Self-regulation of Lipases by Changes to Quaternary Structure

通过四级结构的变化进行脂肪酶的自我调节

• 批准号: 10703368
• 负责人: Kathryn Harris Gunn
• 金额: $ 4.71万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2024-01-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10703368
• 关键词: Acinar Cell; Address; Adipocytes; Adopted; Affect; Amylases; Antibodies; Binding; Blood; Blood capillaries; Cell Death; Cell Line; Cells; Centrifugation; Chylomicrons; Cryo-electron tomography; Cryoelectron Microscopy; Cues; Cytoplasmic Granules; Data; Development; Diagnostic; Disease; Enzyme Precursors; Enzymes; Equilibrium; Feedback; Filament; Foundations; Freezing; Gastrointestinal tract structure; Health; Heparan Sulfate Proteoglycan; Heparin Binding; Human body; Immunofluorescence Microscopy; In Situ; In Vitro; Inflammation; Informal Social Control; Learning; Life; Lipase; Lipids; Lipoprotein (a); Mass Spectrum Analysis; Metabolic; Metabolic syndrome; Metabolism; Microscopy; Molecular Conformation; Monitor; Negative Staining; Nutritional; Pain; Pancreas; Pancreatic enzyme; Pancreatitis; Phase; Phenotype; Phospholipase; Phospholipids; Play; Proteins; Regulation; Research; Research Personnel; Role; Route; Secretory Vesicles; Signal Transduction; Small Intestines; Solid; Structure; Techniques; Tomogram; Training; Triglycerides; Very low density lipoprotein; Vesicle; Visualization; Western Blotting; Work; Yeasts; acute pancreatitis; enzyme activity; in vivo; innovation; knock-down; lipoprotein lipase; monomer; nanobodies; novel; novel therapeutics; pancreatic cell line; prevent; skills; spatiotemporal; success; trafficking

Abstract Lipases are a key regulator of metabolic equilibrium in the human body. Examples of conditions in which lipases are dysregulated include pancreatitis, metabolic syndrome, and lipid storages diseases. One of the hallmarks of pancreatitis is the secretion of digestive enzymes, such as pancreatic triacylglycerol lipase (PTL), into the capillaries, rather than the digestive tract, which damages pancreatic cells. Thus, there is a clear need for precise spatiotemporal regulation of lipase activity in the body. In recent work, we found that lipoprotein lipase (LPL) adopts an inactive helical oligomer for storage in adipocyte vesicles prior to secretion. Lipases, like LPL, have a special need for mechanisms of self-regulation, as many possess phospholipase activity, making it difficult to store them in phospholipid-based vesicles. It is likely that other lipases beyond LPL self-regulate by quaternary structure formation to protect the delicate balance of metabolism in the body. In Aim 1, I will elucidate the in situ structure of inactive oligomers of LPL. I will train to use cryo-electron tomography (cryoET) to study LPL structure inside of vesicles. I will also develop a conformation-specific nanobody to discriminate between helical LPL and monomer LPL for use with immunofluorescence microscopy. I will launch my independent R00 research phase by investigating PTL in Aim 2. Preliminary data suggests that PTL forms filaments inside of vesicles and I will screen PTL in vitro for the ability to form inactive self-regulated oligomers and solve their structure using cryo-electron microscopy (cryoEM). I will then apply the pipeline of cryoET and nanobodies developed for studying LPL in vivo, to look at PTL. Finally in Aim 3, I will use pancreatic acinar cells to examine the secretome of the pancreas with and without an acute pancreatitis phenotype. I will look specifically for enzymes stored in inactive quaternary structures and characterize the role played by heparan-sulfate proteoglycans (HSPGs) in secretion. HSPGs have been shown to stabilize LPL filaments and are top candidates for targeting self-regulated filaments into secretory granules. This research will provide crucial information about the structure of lipases in vesicles during cellular trafficking and identify innovative ways to address dysregulation of enzyme secretion associated with pancreatitis. The skills I acquire using cryoET, developing nanobodies, performing immunofluorescence microscopy, and learning about the pancreas will be essential for setting up my success as an independent researcher. They will allow me to pursue pioneering studies of in situ lipase quaternary structure and uncover mechanisms to prevent dysregulation of enzyme secretion during pancreatitis.

摘要 脂肪酶是人体代谢平衡的关键调节因子。脂肪酶条件的示例 失调包括胰腺炎、代谢综合征和脂肪储存疾病。标志之一就是 胰腺炎是消化酶的分泌，如胰腺三酰甘油脂肪酶(PTL)，进入 破坏胰腺细胞的是毛细血管，而不是消化道。因此，显然有必要准确地 体内脂肪酶活性的时空调节。在最近的工作中，我们发现脂蛋白脂肪酶(LPL) 采用非活性螺旋低聚物，在分泌前储存在脂肪细胞囊泡中。脂肪酶，像LPL一样，有一个 特别需要自我调节机制，因为许多机制具有磷脂酶活性，使其难以 储存在以磷脂为基础的囊泡中。LPL以外的其他脂肪酶很可能受第四纪的自我调节 结构形成，保护体内微妙的新陈代谢平衡。 在目标1中，我将阐明LPL的非活性低聚物的原位结构。我会训练使用低温电子的 体层摄影术(冷冻ET)研究小泡内的LPL结构。我还将开发一种特定于构象的 用于免疫荧光显微镜的区分螺旋LPL和单体LPL的纳米体。 我将通过在AIM 2中调查PTL来启动我的独立R00研究阶段。初步数据表明 PTL在囊泡内形成细丝，我将在体外筛选PTL形成非活性自我调节的能力 并使用低温电子显微镜(Cryo-Electronics显微镜，CryoEM)解析其结构。然后我将应用管道中的 在活体内研究LPL，观察PTL而开发的冷冻ET和纳米体。最后，在《目标3》中，我将使用胰腺 腺泡细胞检查有无急性胰腺炎表型的胰腺分泌体。这就做 专门寻找储存在不活跃的四元结构中的酶，并表征其所起的作用 分泌物中的硫酸乙酰肝素蛋白多糖(HSPGs)。HSPG已被证明可以稳定LPL细丝和 是将自我调节的细丝靶向分泌颗粒的首选。这项研究将提供至关重要的 关于细胞运输过程中小泡中脂肪酶结构的信息，并确定创新的方法 解决与胰腺炎相关的酶分泌失调问题。 我使用冷冻ET获得的技能，开发纳米体，执行免疫荧光显微镜，以及 对胰腺的了解对我成为一名成功的独立研究人员至关重要。他们会 请允许我继续进行原位脂肪酶四级结构的开创性研究，并揭示预防 胰腺炎时酶分泌失调。