Role of the SCF/Fbxo15 ubiquitin ligase in stem cells and cancer biology

描述（由申请人提供）：泛素-蛋白酶体系统是细胞过程的主要调节器，其中速度、特异性和时机是关键的（例如，细胞周期进程、基因转录、细胞分化、凋亡和信号转导）。泛素介导的关键底物的蛋白水解由多个机器介导，包括两个功能：泛素标记（即泛素缀合酶和泛素连接酶）和蛋白质降解（即蛋白酶体）。泛素连接酶，其有助于将泛素转移到预定降解的蛋白质，通常由几个亚基组成（例如，SCF泛素连接酶复合物），其赋予该方法高度的精确度。在人类中，存在69种SCF连接酶，每种SCF连接酶的特征在于不变的结构元件（Skp 1、Cul 1和Rbx 1）和可变的F盒蛋白亚基，所述可变的F盒蛋白亚基通过将底物直接募集到连接酶的核心来提供特异性。值得注意的是，69种人SCF连接酶中只有9种（含有F-box蛋白2 TrCP 1、2 TrCP 2、Fbxw 7、Skp 2、Fbxl 3、Fbxl 5、Fbxo 1、Fbxo 4和Fbxo 6）具有良好建立/接受的底物和功能。剩下的60种F-box蛋白被认为是“孤儿”，它们的底物仍在等待发现。Fbxo 15是一种孤儿F-box蛋白，我们的初步数据和已发表的研究结果表明，它参与了干细胞自我更新和分化的控制。我们的实验室已经成功地利用两种技术的F-盒蛋白底物的无偏鉴定。使用传统的串联亲和纯化，我们已经确定了新的SCF底物，但我们也开发了一种新的免疫亲和/酶法测定，富集泛素化底物的能力的基础上的SCF复合物泛素化共同纯化的底物在体外。基于我们以前在鉴定和表征F-box蛋白底物方面的成功，我们提出了以下两个目标。我们将鉴定人Fbxo 15的生物学重要底物（特异性目的1），并验证这些底物的Fbxo 15依赖性调节（特异性目的2）。 公共卫生相关性：许多增殖性疾病，如癌症，由于分化过程中的缺陷而发展。初步数据表明，泛素介导的Fbxo 15底物的蛋白水解在控制哺乳动物细胞的分化状态中非常重要，该团队致力于将其基础研究整合到Fbxo 15底物降解的分子机制中，并了解恶性转化。对调节细胞自我更新和分化的途径的详细了解可能会导致癌症的新疗法。

DESCRIPTION (provided by applicant): The ubiquitin-proteasome system is a major regulator of cellular processes in which speed, specificity, and timing are critical (e.g., cell cycle progression, gene transcription, cell differentiation, apoptosis, and signal transduction). Ubiquitin-mediated proteolysis of key substrates is mediated by multiple machines encompassing two functions: ubiquitin-tagging (i.e. ubiquitin conjugating enzymes and ubiquitin ligases) and protein degradation (i.e. the proteasome). Ubiquitin ligases, which contribute to transfer ubiquitin to proteins destined for degradation, are often composed of several subunits (e.g., the SCF ubiquitin ligase complexes) that impart a high degree of precision to the process. In humans, there are 69 SCF ligases, each characterized by invariable, structural elements (Skp1, Cul1, and Rbx1) and a variable F- box protein subunit that provides specificity by directly recruiting the substrate to the core of the ligase. Notably, only 9 of the 69 human SCF ligases (containing the F-box proteins 2TrCP1, 2TrCP2, Fbxw7, Skp2, Fbxl3, Fbxl5, Fbxo1, Fbxo4, and Fbxo6) have well-established/accepted substrates and functions. The remaining 60 F-box proteins are considered "orphans," and their substrates still await discovery. Fbxo15 is an orphan F-box protein that our preliminary data and published findings suggest to be involved in the control of stem cell self-renewal and differentiation. Our laboratory has successfully utilized two techniques for unbiased identification of F-box protein substrates. Using traditional tandem affinity purifications, we have identified novel SCF substrates, but we also developed a novel immunoaffinity/enzymatic assay that enriches for ubiquitylated substrates based on the ability of SCF complexes to ubiquitylate co-purified substrates in vitro. Based on our previous success in identifying and characterizing F-box protein substrates, we propose the following two aims. We will identify biologically significant substrates of human Fbxo15 (Specific Aim 1) and validate the Fbxo15-dependent regulation of these substrates (Specific Aim 2). PUBLIC HEALTH RELEVANCE: Many proliferative diseases, such as cancer, develop due to defects in differentiation processes. Preliminary data suggests that the ubiquitin-mediated proteolysis of Fbxo15 substrates is important in controlling the differentiation state of mammalian cells, and this team is committed to the integration of its basic research into the molecular mechanisms of Fbxo15 substrate degradation with an understanding of malignant transformation. A detailed understanding of the pathways that regulate the self-renewal and differentiation of cells may lead to novel therapies for cancer.