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

• 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
• 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

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.

项目总结/摘要 肥胖是一种持续的流行病，美国超过三分之二的成年人被认为超重或肥胖。 肥胖这些患者的治疗选择，包括所有FDA批准的抗肥胖药物，对 长期减肥。缺乏有效的治疗肥胖症的方法标志着知识的差距。白色脂肪 组织负责维持能量储存和释放以维持体内的燃料稳态。 由于过量的能量摄入而导致的白色脂肪细胞功能的破坏是肥胖的发病机制的主要因素。 肥胖，导致胰岛素抵抗，2型糖尿病和其他代谢疾病。的最新进展 基因测序和质谱技术使科学家能够识别小的开放阅读 框架（smORFs），编码具有广泛生物学作用的功能性微蛋白。的 Saghatelian实验室开创了一套多组学平台，整合了蛋白质组学、核糖体分析 （Ribo-Seq）和RNA测序来准确地注释少于150个密码子的smORF。通过表演里波- 通过对鼠脂肪细胞模型的测序，申请人在小鼠基因组中鉴定了1721个新的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一问题 通过表征参与脂肪形成（目标1）和胰岛素的新型脂肪细胞smORF， 信号（目标2）。使用特异性靶向1721个新smORF的CRISPR/Cas9文库筛选， 申请人已经鉴定了约20种特异性抑制脂肪生成的smORF。Subaim 1.1将验证 这些发现通过产生smORF敲除3 T3-L1前脂肪细胞来评估特异性smORF 影响脂肪细胞分化，然后通过敲除中过表达smORF进行拯救实验 细胞在Subaim 1.2中，申请人将深入研究Subaim中鉴定的特定微蛋白的功能。 1.1通过表征其细胞位置和微蛋白-蛋白质相互作用。在目标2中，申请人将利用 来自Diversity Outbred（DO）小鼠的基因组和生理学数据，以鉴定与以下相关的smORF： 生理变化，如体重、血糖和甘油三酯。申请人将决定 在3 T3-L1细胞中敲除从该分析中鉴定的smORF是否会影响胰岛素信号传导， 在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和微蛋白如何调节脂肪细胞分化和胰岛素信号传导。