Phosphatidylserine Translocase and Calcium Channels

磷脂酰丝氨酸转位酶和钙通道

• 批准号: 6561733
• 负责人: PROBAL BANERJEE
• 金额: $ 2.18万
• 依托单位: COLLEGE OF STATEN ISLAND
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/6561733
• 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

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，然后通过吞噬作用清除。在外周组织中， 吞噬作用之前是一个识别过程， 巨噬细胞选择性结合磷脂酰丝氨酸（PS）分子， 暴露在凋亡细胞的表面。在当前授予 期间，申请人的团队已经表明，PS在 凋亡神经瘤细胞的识别由常驻清道夫细胞 大脑，小胶质细胞这种酶在免疫系统中起着核心作用， 在健康细胞中，PS的内膜隔离是氨基磷脂 APTL也是一种Mg ~（2+）-ATP酶，属于新近发现的一种 分类的P型ATP酶亚家族。它在CNS中高度表达，但 其在神经元中的调节和作用知之甚少。其抑制或 下调导致PS外化的典型凋亡特征。 为了研究该酶的调控模式， 从载体pCMV 6在钙通道缺陷的杂交体中过表达 神经母细胞瘤细胞，HN 2。除了显示出15倍的增长， 磷脂酰丝氨酸移位酶活性，所有克隆令人惊讶地显示出 显著水平的电压门控钙通道。另一个细胞系 （HN 2 V32），其通过稳定表达异源基因制备，还 在pCMV 6中，不显示任何电压门控钙电流。的 该更新申请的中心目标是表征近端 APTL基因的启动子，并了解 过表达的APTL和电压门控钙通道的出现。的 使用“cDNA快速扩增”获得近端启动子序列 末端”（5 '-RACE）和S1核酸酶消化分析。功能活性和 启动子的序列特征将使用连续缺失的 启动子序列的片段来驱动氯霉素的表达， 乙酰转移酶（CAT）报告基因。启动子的软件分析 序列将揭示增强子/阻遏子元件的存在。可能 APTL和pCMV 6在成孔α 1表达中的协同作用 电压门控钙通道的亚基将通过表达APTL来测试 从与pCMV 6完全不同的载体中提取cDNA，然后测试 用于表达α 1亚基的APTL过表达克隆。可能 APTL和α 1亚基之间的串扰将通过表达 表位标记的APTL，然后测试APTL表达水平对 钙通道α 1亚单位的表达。此外，APTL表达将 通过反义处理来抑制，以寻找其对表达的影响 和钙通道的活性。该项目的结果将为我们提供新的视角， APTL蛋白在脑神经元和凋亡中的调节和作用 神经瘤细胞