Comprehensive Identification of FXR1 Targets Using pSILAC-BONCAT Proteomics

使用 pSILAC-BONCAT 蛋白质组学全面鉴定 FXR1 靶标

基本信息

批准号：
9241667
负责人：
Viswanathan Palanisamy
金额：
$ 22.43万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-01-01 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9241667
关键词：
Affect Amino Acids Apoptosis Architecture Bioinformatics Biological Biological Assay Biological Process Bypass CDKN1A gene Cell Aging Cell Culture Techniques Cell Cycle Arrest Cell Line Cells Chemistry Clinic Data Development Disease Dissection Embryo Eukaryotic Cell Event FMR1 FXR1 gene Family Fibroblasts Foundations Fragile X Syndrome G1 Phase Gene Expression Gene Expression Alteration Gene Expression Profile Genes Genetic Genetic Transcription Genetic Translation Goals Head and Neck Cancer Human Immunoprecipitation Laboratories Malignant - descriptor Malignant Epithelial Cell Malignant Neoplasms Mass Spectrum Analysis Mediating Messenger RNA Molecular Molecular Profiling Mouth Carcinoma Mus Non-Small-Cell Lung Carcinoma Ontology Oral Pathway interactions Pattern Phenotype Physiologic pulse Play Polyribosomes Post-Transcriptional Regulation Predisposition Prevalence Proteins Proteome Proteomics Publishing RNA RNA Processing RNA-Binding Proteins Recruitment Activity Regulation Regulator Genes Reporting Research Role Stable Isotope Labeling Stimulus Stress Survival Rate Systems Biology TP53 gene Telomerase RNA Component Testing Transcription Process Translating Translations Tumor Tissue Validation Western Blotting aggressive therapy base cancer cell carcinogenesis conventional therapy experience genetic signature genome-wide head and neck cancer patient high throughput analysis keratinocyte knock-down loss of function mRNA Decay mRNA Stability malignant mouth neoplasm mouth squamous cell carcinoma mutant neoplastic cell new therapeutic target novel marker overexpression prevent protein expression screening senescence small hairpin RNA targeted treatment therapeutic target therapy resistant tumor tumor heterogeneity tumorigenesis

项目摘要

The purpose of this project is to determine the post-transcriptional regulation that changes the progression of oral cancer via the RNA-binding protein FXR1 and its mRNA translational control that govern cellular senescence. FXR1 was considered as a critical regulator of gene expression in most of the eukaryotic cells by controlling mRNA stability and translation. Thus, the main goal of this project is to understand the molecular mechanisms of FXR1-mediated mRNA translation and their implications in oral squamous cell carcinoma (OSCC) cellular senescence. Cellular senescence is an important mechanism for preventing the proliferation of potential cancer cells. Hence, underpinning the mechanistic aspects of cellular machinery by dissection the protein expression profiles provides novel biomarkers and/or therapeutic targets. Our laboratory has extensive experience in studying RNA-binding proteins and high throughput analysis of protein expression by pulsed-stable isotope labeling with amino acids in cell culture (pSILAC). We have developed a genome- wide shRNA screen of RBPs to test the cellular senescence in oral cancer cells. By studying oral cancer cells for cellular senescence, we have discovered FXR1 is overexpressed and depletion of FXR1 induces cellular senescence. Interestingly, FXR1 is a major player to control the expression of protein at the translational level that could mediate cellular senescence. Hence, we plan to systematically study the phenotypic changes of cellular senescence in oral cancer cells by understanding the expression of proteins that are controlled by FXR1. Thus, our central hypothesis is to understand how overexpression of FXR1 in oral cancer cells alter the protein expression by modulating RNA processing machinery to control cellular senescence. We propose the following specific aims to test this hypothesis: 1) to determine the critical role of FXR1 in bypassing OSCC senescence. We will test if the gain- and loss-of-function of FXR1 affects senescence in OSCC cells through various cell biological senescence assays, 2) to determine the consequence of FXR1 dysregulation on the cellular proteome. The protein targets of FXR1 will be identified using pulsed-Stable Isotope Labeling of Cells in cultured OSCC cells with click-chemistry Bioorthogonal Noncanonical Amino Acid Tagging (pSILAC- BONCAT) and Mass Spectrometry.

该项目的目的是确定改变转录后调控通过RNA结合蛋白FXR1及其控制的mRNA翻译控制口腔癌的进展细胞衰老。 FXR1被认为是大多数真核生物中基因表达的关键调节因子细胞通过控制 mRNA 稳定性和翻译。因此，该项目的主要目标是了解 FXR1介导的mRNA翻译的分子机制及其对口腔鳞状细胞的影响癌（OSCC）细胞衰老。细胞衰老是预防衰老的重要机制潜在癌细胞的增殖。因此，通过以下方式支撑细胞机器的机械方面：剖析蛋白质表达谱提供了新的生物标志物和/或治疗靶点。我们的实验室在研究RNA结合蛋白和蛋白表达的高通量分析方面拥有丰富的经验通过在细胞培养物中用脉冲稳定同位素标记氨基酸 (pSILAC)。我们开发了一个基因组—— RBP 的广泛 shRNA 筛选以测试口腔癌细胞的细胞衰老。通过研究口腔癌细胞对于细胞衰老，我们发现 FXR1 过度表达，并且 FXR1 的耗竭会诱导细胞衰老衰老。有趣的是，FXR1 是翻译水平上控制蛋白质表达的主要参与者可以介导细胞衰老。因此，我们计划系统地研究表型变化通过了解受控制的蛋白质的表达来研究口腔癌细胞的细胞衰老 FXR1。因此，我们的中心假设是了解口腔癌细胞中 FXR1 的过度表达如何改变通过调节RNA加工机制来控制细胞衰老的蛋白质表达。我们建议以下具体目的是检验这一假设：1) 确定 FXR1 在绕过 OSCC 中的关键作用衰老。我们将通过以下方式测试 FXR1 功能的获得和丧失是否会影响 OSCC 细胞的衰老：各种细胞生物衰老测定，2) 确定 FXR1 失调对细胞的影响细胞蛋白质组。 FXR1 的蛋白质靶标将使用细胞脉冲稳定同位素标记来识别在培养的 OSCC 细胞中使用点击化学生物正交非规范氨基酸标签 (pSILAC- BONCAT）和质谱分析。