Phosphatidylserine Translocase and Calcium Channels

描述(申请人提供)：化疗导致脑细胞凋亡 肿瘤细胞，然后通过吞噬清除这些细胞。在外周组织中， 吞噬作用之前有一个识别过程，在这个过程中清道夫 巨噬细胞选择性地与磷脂酰丝氨酸(PS)分子结合 暴露在凋亡细胞的表面。在当前授予期间 在此期间，申请人的团队已经证明PS在 小鼠常驻清道夫细胞对神经瘤细胞的识别作用 大脑，小胶质细胞。一种在体内起核心作用的酶 在健康细胞中PS的膜内隔离是氨基磷脂 转位酶(APTL)，也是一种镁离子-ATPase，属于最近的 P型ATPase亚家族的分类。它在中枢神经系统中高度表达，但 它在神经元中的调节和作用还知之甚少。它的抑制或 下调导致PS外化的典型的细胞凋亡特征。 为了研究该酶的调控特性，小鼠APTL的cDNA 在钙通道缺陷型杂交种中从载体pCMV6中过表达 神经母细胞瘤细胞，HN2。除了显示出15倍的增长外， 磷脂酰丝氨酸转移酶活性，所有克隆都意外地显示出 显著水平的电压门控钙通道。另一株细胞系 (HN2V32)也是通过稳定表达外源基因而制备的 在pCMV6中，不显示任何电压门控性钙电流。这个 此续订申请的主要目标是描述近端 APTL基因的启动子，并了解两者之间的相关性 APTL过表达和电压门控性钙通道的出现。这个 近端的启动子序列将被用“快速扩增的cDNA”获得 Ends“(5‘-RACE)和S1核酸酶消化分析。功能活性和 将用序列缺失的方法检测启动子的序列特征 驱动氯霉素表达的启动子序列片段 乙酰基转移酶(CAT)报告基因。启动子的软件分析 序列将揭示增强子/抑制子元件的存在。可能的 APTL和pCMV6在成孔蛋白α1表达中的协同作用 电压门控性钙通道亚单位将通过表达APTL进行测试 从与pCMV6完全不同的载体中提取cDNA，然后检测 用于α1亚基表达的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表达水平的影响 钙通道α1亚基的表达。此外，APTL表达式将 被反义治疗抑制，观察其对表达的影响 和钙通道的活性。这个项目的结果将带来新的曙光 APTL蛋白在脑神经元和细胞凋亡中的调节和作用 神经肿瘤细胞。

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.