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.

项目概要 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均以 PLC 活性导致的 Ca2+ 流入为目标。然而，关于 10% 的高血压患者表现出顽固性高血压，即血压仍未得到控制 尽管使用了 3 种以上不同的抗高血压药物类别。迫切需要寻找替代方案 PLC 活性调节剂不易受到耐药性的影响。 PLC生产的下游脂质， 磷脂酸（PA），可以通过补充PLC的底物磷脂酰肌醇4,5-来调节PLC信号传导 二磷酸盐（PIP2）。 PA 是由二酰基甘油激酶（DGK 至 P​​LC 活性下游）产生的 DGK）或磷脂酶 D 酶（PLD1 和 PLD2）。然后 PA 通过以下方式帮助重新合成 PIP2： 代谢途径并通过激活磷脂酰肌醇 4-磷酸 5-激酶 (PIP5K)。基于此， 该提案的目的是确定各种 DGK 和 PLD 同工酶如何影响 PIP2 水平 和PLC调节。拟议研究的基本原理是确定新的、可成药的、异构体特异性的靶标 调节难治性高血压中的 PLC 活性。中心假设是 PLC 信号激活 DGK 和 PLD 同工酶的特定子集，分别是 PI 回收和 PIP5K 活性所必需的。 我们将使用两个具体目标来检验这一假设：1) 定义在以下功能中发挥作用的 DGK 同工酶： 磷脂酰肌醇回收和 2) 确定 DGK 和 PLD 同工酶在 PIP5K 激活、PIP2 中的作用 补充和下游 Ca2+ 信号传导。我们的研究设计包括对 DGK 进行内源标记 和 PLD 同工酶，使用 CRISPR/Cas9 方法观察 PLC 如何激活不同的同工酶 发信号。然后我们将敲除 PLC 激活的亚型，看看它们如何影响脂质水平、PIP5K 活性、 和Ca2+流入。拟议的研究意义重大，因为它将证明特定亚型如何 DGK 和 PLD 调节 PLC 活动。这是确定亚型特异性靶标的关键的第一步 抗高血压药物治疗。