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资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。P型ATP酶包含蛋白质的大超家族，存在于原核生物和真核生物中，其跨细胞膜运输无机阳离子和其它底物。根据其保守的核心序列，它们被分为5个亚家族，称为P1-P5。P1-P3亚家族的成员已经使用生物化学、分子生物学和遗传技术充分表征，并且包括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种哺乳动物泵，其似乎起氨基磷脂转运蛋白的作用。了解最少的P型ATP酶是P5亚家族的那些，其仅在真核生物中表达。尽管在酵母、蠕虫、鱼类和人类等不同的生物体中，它们在进化过程中被保留下来，但人们对其功能知之甚少。目前建议的总体目标是进行小鼠中P5-ATP酶的基本表征。 具体而言，目的是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其体内细胞和生理作用。 到目前为止，我们几乎已经完成了目标1。已经确定了五个家族成员的全长cDNA克隆及其组织分布。这项工作于2004年10月出版（见出版物）。自上次进展报告以来，我们产生了针对每种转运蛋白的同种型特异性肽的抗体，目前正在通过Western印迹分析对其进行表征。我们还产生了表达V5-组氨酸和GFP标记形式的转运蛋白的稳定细胞系。这些细胞系被用于免疫荧光和共定位实验，以确定每个家庭成员的膜位置。还将通过镍亲和色谱法纯化该组氨酸标记的转运蛋白，并将其用于生物化学研究，以确定转运蛋白的离子特异性。针对两种P5-ATP酶Atp 13 a1和Atp 13 a2的靶向载体的构建工作也已开始。 一项名为“最近已提交给NIH”的R15赠款。