A Luciferase Fusion Protein Library to Identify & Monitor Ubiquitylation Targets

调节蛋白水解在细胞内稳态中起着关键作用。许多蛋白质被作为破坏的目标 通过多聚泛素化，标记它们的蛋白酶体降解。人类的多泛素化涉及 E1泛素活化酶、E2泛素结合酶和E3泛素结合酶的顺序作用， 泛素连接酶估计有数百个潜在的E3，因此有数百个（如果不是数千个） 在人类蛋白质组中潜在的泛素化靶点。由E3底物组成的融合蛋白， 生物发光（或荧光）蛋白通常被相应的E3识别，并被靶向 降解这样的报告物然后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用于监测cdk 2 活性和氧的可用性，分别在完整的细胞。cdk 2对p27的磷酸化产生了一种 含有Skp 2的E3的结合位点和HIF的脯氨酰羟基化，其需要氧和2- 氧代戊二酸产生含E3的pVHL的结合位点。 在具体目标1中，我们将~ 12，000个开放阅读框（ORF）穿梭到ORF-荧光素酶融合载体中。 我们将询问这些ORF中有多少百分比的行为似乎是基于1）在2000 - 2005年的积累而聚泛素化的。 ts 20细胞中限制性温度，其具有温度敏感性E1等位基因和2）积累 在蛋白酶体抑制剂的存在下。这一目标应产生ORF-荧光素酶融合蛋白文库 它富含E3底物。这个图书馆将是发现小说的资源 目的E3（包括与疾病相关的E3）的底物和发现可 用于监测各种分子途径及其对药理学试剂的反应。非特异性 目的2和3我们将使用该文库来寻找新的pVHL底物（目的2）或用于小分子的新的报告基因。 分子羟化酶抑制剂（AIM 3）。在目标2和3中重新分离HIF将构成概念验证 关于这种方法的潜在效用。

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.