Phosphatidylserine Translocase and Calcium Channels

磷脂酰丝氨酸转位酶和钙通道

• 批准号: 6460322
• 负责人: PROBAL BANERJEE
• 金额: $ 3.3万
• 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/6460322
• 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。