CHARACTERIZATION OF PUTATIVE MAGNESIUM TRANSPORTING P-TYPE ATPASES

假定的镁转运 P 型ATP酶的表征

• 批准号: 7381777
• 负责人: PATRICK John SCHULTHEIS
• 金额: $ 15.51万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381777
• 关键词: CHARACTERIZATION; PUTATIVE; MAGNESIUM; TRANSPORTING; TYPE

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. P-type ATPases comprise a large superfamily of proteins, present in both prokaryotes and eukaryotes, that transport inorganic cations and other substrates across cell membranes. Based on their conserved core sequences, they have been classified into 5 subfamilies, termed P1-P5. Members of the P1-P3 subfamilies have been well characterized using biochemical, molecular biological, and genetic techniques and include Na,K-ATPases, Ca-ATPases, and H,K-ATPases among others. P4-ATPases are expressed in all eukaryotes and include ~20 mammalian pumps that appear to function as aminophospholipid transporters. The most poorly understood P-type ATPases are those of the P5 subfamily, which are expressed only in eukaryotes. Despite being retained over evolutionary time in organisms as diverse as yeast, worms, fish and humans little is known about their function. The overall goal of the current proposal is to carry out a basic characterization of the P5-ATPases in mice. Specifically, the aims are to 1) isolate full-length cDNA clones for each of the family members and determine their tissue distribution and membrane location, 2) overexpress the novel P-type ATPases in the appropriate cell lines in order to assess their ion specificity, and 3) determine their cellular and physiological roles in vivo using RNA interference (RNAi) and gene targeting technologies. To date, we have almost completed aim 1. Full-length cDNA clones of each of the five family members and their tissue distributions have been determined. This work was published in October of 2004 (see publications). Since the last progress report we generated antibodies to isoform specific peptides of each transporter and these are currently being characterized by Western blot analysis. We have also generated stable cell lines expressing V5-histidine and GFP tagged versions of the transporters. These cell lines are being used in immunofluoresence and colocalization experiments to identify the membrane location of each family member. This histidine tagged transporters will also be purified by nickel affinity chromatography and will be used in biochemical studies to determine the ion specificity of the transporters. Work has also begun on the construction of targeting vectors for two of the P5-ATPases, Atp13a1 and Atp13a2. An R15 grant entitled, " has been submitted to NIH recently.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。 P 型 ATP 酶包含一个大的蛋白质超家族，存在于原核生物和真核生物中，可跨细胞膜运输无机阳离子和其他底物。根据其保守的核心序列，它们被分为5个亚科，称为P1-P5。 P1-P3 亚家族的成员已通过生化、分子生物学和遗传技术得到了很好的表征，其中包括 Na,K-ATP 酶、Ca-ATP 酶和 H,K-ATP 酶等。 P4-ATP 酶在所有真核生物中表达，包括约 20 个哺乳动物泵，这些泵似乎起到氨基磷脂转运蛋白的作用。人们最了解的 P 型 ATP 酶是 P5 亚家族的酶，它们仅在真核生物中表达。尽管在进化过程中被保留在酵母、蠕虫、鱼类和人类等多种生物体中，但人们对它们的功能知之甚少。当前提案的总体目标是对小鼠中的 P5-ATP 酶进行基本表征。 具体来说，目标是 1) 分离每个家族成员的全长 cDNA 克隆，并确定其组织分布和膜位置，2) 在适当的细胞系中过表达新型 P 型 ATP 酶，以评估其离子特异性，3) 使用 RNA 干扰 (RNAi) 和基因靶向技术确定它们在体内的细胞和生理作用。 到目前为止，我们几乎完成了目标1。五个家族成员的全长cDNA克隆及其组织分布已经确定。该作品于 2004 年 10 月出版（参见出版物）。自上次进展报告以来，我们生成了针对每种转运蛋白同工型特异性肽的抗体，目前正在通过蛋白质印迹分析对这些抗体进行表征。我们还生成了表达 V5-组氨酸和 GFP 标记版本的转运蛋白的稳定细胞系。这些细胞系用于免疫荧光和共定位实验，以识别每个家族成员的膜位置。这种组氨酸标记的转运蛋白也将通过镍亲和层析进行纯化，并将用于生化研究以确定转运蛋白的离子特异性。两种 P5-ATP 酶 Atp13a1 和 Atp13a2 的靶向载体的构建工作也已开始。 最近已向 NIH 提交了题为“的 R15 资助”。