Trimming the fat with small proteins: Micropeptides in adipogenesis

项目摘要/摘要 肥胖是一种持续的流行病，在美国，超过三分之二的成年人被认为超重或 太胖了。这些患者的治疗选择，包括FDA批准的所有减肥药物，对 长期减肥。缺乏有效的肥胖症治疗方法标志着知识的差距。白色脂肪 组织负责维持能量的储存和释放，以维持体内的燃料平衡。 能量摄入过多导致白色脂肪细胞功能紊乱是糖尿病发病的主要原因。 肥胖，导致胰岛素抵抗、2型糖尿病和其他代谢性疾病。最新进展 基因测序和质谱学技术使科学家能够识别出小开放阅读 框架(SmORF)，编码具有广泛生物学作用的功能微蛋白。这个 Saghatelian实验室率先开发了一套多组学平台，将蛋白质组学、核糖体图谱 (Ribo-Seq)和RNA测序，以准确注释少于150个密码子的smORF。通过表演Ribo- 在小鼠脂肪细胞模型上，申请人已经在小鼠基因组中鉴定出1721个新的smORF。 这些smORF中的绝大多数都是没有特征的。这项提案的主要目标就是解决这个问题 通过鉴定参与脂肪形成的新的脂肪细胞smORF(目标1)和胰岛素的知识差距 信令(目标2)。使用CRISPR/Cas9文库屏幕，专门针对1721个新的smORF， 申请者已经确定了大约20个特定抑制脂肪生成的smORF。Subaim 1.1将验证 这些发现通过生成smORF基因敲除的3T3-L1前脂肪细胞来评估特异性smORF 影响脂肪细胞分化，随后通过在基因敲除中过表达smORFs进行救援实验 细胞。在Subaim 1.2中，申请者将深入研究在Subaim中确定的特定微蛋白的功能 1.1通过鉴定其细胞定位和微蛋白与蛋白质的相互作用。在目标2中，申请者将利用 差异杂交(DO)小鼠的基因组和生理学数据以确定与 生理变化，如体重、血糖和甘油三酯。申请者将决定 在3T3-L1细胞中敲除分析确定的smORF是否会影响胰岛素信号转导和 正常条件下和胰岛素抵抗体外模型中的葡萄糖摄取。完成这些研究 将揭示smORF和微蛋白如何调控脂肪细胞分化和胰岛素信号转导。

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.