A Luciferase Fusion Protein Library to Identify & Monitor Ubiquitylation Targets

受调控的蛋白分解在细胞内稳态中起着关键作用。许多蛋白质都是被破坏的目标 通过多泛素化，这标志着它们的蛋白酶体降解。人类中的多泛素化涉及 E_1泛素活化酶、E_2泛素结合酶和E_3的顺序作用 泛素连接酶。据估计，有数百个潜在的E3，因此有数百个(如果不是数千个) 人类蛋白质组中潜在的泛素化靶标。由E3底物和 生物发光(或荧光)蛋白通常被相应的E3识别并被靶向 退化。这样的记录器然后可以用来监控影响衬底稳定性的信号 半个月。例如，p27-荧光素酶和hif-荧光素酶融合蛋白已被用来监测cdk2。 活性和氧气利用率，分别在完整的细胞。CDK2使p27磷酸化产生一种 含有Skp2和HIF的Pro羟基化的E3的结合部位，这需要氧气和2- 草戊二酸，为含有pVHL的E3产生结合位点。 在特定的目标1WE将穿梭-12,000个开放阅读框架(ORF)到一个开放阅读框架-荧光素酶融合载体。 我们会问，这些ORF中有多少%的行为就像它们是基于1)积累的多泛素化的 TS20细胞的限制性温度，它含有温度敏感的E1等位基因和2)积聚 在蛋白酶体抑制剂存在的情况下。这个目标应该会产生一个ORF-荧光素酶融合蛋白的文库。 这对于E3底物来说是高度浓缩的。这个图书馆将成为发现小说的资源 感兴趣的E3的底物(包括与疾病有关的E3)和发现可以 用于监测各种分子途径及其对药物的反应。不特定的 目标2和3我们将使用这个文库来寻找新的pVHL底物(目标2)或新的小报告 分子羟基酶抑制剂(目标3)。在AIMS 2和3中重新隔离HIF将构成概念证明 关于这一方法的潜在效用。

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.