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Metabolism, regulation and function of phosphatidylserine in mammalian cells.

哺乳动物细胞中磷脂酰丝氨酸的代谢、调节和功能。

基本信息

批准号：
16390028
负责人：
NISHIJIMA Masahiro
金额：
$ 8.9万
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 (B)
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/grant/KAKENHI-PROJECT-16390028/
关键词：
phosphatidylserine CHO cells Sindbis virus phosphatidylserine synthase

项目摘要

(1)PtdSer (phosphatidylserine) synthesis in mammalian cells occurs through the exchange of L-serine with the base moieties of phosphatidylcholine and phosphatidylethanolamine, which is catalysed by PSS (PtdSer synthase) 1 and 2 respectively. PtdSer synthesis in intact cells and an isolated membrane fraction was inhibited by exogenous PtdSer, indicating that feedback control is involved in the regulation of PtdSer biosynthesis. PSS 1 and 2 are similar in amino acid sequence, with an identity of 32%; however, due to a lack of homology with other known enzymes, their amino acid sequences do not provide information on their catalytic and regulatory mechanisms. In the present study, to identify amino acid residues crucial for the activity and/or regulation of PSS 1, we systematically introduced mutations into a Chinese hamster PSS 1 cDNA clone ; namely, each of the 66 polar amino acid residues common to PSS 2 was replaced with an alanine residue. On analysis of Chinese hamster ovary cells t … More ransfected with each of the alanine mutant clones, we identified eight amino acid residues (His-172, Glu-197, Glu-200, Asn-209, Glu-212, Asp-216, Asp-221 and Asn-226) as those crucial for the enzyme reaction or the maintenance of the correct structure required for serine base-exchange activity. Among these residues, Asn-209 was suggested to be involved in the recognition and/or binding of free L-serine. We also identified six amino acid residues (Arg-95, His-97, Cys-189, Arg-262, Gln-266 and Arg-336) as those important for regulation of PSS 1. In addition, we found that the alanine mutations at Tyr-111, Asp-166, Arg-184, Arg-323, and Glu-364 affected the production and/or stability of PSS 1 in Chinese hamster ovary cells.(2)Sindbis virus replicon-mediated gene expression involves RNA synthesis by viral replicase on cytoplasmic membranes. Here we examined the involvement of a membrane lipid, phosphatidylserine, in the replicon-mediated gene expression by using Chinese hamster ovary cell mutants defective in phosphatidylserine biosynthesis. When the mutant cells were transfected with a replicon RNA carrying a viral replicase gene followed by a subgenomic promoter-driven lacZ gene, expression of β-galactosidase from the replicon subgenomic RNA was inhibited under phosphatidylserine-deficient conditions. In contrast, expression of a replicase protein, nsP1, from the replicon genomic RNA and accumulation of the subgenomic RNA were not inhibited by the phosphatidylserine deficiency, indicating that inhibition of the reporter expression was not due to defects in production and function of the replicase. The reporter expression from an SV40 promoter-driven construct was found to be normal under similar conditions, implying the phosphatidylserine deficiency does not affect general translation and protein degradation. These results indicate that phosphatidylserine is specifically involved in a posttranscriptional event of Sindbis virus subgenomic promoter-driven gene expression. Less

（1）哺乳动物细胞中的PtdSer（磷脂酰丝氨酸）合成通过L-丝氨酸与磷脂酰胆碱和磷脂酰乙醇胺的碱基部分的交换发生，其分别由PSS（PtdSer合酶）1和2催化。完整细胞和分离的膜部分中的PtdSer合成被外源性PtdSer抑制，表明反馈控制参与了PtdSer生物合成的调节。PSS 1和2在氨基酸序列上相似，具有32%的同一性;然而，由于与其他已知酶缺乏同源性，它们的氨基酸序列不提供关于它们的催化和调节机制的信息。在本研究中，以确定氨基酸残基的活性和/或调节的PSS 1的关键，我们系统地引入突变到中国仓鼠PSS 1的cDNA克隆，即，每个66个极性氨基酸残基的PSS 2的共同替换为丙氨酸残基。中国仓鼠卵巢细胞的分析 ...更多信息用每个丙氨酸突变体克隆进行转染，我们鉴定了8个氨基酸残基（His-172、Glu-197、Glu-200、Asn-209、Glu-212、Asp-216、Asp-221和Asn-226），它们对于酶反应或维持丝氨酸碱基交换活性所需的正确结构至关重要。在这些残基中，Asn-209被认为参与游离L-丝氨酸的识别和/或结合。我们还鉴定了六个氨基酸残基（Arg-95、His-97、Cys-189、Arg-262、Gln-266和Arg-336）作为对PSS 1的调节重要的那些。此外，我们发现Tyr-111、Asp-166、Arg-184、Arg-323和Glu-364处的丙氨酸突变影响PSS 1在中国仓鼠卵巢细胞中的产生和/或稳定性。（2）辛德毕斯病毒复制子介导的基因表达涉及细胞质膜上病毒复制酶的RNA合成。在这里，我们研究了参与的膜脂质，磷脂酰丝氨酸，在复制子介导的基因表达，通过使用中国仓鼠卵巢细胞突变体缺陷的磷脂酰丝氨酸生物合成。当用携带病毒复制酶基因的复制子RNA转染突变体细胞时，随后是亚基因组启动子驱动的lacZ基因，在磷脂酰丝氨酸缺陷条件下，来自复制子亚基因组RNA的β-半乳糖苷酶的表达被抑制。相反，复制酶蛋白，nsP 1，从复制子基因组RNA的表达和积累的亚基因组RNA不受磷脂酰丝氨酸缺乏症的抑制，这表明抑制报告基因的表达是不是由于复制酶的生产和功能的缺陷。在类似条件下，发现SV 40启动子驱动的构建体的报告基因表达是正常的，这意味着磷脂酰丝氨酸缺乏不影响一般的翻译和蛋白质降解。这些结果表明，磷脂酰丝氨酸是专门参与辛德毕斯病毒亚基因组启动子驱动的基因表达的转录后事件。少

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Hydrolysis of sphingosylphosphocholine by neutral sphingomyelinases

中性鞘磷脂酶水解鞘氨酰磷酸胆碱

DOI：
发表时间：
2004
期刊：
FEBS Letters 557
影响因子：
0
作者：
Y.Miura;E.Gotoh;F.Nara;M.Nishijima;K.Hanada
通讯作者：
K.Hanada

An lkB-b COOH Terminal Region Protein ls Essential for the Proliferation of CHO Cells Under Acidic stress

lkB-b COOH 末端区域蛋白对于酸性胁迫下 CHO 细胞的增殖至关重要

DOI：
发表时间：
2005
期刊：
J. Cell. Physiol. 203
影响因子：
0
作者：
Q.Lao;O.Kuge;T.Fukamachi;T.Kakegawa;H.Saito;M.Nishijima;H.Kobayashi
通讯作者：
H.Kobayashi

An IkB-b COOH Terminal Region Protein Is Essential for the Proliferation of CHO Cells Under Acidic Stress

IkB-b COOH 末端区域蛋白对于酸性胁迫下 CHO 细胞的增殖至关重要

DOI：
发表时间：
2005
期刊：
J.Cell.Physiol. 203
影响因子：
0
作者：
Q.Lao;O.Kuge;T.Fukamachi;T.Kakegawa;H.Saito;M.Nishijima;H.Kobayashi
通讯作者：
H.Kobayashi

Infection Route-Independent Accumulation of Splenic Abnormal Prion Protein

脾脏异常朊病毒蛋白的感染途径独立积累

DOI：
发表时间：
2005
期刊：
Jpn.J.Infect.Dis. 58
影响因子：
0
作者：
Y.Inoue;Y.Yamakawa;A.Sakudo;T.Kinumi;Y Nakamura;Y.Matsumoto;K.Saeki;T.Kamiyama;T.Onodera;M.Nishijima
通讯作者：
M.Nishijima

Non-glycosylphosphatidylinositol (GPI)-anchored recombinant prion protein with dominant-negative mutation inhibits PrPSc replication in vitro