REGULATION OF PHOSPHOLIPASE A2 NUCLEAR TARGETING

磷脂酶 A2 核靶向的调控

• 批准号: 6139297
• 负责人: CHRISTINA Carroll LESLIE
• 金额: $ 27.24万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6139297
• 关键词: arachidonate; binding sites; biological signal transduction; calcium binding protein; cell line; eicosanoid metabolism; endoplasmic reticulum; enzyme activity; intracellular transport; nuclear membrane; phospholipase A2; protein binding; protein localization; protein structure function; protein transport

Cytosolic phospholipase A2 (cPLA2) is a key regulatory enzyme that mediates agonist-induced arachidonic acid release. Arachidonic acid itself is an important intracellular regulator and can also be converted to a variety of potent lipid metabolites, the eicosanoids. This occurs through the lipoxygenase and cyclooxygenase pathways for the production of leukotrienes, and prostaglandins. These oxygenated metabolites of arachidonic acid play crucial roles in normal homeostasis and in mediating the inflammatory response. Intracellular levels of arachidonic acid are tightly controlled and cPLA2 is subject to complex mechanisms of activation that are poorly understood. cPLA2 is post-translationally regulated by phosphorylation and by levels of intracellular calcium. Agonist-induced serine phosphorylation of cPLA2 on Ser505 by mitogen activated protein kinase enhances cPLA2 activity. An increase in intracellular calcium triggers cPLA2 activation by inducing its translocation from cytosol to the nuclear envelope/endoplasmic reticulum (ER), where 5-lipoxygenase and cyclooxygenase are also localized. Calcium-induced binding of cPLA2 to membrane is mediated by the calcium-dependent phospholipid binding domain (CaLB or C2 domain) on the amino terminus of cPLA2. Although the role of the CaLB domain in membrane binding is established, the mechanisms involved in the specific targeting of cPLA2 to nuclear membrane/ER are not known, and their elucidation is the main objective of this proposal. The specific aims of the proposal include defining the role of the C2 domain and regions downstream of the C2 domain (including the plekstrin homology domain of cPLA2) in regulating nuclear targeting, and identifying proteins that bind cPLA2 and determining their role in regulating cPLA2 activity and translocation. Potential cPLA2 binding proteins have been identified using a biochemical approach and screening a phage display library. Domains of cPLA2 that are important in regulating nuclear targeting will be identified using site-directed mutagenesis. Effects of the mutations on agonist-induced arachidonic acid release and translocation of cPLA2 to the nucleus will be evaluated by expression in mammalian cells and in insect cells using baculovirus. Purified mutant constructs will also be evaluated for effects on enzymatic activity and lipid vesicle binding. Understanding the regulation of cPLA2 will provide greater insight into the signal transduction mechanisms involved in lipid mediator production, and lead to potential methods for pharmacological modulation of the inflammatory process.

胞浆磷脂酶 A2 (cPLA2) 是介导激动剂诱导的花生四烯酸释放的关键调节酶。花生四烯酸本身是一种重要的细胞内调节剂，也可以转化为多种有效的脂质代谢物，即类二十烷酸。 这是通过产生白三烯和前列腺素的脂氧合酶和环氧合酶途径发生的。 这些花生四烯酸的氧化代谢物在正常体内平衡和介导炎症反应中发挥着至关重要的作用。 细胞内花生四烯酸的水平受到严格控制，并且 cPLA2 受到复杂的激活机制的影响，但人们对此知之甚少。 cPLA2 受磷酸化和细胞内钙水平的翻译后调节。 促分裂原激活蛋白激酶激动剂诱导 cPLA2 Ser505 上的丝氨酸磷酸化可增强 cPLA2 活性。 细胞内钙的增加通过诱导 cPLA2 从细胞质易位到核膜/内质网 (ER) 来触发 cPLA2 激活，其中 5-脂氧合酶和环氧合酶也位于其中。钙诱导的 cPLA2 与膜的结合是由 cPLA2 氨基末端的钙依赖性磷脂结合域（CaLB 或 C2 域）介导的。 尽管CaLB结构域在膜结合中的作用已经确定，但cPLA2特异性靶向核膜/ER的机制尚不清楚，阐明它们是本提案的主要目标。 该提案的具体目标包括定义 C2 结构域和 C2 结构域下游区域（包括 cPLA2 的 plekstrin 同源结构域）在调节核靶向中的作用，以及鉴定结合 cPLA2 的蛋白质并确定它们在调节 cPLA2 活性和易位中的作用。 潜在的 cPLA2 结合蛋白已通过生化方法和筛选噬菌体展示文库进行了鉴定。 将使用定点诱变来鉴定对调节核靶向很重要的 cPLA2 结构域。 将通过使用杆状病毒在哺乳动物细胞和昆虫细胞中的表达来评估突变对激动剂诱导的花生四烯酸释放和cPLA2易位至细胞核的影响。 还将评估纯化的突变体构建体对酶活性和脂质囊泡结合的影响。 了解 cPLA2 的调节将有助于更深入地了解脂质介质产生中涉及的信号转导机制，并产生炎症过程的药理学调节的潜在方法。