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：
8168513
负责人：
MICHELE PAGANO
金额：
$ 22.05万
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 2013-06-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8168513
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). 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.

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剂，其中速度，特异性和时机至关重要（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的蛋白水解是由多个机器介导的，其中包括两个功能：泛素蛋白 - 诱使（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原来降解的蛋白质中，通常由几个亚基（例如SCF泛素连接酶复合物）组成，这些亚基赋予了高度的高度精度。在人类中，有69个SCF连接酶，每个连接酶以不变的结构元素（SKP1，CUL1和RBX1）为特征，以及一个可变的F-Box蛋白亚基，可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，在69个人类SCF连接酶（包含F-box蛋白2TRCP1、2TRCP2，FBXW7，SKP2，FBXL3，FBXL3，FBXL5，FBXO1，FBXO1，FBXO4和FBXO6）中，只有9个具有良好的/公认的基础和功能。其余的60个F-box蛋白被认为是“孤儿”，其底物仍在等待发现。 FBXO15是一种孤儿F-box蛋白，我们的初步数据和发表的发现表明与干细胞自我更新和分化的控制有关。我们的实验室已成功利用了两种技术来无偏鉴定F-box蛋白底物。使用传统的串联亲和力净化，我们已经确定了新型的SCF底物，但我们还开发了一种新型的免疫亲和力/酶促测定，该测定法基于SCF复合物对泛素化合物在体外的泛素化底物的能力而富含泛素化的底物。基于我们以前在识别和表征F-box蛋白底物方面的成功，我们提出了以下两个目标。我们将确定人类FBXO15的生物学意义（特定目标1），并验证对这些底物的FBXO15依赖性调节（特定目标2）。公共卫生相关性：由于分化过程中的缺陷，许多增生性疾病（例如癌症）出现。初步数据表明，FBXO15底物的泛素介导的蛋白水解对于控制哺乳动物细胞的分化状态很重要，并且该团队致力于将其基础研究整合到FBXO15底物去分解的分子机制中，以了解对恶性转化的理解。对调节细胞自我更新和分化的途径的详细理解可能会导致对癌症的新疗法。