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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

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.

摘要