCRIPTION (provided by applicant): 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 1 we 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. In specific 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.