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

基本信息

批准号：
10429286
负责人：
Kathryn Harris Gunn
金额：
$ 9.78万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
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：
2022-09-15 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10429286
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 Lead Learning Life Light 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 Western Blotting Work Yeasts acute pancreatitis base enzyme activity in vivo innovation knock-down lipoprotein lipase monomer nanobodies novel novel therapeutics pancreatic cell line prevent skills spatiotemporal success trafficking

项目摘要

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.

抽象的脂肪酶是人体内代谢平衡的关键调节剂。脂肪酶发挥作用的条件示例失调的疾病包括胰腺炎、代谢综合征和脂质沉积病。的标志之一胰腺炎是消化酶（例如胰腺三酰甘油脂肪酶（PTL））分泌到胰腺中毛细血管，而不是消化道，会损害胰腺细胞。因此，显然需要精确的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）采用非活性螺旋寡聚物在分泌前储存在脂肪细胞囊泡中。脂肪酶，如 LPL，具有特别需要自我调节机制，因为许多具有磷脂酶活性，因此很难将它们储存在基于磷脂的囊泡中。 LPL 之外的其他脂肪酶很可能通过季铵盐进行自我调节结构形成，保护体内新陈代谢的微妙平衡。在目标 1 中，我将阐明 LPL 无活性低聚物的原位结构。我将训练使用冷冻电子断层扫描 (cryoET) 用于研究囊泡内部的 LPL 结构。我还将开发一种特定构象的纳米抗体可区分螺旋 LPL 和单体 LPL，用于免疫荧光显微镜。我将通过调查目标 2 中的 PTL 来启动我的独立 R00 研究阶段。初步数据表明 PTL 在囊泡内部形成细丝，我将在体外筛选 PTL 形成非活性自我调节的能力低聚物并使用冷冻电子显微镜 (cryoEM) 解析其结构。然后我将应用管道开发冷冻ET和纳米抗体是为了研究体内LPL，以观察PTL。最后在目标 3 中，我将使用胰脏腺泡细胞来检查有或没有急性胰腺炎表型的胰腺分泌组。我会专门寻找储存在非活性四级结构中的酶，并表征其所起的作用分泌中的硫酸乙酰肝素蛋白多糖（HSPG）。 HSPG 已被证明可以稳定 LPL 细丝并是将自我调节细丝靶向分泌颗粒的最佳候选者。这项研究将提供至关重要的有关细胞运输过程中囊泡中脂肪酶结构的信息，并确定创新方法解决与胰腺炎相关的酶分泌失调。我使用 CryoET 获得的技能、开发纳米抗体、进行免疫荧光显微镜检查以及了解胰腺对于我作为一名独立研究者的成功至关重要。他们会请允许我对原位脂肪酶四级结构进行开创性研究，并揭示防止其发生的机制胰腺炎期间酶分泌失调。