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

SCF/Fbxo15 泛素连接酶在干细胞和癌症生物学中的作用

基本信息

批准号：
8274655
负责人：
MICHELE PAGANO
金额：
$ 18.38万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2014-06-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8274655
关键词：
Affinity Chromatography Apoptosis Basic Science Biochemical Biological Assay Bortezomib Cancer Biology Cell Cycle Progression Cell Differentiation process Cell physiology Cells Clinic Clinical Commit Complex Data Defect Development Disease Elements Embryo Ensure Equilibrium F-Box Proteins Funding Opportunities Genetic Genetic Transcription Grant Human In Vitro Individual Investigation Laboratories Lead Ligase Link Malignant - descriptor Malignant Neoplasms Mammalian Cell Mediating Molecular Oncogene Proteins Orphan Paper Pathway interactions Patients Pharmacologic Substance Phenotype Process Proteasome Inhibitor Protein Subunits Proteins Proteolysis Publishing Recruitment Activity Regulation Research Role Sampling Screening procedure Signal Transduction Specificity Speed Stem cells System Techniques Time Tumor Stem Cells Tumor Suppressor Proteins Ubiquitin Ubiquitin-Conjugating Enzymes Ubiquitin-mediated Proteolysis Pathway United States Food and Drug Administration Work adult stem cell base cancer cell cancer stem cell cancer therapy cell behavior embryonic stem cell genetic regulatory protein inhibitor/antagonist mouse model multicatalytic endopeptidase complex novel overexpression precursor cell protein complex protein degradation protein function protein protein interaction response self-renewal small molecule small molecule libraries stem cell biology stem cell division success tissue culture tumor ubiquitin ligase

项目摘要

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).

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者，其中速度、特异性和时间安排至关重要（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蛋白水解是由多种机器介导的，涵盖两种功能：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）。泛素连接酶有助于将泛素转移到要降解的蛋白质上，通常由多个亚基（例如 SCF 泛素连接酶复合物）组成，为该过程提供了高精度。在人类中，有 69 种 SCF 连接酶，每种都具有不变的结构元件（Skp1、Cul1 和 Rbx1）和可变的 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，69 种人类 SCF 连接酶（包含 F-box 蛋白 2TrCP1、2TrCP2、Fbxw7、Skp2、Fbxl3、Fbxl5、Fbxo1、Fbxo4 和 Fbxo6）中只有 9 种具有成熟/可接受的底物和功能。其余 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干细胞自我更新和分化的控制。我们的实验室已成功利用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，我们已经鉴定了新型 SCF 底物，但我们还开发了一种新型免疫亲和/酶测定，基于 SCF 复合物在体外泛素化共纯化底物的能力，可以富集泛素化底物。基于我们之前在识别和表征 F-box 蛋白底物方面取得的成功，我们提出以下两个目标。我们将鉴定人类 Fbxo15 具有生物学意义的底物（具体目标 1），并验证这些底物的 Fbxo15 依赖性调节（具体目标 2）。