Trimming the fat with small proteins: Micropeptides in adipogenesis

用小蛋白质减少脂肪：脂肪生成中的微肽

基本信息

批准号：
10655394
负责人：
Victor Jieh Pong Pai
金额：
$ 7.43万
依托单位：
SALK INSTITUTE FOR BIOLOGICAL STUDIES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10655394
关键词：
3T3-L1 Cells Address Adipocytes Adipose tissue Adult Affect Alanine Algorithms Anti-Obesity Agents Antibodies Biological Biology Blood Glucose Body Weight Body Weight decreased CRISPR library CRISPR screen CRISPR/Cas technology Cell Line Cells Code Codon Nucleotides Computational algorithm DNA sequencing Data Databases Drug Targeting Energy Intake Epidemic FDA approved Fatty Liver Fatty acid glycerol esters Generations Genes Genetic Genetic Variation Genome Genomics Goals Homeostasis Human Genome Hypertrophy Immunofluorescence Immunologic Immunoprecipitation Individual Inflammation Insulin Resistance Knock-out Knowledge Laboratories Liver Location Mass Spectrum Analysis Metabolic Diseases Methods Molecular Biology Monitor Mouse Strains Mus Mutagenesis Non-Insulin-Dependent Diabetes Mellitus Nonesterified Fatty Acids Obesity Open Reading Frames Output Overweight Pathogenesis Pathway interactions Patients Peptides Phenotype Phosphorylation Physiological Physiology Play Proteins Proteomics Publishing Retinal blind spot Role Scanning Scientist Serine Signal Transduction Technology Testing Translating Triglycerides Validation Work adipocyte biology adipocyte differentiation candidate validation cohort diverse data effective therapy experience experimental study glucose uptake in vitro Model insulin signaling interest lipid biosynthesis live cell imaging mouse genome mouse model multiple omics new therapeutic target next generation novel obesity treatment overexpression protein protein interaction ribosome profiling transcriptome sequencing

项目摘要

Project Summary/Abstract Obesity is an ongoing epidemic, with more than two-thirds of adults in the US considered to be overweight or obese. Treatment options for these patients, including all FDA approved anti-obesity drugs, are ineffective for long term weight loss. The lack of effective treatments for obesity signals a gap in knowledge. The white adipose tissue is responsible for maintaining energy storage and release to maintain fuel homeostasis in the body. Disruption of white adipocyte function due to excess energy intake is a major contributor to the pathogenesis of obesity, resulting in insulin resistance, type 2 diabetes, and other metabolic diseases. Recent advancements in gene sequencing and mass spectrometry technology have allowed scientists to identify small Open Reading Frames (smORFs), which encodes functional microproteins that have a wide range of biological roles. The Saghatelian laboratory has pioneered a suite of multi-omic platforms that integrate proteomics, ribosome profiling (Ribo-Seq), and RNA sequencing to accurately annotate smORFs of less than 150 codons. By performing Ribo- Seq on models of murine adipocytes, the applicant has identified 1721 novel smORFs in the mouse genome. The great majority of these smORFs are uncharacterized. This major goal in this proposal is to address this knowledge gap by characterizing novel adipocyte smORFs that are involved in adipogenesis (Aim 1) and insulin signaling (Aim 2). Using a CRISPR/Cas9 library screen specifically targeting the 1721 novel smORFs, the applicant has already identified ~20 smORFs that specifically inhibit adipogenesis. Subaim 1.1 will validate these findings through generation of smORF knockout 3T3-L1 preadipocytes to assess how specific smORFs affect adipocyte differentiation, followed by rescue experiments through overexpressing smORFs in knockout cells. In Subaim 1.2, the applicant will dive deep into the function of a specific microprotein identified in Subaim 1.1 by characterizing its cellular location and microprotein-protein interaction. In Aim 2, the applicant will exploit the genomic and physiology data from the Diversity Outbred (DO) mice to identify smORFs that correlates with physiological changes, such as body weight, blood glucose, and triglycerides. The applicant will determine whether knocking out the smORFs identified from this analysis in 3T3-L1 cells will affect insulin signaling and glucose uptake under normal conditions and in in vitro models of insulin resistance. Completion of these studies will reveal how smORF and microproteins regulate adipocyte differentiation and insulin signaling.

项目概要/摘要肥胖是一种持续存在的流行病，超过三分之二的美国成年人被认为体重超重或肥胖。这些患者的治疗选择，包括 FDA 批准的所有抗肥胖药物，对以下患者无效长期减肥。缺乏有效的肥胖治疗方法标志着知识上的差距。白色脂肪组织负责维持能量储存和释放，以维持体内燃料稳态。由于能量摄入过多而导致的白色脂肪细胞功能的破坏是导致肥胖症发病的主要原因肥胖，导致胰岛素抵抗、2型糖尿病和其他代谢性疾病。最近的进展基因测序和质谱技术使科学家能够识别小型开放阅读框架（smORF），编码具有广泛生物学作用的功能性微生物蛋白。这 Saghatelian 实验室率先推出了一套集成蛋白质组学、核糖体分析的多组学平台 (Ribo-Seq) 和 RNA 测序，以准确注释少于 150 个密码子的 smORF。通过执行 Ribo- 通过对小鼠脂肪细胞模型进行 Seq，申请人在小鼠基因组中鉴定出了 1721 个新型 smORF。这些 smORF 中的绝大多数是未表征的。本提案的主要目标是解决这个问题通过表征参与脂肪生成（目标 1）和胰岛素的新型脂肪细胞 smORF 来弥补知识差距信号（目标 2）。使用专门针对 1721 个新型 smORF 的 CRISPR/Cas9 文库筛选，申请人已经鉴定出约 20 个特异性抑制脂肪生成的 smORF。 Subaim 1.1 将验证这些发现是通过生成 smORF 敲除的 3T3-L1 前脂肪细胞来评估特异性 smORF 的作用影响脂肪细胞分化，然后通过敲除中过度表达 smORF 进行拯救实验细胞。在 Subaim 1.2 中，申请人将深入研究 Subaim 中识别的特定微生物蛋白的功能 1.1 通过表征其细胞位置和微生物蛋白质-蛋白质相互作用。在目标 2 中，申请人将利用来自多样性远交 (DO) 小鼠的基因组和生理学数据，以识别与相关的 smORF 生理变化，例如体重、血糖和甘油三酯。申请人将决定敲除 3T3-L1 细胞中通过分析确定的 smORF 是否会影响胰岛素信号传导以及正常条件下和体外胰岛素抵抗模型中的葡萄糖摄取。完成这些研究将揭示 smORF 和微生物蛋白如何调节脂肪细胞分化和胰岛素信号传导。