Perilipin Phosphorylation as a Scaffold for 14-3-3 Proteins and Lipolytic Enzymes

Perilipin 磷酸化作为 14-3-3 蛋白质和脂肪分解酶的支架

• 批准号: 9430683
• 负责人: Andrew Reeves
• 金额: $ 0.19万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-14 至 2019-09-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9430683
• 关键词: Adaptor Signaling Protein; Adipocytes; Adipose tissue; Amino Acid Motifs; Amino Acids; Binding; Binding Proteins; Binding Sites; Blood; Body Composition; Body measure procedure; Brown Fat; CRISPR/Cas technology; Cartoons; Catecholamines; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Computer Simulation; Confocal Microscopy; Consensus; Cyclic AMP-Dependent Protein Kinases; Development; Diabetes Mellitus; Distal; Dominant-Negative Mutation; Energy Metabolism; Enzymes; Fasting; Fatty Acids; Fluorescence Resonance Energy Transfer; Genes; Glucose tolerance test; Goals; Grant; Image; Immunoprecipitation; In Vitro; Individual; Insulin; Insulin Resistance; Lead; Link; Lipase; Lipids; Lipolysis; Mass Spectrum Analysis; Measures; Mediating; Metabolism; Mus; Mutate; Mutation; Nonesterified Fatty Acids; Obesity; Peripheral; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphoserine; Phosphothreonine; Physiological; Physiology; Prevalence; Process; Protein Family; Protein Isoforms; Protein Kinase; Proteins; Proteomics; Recruitment Activity; Role; Sequence Analysis; Serine; Serum; Site; Surface; Techniques; Thermogenesis; Tissues; Training; Triglycerides; Western Blotting; Work; base; blood glucose regulation; experimental study; in vivo; insulin tolerance; mutant; new therapeutic target; novel; perilipin; perilipin A; protein complex; protein protein interaction; protein transport; scaffold; sterol esterase

PROJECT SUMMARY Due to the increasing prevalence of obesity in the world, understanding the mechanisms involved in adipocyte metabolism and the conditions that lead to insulin resistance and diabetes is important. In adipose tissue lipids are stored as triglyceride (TAG) within lipid droplets (LD) that are tightly regulated by an assortment of proteins. When the body needs energy, TAG from the LD is hydrolyzed to release free fatty acids (FFA) in a process called lipolysis. In fasting states, circulating FFAs are utilized by distal tissues for energy, whereas in brown adipocytes lipolysis provides fatty acids to be used for adaptive thermogenesis. In obese individuals, dysregulated lipolysis causes the release of an excess of FFA, which can lead to insulin resistance. Perilipin (plin1) is a protein found on the surface of LD that regulates lipolysis by acting as scaffold for many of the enzymes and adapter proteins involved with hydrolyzing TAG into FFA. The goal of this grant is to provide training for the applicant in order to study how activation of cyclic-AMP dependent protein kinase (PKA) regulates plin1 activity and the mechanisms by which plin1 recruits the lipolytic machinery to the LD surface, as well as its role in systemic metabolism. Specifically, our preliminary results show that a family of proteins called the 14-3-3 proteins associate with plin1 at a novel phosphorylation site that we hypothesize is a critical regulator of stimulated-lipolysis. We will study the role of 14-3-3 proteins binding to plin1 in regulating PKA- stimulated lipolysis using two different experimental approaches: 1) mutating the binding site within plin1 and 2) by expressing a dominant negative form of the 14-3-3 proteins, thereby sequestering the 14-3-3 proteins, within cultured adipocytes. We will determine the specific proteins that are associated with plin1 during lipolysis as well as the changes in proteins that are bound after mutation of the 14-3-3 binding site within plin1 by using mass spectrometry based proteomics. As confirmation of the proteomics work, we will investigate direct interactions of the various proteins bound to plin1 using confocal microscopy and fluorescence resonance energy transfer. Finally, we will generate a mouse that has a mutation in the 14-3-3 binding site within plin1 in vivo using CRISPR-mediated gene editing. These mice will be used to evaluate the effects of the mutation on changes in body composition, brown adipocyte thermogenesis, insulin-glucose homeostasis, adipocyte physiology including lipolysis, and the lipolytic complex of proteins that associate with plin1 to regulate lipolysis.

项目概要 由于世界上肥胖症的患病率日益增加，了解脂肪细胞所涉及的机制 新陈代谢以及导致胰岛素抵抗和糖尿病的条件很重要。脂肪组织中的脂质 以甘油三酯 (TAG) 的形式储存在脂滴 (LD) 中，并受到各种蛋白质的严格调控。 当身体需要能量时，LD 中的 TAG 会被水解，释放游离脂肪酸 (FFA) 称为脂肪分解。在禁食状态下，循环的 FFA 被远端组织用来获取能量，而在棕色状态下 脂肪细胞脂解提供用于适应性产热的脂肪酸。在肥胖者中， 脂肪分解失调会导致释放过量的 FFA，从而导致胰岛素抵抗。周利平 (plin1) 是一种在 LD 表面发现的蛋白质，通过充当许多脂质体的支架来调节脂肪分解。 参与将 TAG 水解为 FFA 的酶和接头蛋白。这笔赠款的目标是提供 为申请人提供培训，以研究如何激活环AMP依赖性蛋白激酶（PKA） 调节 plin1 活性以及 plin1 将脂肪分解机制招募到 LD 表面的机制，如 及其在全身代谢中的作用。具体来说，我们的初步结果表明，一个称为 14-3-3 蛋白在一个新的磷酸化位点与 plin1 结合，我们假设该位点是一个关键 刺激脂肪分解调节剂。我们将研究 14-3-3 蛋白与 plin1 结合在调节 PKA 中的作用 使用两种不同的实验方法刺激脂肪分解：1）突变 plin1 内的结合位点和 2)通过表达14-3-3蛋白的显性失活形式，从而隔离14-3-3蛋白， 在培养的脂肪细胞内。我们将确定脂肪分解过程中与 plin1 相关的特定蛋白质 以及 plin1 内 14-3-3 结合位点突变后结合的蛋白质的变化 基于质谱的蛋白质组学。作为蛋白质组学工作的确认，我们将直接研究 使用共焦显微镜和荧光共振观察与 plin1 结合的各种蛋白质的相互作用 能量转移。最后，我们将生成一只在 plin1 内的 14-3-3 结合位点发生突变的小鼠 体内使用 CRISPR 介导的基因编辑。这些小鼠将用于评估突变对 身体成分的变化，棕色脂肪细胞产热，胰岛素-葡萄糖稳态，脂肪细胞 生理学，包括脂肪分解，以及与 plin1 结合调节脂肪分解的蛋白质脂肪分解复合物。