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、特异性和时间至关重要（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质水解是由包含两个功能的多个机器介导的：泛素标记（即泛素偶联酶和泛素连接酶）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用于降解的蛋白质中，通常由几个亚基（例如，SCF泛素连接酶复合物）组成，这些亚基赋予该过程高度的精度。在人类中，有69个SCF连接酶，每个都具有不变的结构元件（Skp1， Cul1和Rbx1）和一个可变的F- box蛋白亚基，该亚基通过直接将底物募集到连接酶的核心来提供特异性。值得注意的是，在69个人类SCF连接酶（包含F-box蛋白2TrCP1、2TrCP2、Fbxw7、Skp2、Fbxl3、fbx15、Fbxo1、Fbxo4和Fbxo6）中，只有9个具有确定/接受的底物和功能。剩下的60个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了两种技术来公正地鉴定F-box蛋白底物。使用传统的串联亲和纯化，我们已经鉴定出了新的SCF底物，但我们也开发了一种新的免疫亲和/酶分析，基于SCF复合物在体外泛素化共纯化底物的能力，丰富了泛素化底物。基于我们之前在鉴定和表征F-box蛋白底物方面的成功，我们提出以下两个目标。我们将鉴定具有生物学意义的人类Fbxo15底物（Specific Aim 1），并验证这些底物的Fbxo15依赖性调节（Specific Aim 2）。

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.