Self-regulation of Lipases by Changes to Quaternary Structure

摘要 脂肪酶是人体代谢平衡的关键调节剂。其中脂肪酶 包括胰腺炎、代谢综合征和脂质储存疾病。的标志之一 胰腺炎是消化酶如胰三酰甘油脂肪酶（PTL）分泌到胰腺中的一种疾病。 毛细血管，而不是消化道，这会损害胰腺细胞。因此，显然需要精确的 脂肪酶活性的时空调节。在最近的工作中，我们发现脂蛋白脂酶（LPL） 采用无活性的螺旋低聚物，在分泌前储存在脂肪细胞囊泡中。脂肪酶，如LPL，具有 对自我调节机制的特殊需要，因为许多人具有磷脂酶活性， 储存在磷脂囊泡中。很可能LPL以外的其他脂肪酶通过季铵盐自我调节。 结构的形成，以保护体内新陈代谢的微妙平衡。 在目标1中，我将阐明LPL的无活性寡聚体的原位结构。我会训练使用低温电子 断层扫描（cryoET）以研究囊泡内部的LPL结构。我还将开发一个特定的构象 纳米抗体以区分螺旋LPL和单体LPL，用于免疫荧光显微术。 我将通过调查目标2中的PTL来启动我的独立R00研究阶段。初步数据提示 PTL在囊泡内形成细丝，我将在体外筛选PTL形成无活性自我调节的细胞的能力。 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 cryoET和为在体内研究LPL而开发的纳米抗体，以观察PTL。最后，在目标3中，我将使用胰腺 腺泡细胞，以检查有和没有急性胰腺炎表型的胰腺分泌物组。我会 特别寻找储存在非活性四级结构中的酶，并表征 硫酸乙酰肝素蛋白聚糖（HSPG）分泌。HSPG已经显示出稳定LPL细丝， 是靶向自我调节丝进入分泌颗粒的首选。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。 我使用冷冻ET获得的技能，开发纳米抗体，进行免疫荧光显微镜检查， 了解胰腺对我成为一名成功的独立研究者至关重要。他们将 允许我继续进行原位脂肪酶四级结构的开创性研究，并揭示防止 胰腺炎期间酶分泌失调。

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.