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Biosymthetic Regulation and Intracellular Transport of Phosphatidylserine

磷脂酰丝氨酸的生物合成调节和细胞内运输

基本信息

批准号：
11680644
负责人：
KUGE Osamu
金额：
$ 2.18万
依托单位：
National Institute of Infectious Diseases
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680644/
关键词：
Phospholipid Phosphatidylserine Biosynthesis Transport

项目摘要

1.Biosynthetic regulation of phosphatidylserine (PS)The PS biosynthesis in Chinese hamster ovary cells is catalyzed by at least two different PS synthases named PSS I and PSS II. In this study, we showed that (1) PSS II in CHO-K1 cells is inhibited by exogenous PS, (2) overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PS, (3) the activity of over produced PSS II in CHO Kl cells is depressed for maintenance of the normal PS biosynthetic rate, probably through molecular mechanisms different from those for the exogenous PS-mediated inhibition, and (4) the Arg97 of PSS II is a critical residue for both the exogenous PS mediated inhibition of PSS II and the depression of overproduced PSS II activity.2.Intracellular transport of PSPhosphatidylethanolamine synthesis through the phosphatidylserine (PS) decarboxylation pathway requires PS transport from the endoplasmic reticulum or mitochondria-associated membrane to the mitochondrial inner membrane in mammalian cells. The transport-dependent PS decarboxylation in permeabilized Chinese hamster ovary (CHO) cells was enhanced by cytosolic factors from bovine brain. A cytosolic protein factor exhibiting this enhancing activity was purified, and its amino acid sequence was partially determined. The sequence determined was identical with part of the amino acid sequence of an EF-hand type calcium-binding protein, S100B. A His_<6>-tagged recombinant CHO S100B protein was able to remarkably enhance the transport-dependent PS decarboxylation in permeabilized CHO cells. Under the standard assay conditions for PS decarboxylase, the recombinant S100B protein did not stimulate PS decarboxylase activity and exhibited no PS decarboxylase activity. These results implicated the S100B protein in the transport of PS to the mitochondrial inner membrane.

1.磷脂酰丝氨酸（PS）的生物合成调控中国仓鼠卵巢细胞中的PS生物合成由至少两种不同的PS酶（PSS I和PSS II）催化。在本研究中，我们发现（1）外源PS抑制CHO-K1细胞中的PSS II，（2）过量产生的PSS II导致外源PS失去对PSS II活性的正常控制，（3）抑制CHO-K1细胞中过量产生的PSS II活性以维持正常的PS生物合成速率，这可能是通过与外源PS介导的抑制不同的分子机制，（4）PSS Ⅱ的Arg 97是外源性PS抑制PSS Ⅱ和抑制过度产生的PSS Ⅱ活性的关键残基。2.在哺乳动物细胞中，通过磷脂酰丝氨酸（PS）脱羧途径合成PS磷脂酰乙醇胺的细胞内转运需要PS从内质网或线粒体相关膜转运到线粒体内膜。牛脑胞浆因子可增强透化中国仓鼠卵巢（CHO）细胞中转运依赖性PS脱羧。表现出这种增强活性的胞质蛋白因子被纯化，其氨基酸序列被部分确定。测定的序列与EF-手型钙结合蛋白S100 B的部分氨基酸序列相同。在<6>透性化CHO细胞中，带His标签的重组CHO S100 B蛋白能够显著增强依赖转运的PS脱羧。在PS脱羧酶的标准测定条件下，重组S100 B蛋白不刺激PS脱羧酶活性，并且不表现出PS脱羧酶活性。这些结果表明，S100 B蛋白参与了PS向线粒体内膜的转运。