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Dissection of the molecular mechanism of the novel neurotrophin-like effects of secretory phospholipase A_2

分泌型磷脂酶A_2新型神经营养素样作用的分子机制剖析

基本信息

批准号：
16580054
负责人：
ARIOKA Manabu
金额：
$ 2.3万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16580054/
关键词：
secretory phospholipase A_2 neurotrophic factor L-type Ca^<2+> channel apoptosis PC12 cerebellar granule neuron neurite outgrowth Aspergillus oryzae 神経突起伸張

项目摘要

We previously demonstrated that secretory phospholipase A_2 (sPLA_2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. Furthermore, sPLA_2 and LPC rescued cerebellar granule neurons from apoptotic cell death induced by deprivation of depolarizing stimulus. In the current study, we further analyzed the mechanism whereby sPLA_2 displays neurite-inducing activity. Exogenously-added mammalian group X sPLA_2 (sPLA_2-X), but not group IB and IIA sPLA_2s, induced neuritogenesis, which correlated with the ability of sPLA_2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA_2 or LPC. Overproduction or suppression of G2A, a G protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA_2 treatment. These results indicate that neu … More ritogenic effect of sPLA_2 is mediated by generation of LPC and subsequent activation of G2A.In contrast to mammalian sPLA_2s, much less is known for the physiological function of microbial sPLA_2. We cloned and characterized spaA and spaB encoding putative sPLA_2s from a filamentous fungus Aspergillus oryzae. PLA_2 activity assays showed that recombinant SpaA and SpaB had different enzymatic characteristics. Western blot and immunofluorescence analyzes demonstrated that, while SpaA was mainly secreted to culture medium, SpaB was associated with the intracellular structures reminiscent of the endoplasmic reticulum and/or lipid body. The expression of spaA was induced in response to carbon starvation, oxidative stress and conidiation, while spaB was constitutively expressed at very low steady level and upregulated in the cold-stressed condition. spaA- and spaB-overexpressing strains showed poor-conidiation phenotype which was remarkable in the latter. Although no morphological abnormalities were found in spaA, spaB, and spaA/spaB disruptants, we found that conidia of spaA disruptant and hyphae of spaB disruptant became sensitive to oxidative stress. These results suggest that fungal sPLA_2s play roles in defense system to oxidative stress. Less

我们之前证明，分泌性磷脂酶 A_2 (sPLA_2) 和溶血磷脂酰胆碱 (LPC) 在大鼠嗜铬细胞瘤细胞系 PC12 中表现出神经营养蛋白样神经突形成活性。此外，sPLA_2 和 LPC 将小脑颗粒神经元从去极化刺激剥夺引起的细胞凋亡中拯救出来。在本研究中，我们进一步分析了 sPLA_2 表现出神经突诱导活性的机制。外源添加的哺乳动物 X 组 sPLA_2 (sPLA_2-X)（而非 IB 组和 IIA 组 sPLA_2s）诱导神经突发生，这与 sPLA_2-X 将 LPC 释放到培养基中的能力相关。因此，通过补充牛血清白蛋白或磷脂酶 B 来阻断 LPC 的作用会减弱 sPLA_2 或 LPC 的神经突发生。 G2A（一种参与 LPC 信号转导的 G 蛋白偶联受体）的过量产生或抑制会导致 sPLA_2 治疗诱导的神经突发生增强或减少。这些结果表明，sPLA_2 的神经刺激作用是通过 LPC 的生成和随后的 G2A 激活介导的。与哺乳动物 sPLA_2 相比，人们对微生物 sPLA_2 的生理功能知之甚少。我们从丝状真菌米曲霉中克隆并表征了编码假定的 sPLA_2 的 spaA 和 spaB。 PLA_2活性测定表明重组SpaA和SpaB具有不同的酶学特征。蛋白质印迹和免疫荧光分析表明，虽然 SpaA 主要分泌到培养基中，但 SpaB 与细胞内结构相关，例如内质网和/或脂质体。 spaA 的表达是响应碳饥饿、氧化应激和分生孢子形成而诱导的，而 spaB 则以非常低的稳定水平组成型表达，并在冷应激条件下上调。 spaA 和 spaB 过表达菌株表现出不良分生孢子表型，后者尤为显着。尽管在spaA、spaB和spaA/spaB破坏体中没有发现形态异常，但我们发现spaA破坏体的分生孢子和spaB破坏体的菌丝对氧化应激变得敏感。这些结果表明真菌sPLA_2s在氧化应激的防御系统中发挥作用。较少的