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

描述(申请人提供)：泛素-蛋白酶体系统是细胞过程的主要调节者，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、细胞凋亡和信号转导)。泛素介导的关键底物的蛋白分解是由多个机器介导的，包括两个功能：泛素标记(即泛素结合酶和泛素连接酶)和蛋白质降解(即蛋白酶体)。泛素连接酶有助于将泛素转移到最终要降解的蛋白质中，通常由几个亚基(例如，SCF泛素连接酶复合体)组成，使这一过程具有高度的精确度。在人类中，有69个SCF连接酶，每个连接酶都有不变的结构元件(Skp1，Cul1和Rbx1)和一个可变的F-box蛋白亚基，通过直接将底物招募到连接酶的核心来提供特异性。值得注意的是，69个人SCF连接酶中只有9个(包含F-box蛋白2TrCP1、2TrCP2、Fbxw7、Skp2、Fbx13、Fbx15、Fbxo1、Fbxo4和Fbxo6)具有良好的底物和功能。剩下的60种F-box蛋白被认为是“孤儿”，它们的底物仍有待发现。Fbxo15是一种孤立的F-box蛋白，我们的初步数据和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。我们实验室已经成功地利用两种技术对F-box蛋白底物进行了无偏鉴定。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/酶分析方法，该方法基于SCF复合体在体外对共纯化底物泛素化的能力来丰富泛素化底物。在我们之前成功鉴定和鉴定F-box蛋白底物的基础上，我们提出了以下两个目标。我们将鉴定具有生物学意义的人Fbxo15底物(特定目标1)，并验证这些底物的Fbxo15依赖调节(特定目标2)。 公共卫生相关性：许多增殖性疾病，如癌症，都是由于分化过程中的缺陷而发展起来的。初步数据表明，泛素介导的Fbxo15底物的蛋白分解对控制哺乳动物细胞的分化状态很重要，该团队致力于将其基础研究整合到Fbxo15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.