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 当前的降压药的一线靶标Ca2+ PLC活性导致的靶标Ca2+影响。但是，关于 高血压患者中有10％表现出抗高血压，血压保持不受控制 尽管使用了3种不同的降压药类别。很需要找到替代方案 PLC活性的调节剂不易受阻。 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-- 双磷酸盐（PIP2）。 PA由二酰基甘油激酶（通过DGK通过 DGK）或磷脂酶D酶（PLD1和PLD2）。然后，PA可以帮助通过PIP2重新合成 代谢途径以及通过4-磷酸5-激酶（PIP5K）的磷脂酰肌醇的激活。基于此， 该提案的目的是确定各种DGK和PLD同工酶如何促进PIP2水平 和PLC调节。拟议的研究的基本原理是确定新的，可吸毒的同工型特异性目标 调节耐药性高血压中的PLC活性。中心假设是PLC信号激活A DGK和PLD同工酶的特定子集分别用于PI回收和PIP5K活性。 我们将使用两个特定目的检验该假设：1）定义在功能中起作用的DGK同工酶 磷脂酰肌醇回收和2）确定DGK和PLD同工酶在PIP5K激活中的作用，PIP2 补充和下游CA2+信号传导。我们的研究设计包括对DGK进行内源性标签 使用CRISPR/CAS9方法来查看不同的同工酶如何被PLC激活 信号。然后，我们将敲除PLC激活的同工型，以了解它们如何影响脂质水平，PIP5K活性， 和CA2+影响。拟议的研究很重要，因为它将证明特定的同工型如何 DGK和PLD调节PLC活性。这是确定同工型特异性目标的关键第一步 降压药疗法。