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.

项目摘要 磷脂酶C（PLC）活性的增加与高血压等疾病有关， 目前的一线抗高血压药物都靶向PLC活性引起的Ca 2+内流。但约 10%的高血压患者表现为顽固性高血压，血压保持不受控制 尽管使用了3+种不同的抗高血压药物类别。我们迫切需要找到替代方案， PLC活性的调节剂，对抗性较不敏感。PLC产生的下游脂质， 磷脂酸（PA），可以通过补充PLC的底物磷脂酰肌醇4，5- 二磷酸盐（PIP 2）。PA在PLC活性的下游由二酰基甘油激酶（DGK）通过 DGK β）或磷脂酶D酶（PLD 1和PLD 2）。然后，PA通过以下方式帮助重新合成PIP 2： 代谢途径和通过激活磷脂酰肌醇4-磷酸5-激酶（PIP 5 K）。在此基础上， 本研究的目的是确定不同的DGK和PLD同工酶对PIP 2水平的影响 PLC控制。这项研究的基本原理是确定新的、可药用的、亚型特异性的靶点 在顽固性高血压中调节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，其分别是PI再循环和PIP 5 K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， 磷脂酰肌醇再循环和2）确定PIP 5 K激活中DGK和PLD同工酶的作用，PIP 2 补充和下游Ca 2+信号传导。我们的研究设计包括内源性标记DGK 和PLD同工酶使用CRISPR/Cas9方法来观察不同的同工酶如何被PLC激活 发信号。然后，我们将敲低PLC激活的亚型，看看它们如何影响脂质水平，PIP 5 K活性， 和Ca 2+内流。这项拟议的研究是重要的，因为它将证明如何特定的亚型的 DGK和PLD调节PLC活性。这是确定针对特定亚型的靶点的关键的第一步。 抗高血压药物治疗。