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Isolation and characterization of mammalian glycerophosphodiester phosphodiesterases

哺乳动物甘油磷酸二酯磷酸二酯酶的分离和表征

基本信息

批准号：
18580094
负责人：
YANAKA Noriyuki
金额：
$ 2.51万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

Bacterial glycerophosphodiester phosphodiesterases are well-characterized to be periplasmic and cytosolic proteins, which play a critical role in the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. In contrast, two novel mammalian GP-PDEs, GDE1 and GDE3, were recently identified, and were shown to be involved in several physiological functions.1. A GP-PDE homolog, GDE2, was widely expressed in brain tissues including, and that the expression of GDE2 in neuroblastoma Neuro2A cells was significantly up-regulated during neuronal differentiation by retinoic acid (RA) treatment. Stable expression of GDE2 resulted in neurite formation in the absence of RA, and GDE2 accumulated at the regions of perinuclear and growth cones in Neuro2A cells. Furthermore, a loss-of-function of GDE2 in Neuro2A cells by RNAi blocked RA-induced neurite formation. These results demonstrate that GDE2 expression during neuronal differentiation plays an important role for growing neurites.2. In this study, seven mammalian GP-PDEs were virtually cloned by the approach using bioinformatics. Analysis of the hydrophobicity profile of a novel GDE, GDE7, protein indicated that two distinct hydrophobic regions are located at the N-terminus and at the C-terminus. GDE7 was expressed in mouse skin, and might be involved in the maintenance of keratinocytes.3. The predicted protein sequence of GDE5 does not contain any transmembrane sequence, suggesting that GDE5 functions as a cytosolic protein. Expression of GDE5 mRNA was shown to be regulated in skeletal muscles of fasted mice and diabetic KK-Ay mice, but its functions in skeletal muscle have remained poorly understood. In this study, I created transgenic mice specifically overexpressing GDE5 in skeletal muscle. These mice had a reduced skeletal muscle mass. Microarray analysis revealed that the expression of several genes related to cellular stress was increased in skeletal muscles of transgenic mice.

细菌甘油磷酸二酯磷酸二酯酶是一类周质和胞质蛋白，在脱酰化甘油磷脂水解为甘油磷酸和乙醇的过程中起着关键作用。相比之下，最近发现了两个新的哺乳动物GP-PDE，GDE1和GDE3，并被证明参与了几种生理功能。GP-PDE同系物GDE2在包括在内的脑组织中广泛表达，在维甲酸(RA)诱导的神经分化过程中，GDE2在神经母细胞瘤Neuro2A细胞中的表达显著上调。在没有RA的情况下，GDE2的稳定表达导致神经突起的形成，GDE2聚集在Neuro2A细胞的核周和生长锥体区域。此外，RNAi使Neuro2A细胞中的GDE2功能丧失，阻断了RA诱导的神经突起的形成。这些结果表明，GDE2在神经元分化过程中的表达对神经元的生长起着重要作用。在本研究中，利用生物信息学的方法虚拟克隆了7个哺乳动物GP-PDE基因。对一种新的GDE蛋白GDE7的疏水性分析表明，在N端和C端有两个不同的疏水区。GDE7在小鼠皮肤中表达，可能参与角质形成细胞的维持。预测的GDE5蛋白序列不包含任何跨膜序列，提示GDE5具有胞质蛋白的功能。GDE5在禁食小鼠和糖尿病KK-Ay小鼠骨骼肌中的表达受到调控，但其在骨骼肌中的功能尚不清楚。在这项研究中，我创造了专门在骨骼肌中过度表达GDE5的转基因小鼠。这些小鼠的骨骼肌量减少了。基因芯片分析显示，转基因小鼠骨骼肌中几个与细胞应激相关的基因表达增加。