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。许多蛋白质被多聚泛素化作用破坏，这标志着它们被蛋白酶体降解。人类的多泛素化涉及E1泛素活化酶、E2泛素缀合酶和E3泛素连接酶的顺序作用。据估计，在人类蛋白质组中有数百个潜在的E3，因此有数百个（如果不是数千个）潜在的泛素化靶点。由E3底物和生物发光（或荧光）蛋白组成的融合蛋白通常被相应的E3识别并靶向降解。这样的报道分子然后可以用于监测影响底物部分的稳定性的信号。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cdk 2活性和氧可用性。cdk 2对p27的磷酸化产生含有Skp 2的E3的结合位点，并且HIF的脯氨酰羟基化（其需要氧和2-酮戊二酸）产生含有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）穿梭到ORF-荧光素酶融合载体中。我们将询问这些ORF中有多少百分比表现得好像它们是聚泛素化的，这是基于1）在限制性温度下在ts 20细胞中的积累，ts 20细胞具有温度敏感性E1等位基因，以及2）在蛋白酶体抑制剂存在下的积累。这个目标应该产生一个文库的ORF-荧光素酶融合蛋白，是高度富集的E3底物。该库将是发现感兴趣的E3（包括与疾病相关的E3）的新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的报告分子的资源。在具体的目标2和3中，我们将使用该文库来搜索新的pVHL底物（目标2）或小分子羟化酶抑制剂的新报告物（目标3）。在目标2和3中重新分离HIF将构成关于该方法的潜在效用的概念证明。

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.