Phosphatidylserine Translocase and Calcium Channels
磷脂酰丝氨酸转位酶和钙通道
基本信息
- 批准号:6460322
- 负责人:
- 金额:$ 3.3万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1998
- 资助国家:美国
- 起止时间:1998-05-01 至 2004-04-30
- 项目状态:已结题
- 来源:
- 关键词:SDS polyacrylamide gel electrophoresis annexins antisense nucleic acid apoptosis calcium channel cysteine endopeptidases enzyme activity flow cytometry genetic promoter element laboratory mouse microglia neuroblastoma northern blottings nucleic acid sequence phagocytosis polymerase chain reaction transport proteins voltage gated channel western blottings
项目摘要
DESCRIPTION (provided by applicant): Chemotherapy causes apoptosis of brain
tumor cells, which are then cleared through phagocytosis. In peripheral tissue,
phagocytosis is preceded by a recognition process in which scavenger
macrophages selectively bind to phosphatidylserine (PS) molecules that are
exposed on the surface of the apoptotic cells. During the current granting
period, the applicant's team has shown that PS plays a central role also in the
recognition of apoptotic neurotumor cells by the resident scavenger cells of
the brain, the microglia. The enzyme that plays a central role in the
inner-membrane sequestering of PS in healthy cells is the aminophospholipid
translocase (APTL), which is also a Mg2+-ATPase and belongs to a recently
classified subfamily of P-type ATPase. It is highly expressed in the CNS, but
its regulation and role in neurons are poorly understood. Its inhibition or
down regulation results in the typical apoptotic feature of PS-externalization.
In order to study the regulation profile of this enzyme, the mouse APTL cDNA
was overexpressed from vector pCMV6 in the calcium channel-deficient hybrid
neuroblastoma cells, HN2. In addition to showing a 15-fold increase in
phosphatidylserine translocase activity, all the clones surprisingly displayed
significant levels of voltage-gated calcium channels. Another cell line
(HN2V32) that was prepared by stable expression of a heterologous gene also
harbored in pCMV6, did not display any voltage-gated calcium current. The
central goal of this renewal application is to characterize the proximal
promoter of the APTL gene, and also understand the correlation between
overexpressed APTL and the appearance of voltage-gated calcium channels. The
proximal promoter sequence will be obtained using "Rapid Amplification of cDNA
Ends" (5'-RACE) and S1 nuclease digestion analysis. Functional activity and
sequence features of the promoter will be tested using serially deleted
segments of the promoter sequence to drive expression of the chloramphenical
acetyl transferase (CAT) reporter gene. Software analysis of the promoter
sequence will reveal the presence of enhancer/repressor elements. Possible
synergism between APTL and pCMV6 in the expression of the pore-forming alpha1
subunit of voltage-gated calcium channels will be tested by expressing APTL
cDNA from a vector completely different from pCMV6 and then testing the
APTL-overexpressing clones for the expression of alpha1 subunits. Possible
cross talk between APTL and the alpha1 subunits will be tested by expressing
epitope-tagged APTL and then testing the effect of APTL expression levels on
the expression of calcium channel alpha1 subunits. Also, APTL expression will
be suppressed by antisense treatment to look for its effect on the expression
and activity of calcium channels. Results from this project will shed new light
on the regulation and role of the protein APTL in brain neurons and apoptotic
neurotumor cells.
描述(申请人提供):化疗导致脑细胞凋亡
肿瘤细胞,然后通过吞噬作用被清除。在外周组织中,
吞噬作用之前是一个识别过程,其中清除剂
巨噬细胞选择性地结合磷脂酰丝氨酸(PS)分子
暴露于凋亡细胞表面。本次授予期间
在此期间,申请人的团队已经表明 PS 在
常驻清道夫细胞对凋亡神经肿瘤细胞的识别
大脑,小胶质细胞。酶在其中起核心作用
健康细胞内 PS 的内膜隔离是氨基磷脂
转位酶(APTL),也是一种 Mg2+-ATP 酶,属于最近发现的一种
P型ATP酶的分类亚家族。它在中枢神经系统中高度表达,但
人们对它在神经元中的调节和作用知之甚少。它的抑制或
下调导致 PS 外化的典型细胞凋亡特征。
为了研究该酶的调控谱,小鼠 APTL cDNA
在钙通道缺陷型杂交体中从载体 pCMV6 过表达
神经母细胞瘤细胞,HN2。除了显示出 15 倍的增长
磷脂酰丝氨酸转位酶活性,所有克隆都令人惊讶地表现出来
电压门控钙通道的显着水平。另一种细胞系
(HN2V32)也是通过稳定表达异源基因而制备的
包含在 pCMV6 中,没有显示任何电压门控钙电流。这
该更新应用程序的中心目标是表征近端
APTL基因的启动子,并了解之间的相关性
APTL 过度表达和电压门控钙通道的出现。这
近端启动子序列将使用“cDNA快速扩增”获得
Ends” (5'-RACE) 和 S1 核酸酶消化分析。功能活性和
将使用串行删除来测试启动子的序列特征
驱动氯霉素表达的启动子序列片段
乙酰转移酶(CAT)报告基因。启动器软件分析
序列将揭示增强子/阻遏子元件的存在。可能的
APTL 和 pCMV6 在成孔 α1 表达中的协同作用
电压门控钙通道亚基将通过表达 APTL 进行测试
来自与 pCMV6 完全不同的载体的 cDNA,然后测试
用于表达 alpha1 亚基的 APTL 过表达克隆。可能的
APTL 和 alpha1 亚基之间的串扰将通过表达来测试
表位标记的 APTL,然后测试 APTL 表达水平对
钙通道α1亚基的表达。此外,APTL 表达将
通过反义处理抑制以寻找其对表达的影响
和钙通道的活性。该项目的结果将带来新的启示
APTL蛋白在脑神经元及细胞凋亡中的调控及作用
神经肿瘤细胞。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('PROBAL BANERJEE', 18)}}的其他基金
A Chemical Strategy of Developing Resistance-Proof, Immune-Friendly Drugs
开发抗耐药性、免疫友好型药物的化学策略
- 批准号:
10113275 - 财政年份:2021
- 资助金额:
$ 3.3万 - 项目类别:
REGULATION OF APOPTOSIS BY THE SEROTONIN 1A RECEPTOR
5-羟色胺 1A 受体对细胞凋亡的调节
- 批准号:
2612538 - 财政年份:1998
- 资助金额:
$ 3.3万 - 项目类别:
Phosphatidylserine Translocase and Calcium Channels
磷脂酰丝氨酸转位酶和钙通道
- 批准号:
6747822 - 财政年份:1998
- 资助金额:
$ 3.3万 - 项目类别:
Regulation of ATPase II and Clearance of Cancer Cells
ATPase II 的调节和癌细胞的清除
- 批准号:
6944110 - 财政年份:1998
- 资助金额:
$ 3.3万 - 项目类别:
Phosphatidylserine Translocase and Calcium Channels
磷脂酰丝氨酸转位酶和钙通道
- 批准号:
6561733 - 财政年份:1998
- 资助金额:
$ 3.3万 - 项目类别:
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