A Luciferase Fusion Library of High Throughput Profiling of Protein Stability

描述（由申请人提供）：通过泛素化-蛋白酶体途径的靶向蛋白质降解在包括癌症生物学在内的许多生物学领域中起着重要作用。蛋白质泛素化由一组三种酶（E1泛素活化酶E1、E2泛素缀合酶和E3泛素蛋白连接酶）进行，其将泛素转移到靶蛋白以发出被26 S蛋白酶体破坏的信号。许多肿瘤抑制基因和癌基因编码E3连接酶（包括pVHL、FBW 7、MDM 2、BRCA 1、Park 2和E6）或泛素化靶标（包括p53、E2 F-1、细胞周期蛋白、c-Myc和c-Jun）。此外，所有癌症相关的E3连接酶的潜在底物从未以高通量方式进行系统研究。建立人类基因组编码的所有E3连接酶的潜在底物的完整列表不仅将促进我们对生物学的理解，而且有助于开发更有效的治疗方法来治疗由异常靶向蛋白降解引起的人类疾病。众所周知，不稳定的遍在蛋白化靶点与生物发光报告基因（例如萤火虫荧光素酶）融合时，可以促进其融合伴侣的降解。因此，测量蛋白-X萤火虫荧光素酶融合蛋白的报道活性可以作为监测响应于给定信号的内源性蛋白-X的蛋白稳定性变化的有效手段。为此，我开发了一种高通量的双荧光素酶报告基因测定法，用于测量蛋白质的稳定性。该提议的目的是通过产生具有内置肾荧光素酶控制的人开放阅读框（ORF）萤火虫荧光素酶融合克隆的文库并进行原理验证实验以建立该文库用于筛选癌症或其他人类疾病中涉及的E3连接酶的底物和/或生物标志物的效用，将该方法的应用扩展到全基因组规模。

DESCRIPTION (provided by applicant): Targeted protein degradation via ubiquitination-proteasome pathway plays an important role in many areas of biology including cancer biology. Protein ubiquitination is carried out by a set of three enzymes (an E1 ubiquitin-activating enzyme E1, an E2 ubiquitin-conjugating enzyme and an E3 ubiquitin protein ligase), which transfers the ubiquitin to the target protein to signal for destruction by the 26S proteasome. A number of tumor suppressor genes and oncogenes encode either E3 ligases (including pVHL, FBW7, MDM2, BRCA1, Park2 and E6) or ubiquitination targets (including p53, E2F-1, the Cyclins, c-Myc and c-Jun). Moreover, the potential substrates of all cancer-related E3 ligases have never systematically investigated in a high throughput manner. Establishing a complete list of potential substrates of all E3 ligases encoded by human genome will not only advance our understanding of biology but also help develop more effective therapeutics for treating human diseases caused by aberrant targeted protein degradation. It is well established that unstable ubiquitination targets, when fused with a bioluminescence reporter (such as firefly luciferase), can promote the degradation of their fusion partners. Therefore, measuring the reporter activity of a Protein-X firefly luciferase fusion protein could serve as an effective means to monitor the change of protein stability of endogenous Protein-X in response to a given signal. To this end, I have developed a high throughput, dual- luciferase reporter assay for measuring protein stability. The objective of this proposal is to extend the application of this approach to a genome-wide scale by generating a library of human open reading frame (ORF) firefly luciferase fusion clones with built-in reniall luciferase control and performing proof-of-principle experiments to establish the utility of this library for screening substrates and/or biomarkers of E3 ligases involved in cancer or other human diseases.