The roles of PLD and DGK isoforms in PIP2 homeostasis during PLC signaling

PLC 信号转导过程中 PLD 和 DGK 亚型在 PIP2 稳态中的作用

• 批准号: 10748698
• 负责人: Claire Weckerly
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10748698
• 关键词: Affect; Angiotensins; Antihypertensive Agents; Biosensor; Blood Pressure; CRISPR/Cas technology; Calcium Channel Blockers; Cell membrane; Cell model; Cells; Diacylglycerol Kinase; Diglycerides; Disease; Enzymes; Feedback; Genetic; Homeostasis; Hypertension; Individual; Inositol; Isoenzymes; Link; Lipids; Mediating; Membrane Lipids; Metabolic Pathway; Metabolism; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phosphatidic Acid; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase C; Phospholipase D; Phosphorylation; Phosphotransferases; Physiological; Predisposition; Process; Production; Protein Isoforms; Proteomics; Public Health; Recycling; Regulation; Research; Research Design; Resistance; Resistant Hypertension; Role; Signal Transduction; Testing; Work; adverse outcome; blood pressure elevation; druggable target; hypertensive; inhibitor; inorganic phosphate; knock-down; new therapeutic target; novel; phosphatidylinositol 4-phosphate; recruit; vasoconstriction

PROJECT SUMMARY Increases in phospholipase C (PLC) activity have been associated with diseases such as hypertension, and the current first-line of antihypertensive drugs all target Ca2+ influx that results from PLC activity. However, about 10% of patients with hypertension show resistant hypertension, where blood pressure remains uncontrolled despite the use of 3+ different antihypertensive drug classes. There is a significant need to find alternative modulators of PLC activity that are less susceptible to resistance. The downstream lipid produced by PLC, phosphatidic acid (PA), can regulate PLC signaling by replenishing PLC’s substrate phosphatidylinositol 4,5- bisphosphate (PIP2). PA is produced downstream of PLC activity by the diacylglycerol kinases (DGK through DGK) or phospholipase D enzymes (PLD1 and PLD2). The PA then helps to resynthesize PIP2 through metabolic pathways and through activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K). Based on this, the objective of this proposal is to determine how the various DGK and PLD isozymes contribute to PIP2 levels and PLC regulation. The rationale of the proposed research is to identify new, druggable, isoform-specific targets to regulate PLC activity in resistant hypertension. The central hypothesis is that PLC signaling activates a particular subset of DGK and PLD isozymes that are necessary for PI recycling and PIP5K activity, respectively. We will test this hypothesis using two specific aims: 1) define the DGK isozymes that function in phosphatidylinositol recycling and 2) determine the role of the DGK and PLD isozymes in PIP5K activation, PIP2 replenishment and downstream Ca2+ signaling. Our research design includes endogenously-tagging the DGK and PLD isozymes using a CRISPR/Cas9 approach to see how the different isozymes are activated by PLC signaling. We will then knockdown the PLC-activated isoforms to see how they affect lipid levels, PIP5K activity, and Ca2+ influx. The proposed research is significant because it will demonstrate how the specific isoforms of DGK and PLD regulate PLC activity. This is a critical first step in identifying isoform-specific targets for antihypertensive drug therapies.

项目总结 磷脂酶C(PLC)活性的增加与高血压等疾病有关，而 目前的一线抗高血压药物都以PLC活性引起的钙内流为靶点。但是，关于 10%的高血压患者表现为顽固性高血压，血压仍未得到控制 尽管使用了3+种不同类别的降压药。很有必要找到替代方案 PLC活性的调节剂，对耐药性不太敏感。PLC生产的下游油脂， 磷脂酸(PA)可以通过补充PLC底物磷脂酰肌醇4，5-来调节PLC信号转导。 二磷酸(PIP2)。磷脂酶C活性下游的PA是由二酰基甘油激酶(DGK至 Dgk)或磷脂酶D酶(pld1和pld2)。然后，PA帮助重新合成PIP2 代谢途径和通过激活磷脂酰肌醇4-磷酸-5-激酶(PIP5K)。在此基础上， 这项建议的目的是确定各种DGK和PLD同工酶是如何影响PIP2水平的 和PLC调节。这项拟议的研究的基本原理是确定新的、可用药的、特定于异构体的靶点。 目的：调节难治性高血压患者的PLC活性。中心假设是PLC信号激活了一个 DGK和PLD同工酶的特定子集，分别是PI循环和PIP5K活性所必需的。 我们将使用两个特定的目标来检验这一假设：1)定义DGK同工酶 磷脂酰肌醇循环和2)DGK和PLD同工酶在PIP5K激活中的作用 补给和下游的钙信号。我们的研究设计包括内源性标记DGK 和PLD同工酶，用CRISPR/Cas9方法观察不同的同工酶是如何被PLC激活的 发信号。然后我们将敲除PLC激活的异构体，看看它们如何影响血脂水平，PIP5K活性， 和钙离子内流。这项拟议的研究具有重要意义，因为它将证明特定的亚型是如何 DGK和PLD调节PLC的活性。这是识别特定异构体靶标的关键第一步 抗高血压药物疗法。