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：
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
项目类别：
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

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

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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代谢平衡的关键调节剂。脂肪酶的示例失调包括胰腺炎，代谢综合征和脂质遗传疾病。的标志之一胰腺炎是消化酶的分泌，例如胰三酰基甘油脂肪酶（PTL）毛细血管，而不是损害胰腺细胞的消化道。因此，显然需要精确人体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）在分泌之前，采用不活跃的螺旋寡聚物来存储在脂肪细胞囊泡中。脂肪酶，例如LPL，有一个特殊需要自我调节机制，因为许多人具有磷脂酶活性，因此很难将它们存放在基于磷脂的囊泡中。 LPL以外的其他脂肪可能会通过第四纪自我调节结构形成，以保护体内代谢的微妙平衡。在AIM 1中，我将阐明LPL的无活跃低聚物的原位结构。我将训练使用冷冻电子层析成像（冷冻）以研究囊泡内部的LPL结构。我还将开发特定于构象的纳米病毒以区分螺旋LPL和单体LPL，以与免疫荧光显微镜一起使用。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形成囊泡内部的细丝，我将在体外筛选PTL，以形成不活跃的自我调节的能力低聚物并使用冷冻电子显微镜（Cryoem）解决结构。然后，我将应用管道为研究PTL而开发了用于研究LPL的冷冻和纳米体。终于在AIM 3中，我将使用胰腺腺泡细胞检查胰腺表型和没有急性胰腺炎表型的胰腺的分泌。我会专门查找存储在非活动季节结构中的酶，并表征了作用分泌中的乙酰硫酸盐蛋白聚糖（HSPGS）。 HSPG已显示可稳定LPL丝和是将自我调节的细丝靶向分泌颗粒的顶级候选者。这项研究将提供至关重要的有关在细胞运输过程中囊泡中脂肪酶结构的信息，并确定创新的方式解决与胰腺炎相关的酶分泌的失调。我使用冷冻，开发纳米体，进行免疫荧光显微镜和了解胰腺对于建立我作为独立研究人员的成功至关重要。他们会的请允许我对原位脂肪酶第四纪结构和发现的机制进行开创性研究胰腺炎期间酶分泌失调。