Structure-Function of a Uterine P-type ATPase

子宫 P 型 ATP 酶的结构-功能

• 批准号: 6370518
• 负责人: Beverly S. Chilton
• 金额: $ 23.04万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6370518
• 关键词: adenosinetriphosphatase; binding proteins; chromatin; confocal scanning microscopy; enzyme activity; enzyme structure; genetic transcription; protein protein interaction; protein sequence; protein structure function; transcription factor; western blottings; yeast two hybrid system

DESCRIPTION: (Provided by the applicant) RUSH proteins are SWI/SNF-related transcription factors with RING-finger signatures near their Ctermini. Long suspected of mediating protein-protein interactions, the RING motif was used to clone a binding partner. The RING-finger binding protein (RFBP) is a Type IV P-type ATPase embedded in the inner nuclear membrane. This putative phospholipid pump has conserved sequences for two loop segments, an ATP-binding site, a phosphorylation domain and transmembrane passes potentially involved in substrate binding and translocation. However, RFBP differs from authentic Type IV P-type ATPases in several important ways. It has only three of four highly conserved NH2-terminal transmembrane passes. Topographically the orientation of the adjacent hydrophilic domains and the determinants of transport specificity are altered suggesting RFBP is not a transport protein. The small, hydrophilic loop extends into the perinuclear space that is contiguous with the lumen of the endoplasmic reticulum. The large, conformationally flexible loop extends into the nucleoplasm to contact euchromatin. Competitive reverse-transcriptase polymerase chain reaction and HPLC analysis showed that endometrial RFBP mRNA expression is hormonally regulated. The goal to more completely characterize RFBP will be achieved with three aims. In aim one, a 3-4-kb fragment of 5'-flanking sequence will be subjected to functional mapping to identify positive as well as negative attenuating elements. Deletion analysis and mutagenesis will help identify combinatorial interactions that govern RFBP transcription. In aim two, subregions of the RING motif and RFBP responsible for protein-protein interactions will be identified with glutathione 5-transferase-pulidown assays and yeast two-hybrid screens. Mutational analysis will be used to map the minimal protein-binding domains. In aim three, the effects of hormones on the dynamic association of RFBP with active chromatin, matrix associated splicing complexes and RING-finger containing factors will be determined by laser scanning confocal microscopy and Western analysis. The physical association of a hormone-dependent RING-finger binding protein with transcriptionally active chromatin suggests that RFBP plays a role in the subnuciear trafficking of transcription factors with RING motifs. The ultimate goal of understanding the regulatory role of hormones in the nuclear partitioning of transcription factors is central to defining the mechanism of hormone action in the uterus, and may be critical to good reproductive health.

描述：（由申请人提供）RUSH 蛋白与 SWI/SNF 相关 Ctermini 附近具有环指签名的转录因子。长的 怀疑介导蛋白质-蛋白质相互作用，RING 基序被用来 克隆结合伙伴。环指结合蛋白 (RFBP) 是 IV 型 P型ATP酶嵌入内核膜。这个假定的 磷脂泵有两个环片段的保守序列，一个 ATP 结合片段 位点、磷酸化结构域和跨膜通道可能参与 底物结合和易位。然而，RFBP 与正品 Type 不同 IV P 型 ATP 酶以几种重要的方式。它只有四个高度中的三个 保守的 NH2 末端跨膜通道。从地形上看，方向 相邻的亲水结构域和运输特异性的决定因素 改变表明 RFBP 不是转运蛋白。体积小，亲水性 环延伸到与管腔相邻的核周空间 内质网。大的、构象灵活的环延伸 进入核质接触常染色质。竞争性逆转录酶 聚合酶链反应和HPLC分析显示子宫内膜RFBP mRNA 表达受激素调节。目标是更完整地表征 RFBP 将通过三个目标来实现。在目标一中，一个 3-4-kb 的片段 5'-侧翼序列将进行功能作图以识别 正衰减元件和负衰减元件。删除分析和 诱变将有助于识别控制 RFBP 的组合相互作用 转录。在目标二中，RING 基序和 RFBP 的子区域负责 对于蛋白质-蛋白质相互作用将用谷胱甘肽进行鉴定 5-转移酶下拉测定和酵母双杂交筛选。突变分析 将用于绘制最小蛋白质结合域图。在目标三中， 激素对 RFBP 与活性染色质动态关联的影响， 基质相关的剪接复合物和包含因子的环指将是 通过激光扫描共焦显微镜和Western分析确定。这 激素依赖性环指结合蛋白与 转录活性染色质表明 RFBP 在 具有 RING 基序的转录因子的亚核运输。终极 目的是了解激素在核中的调节作用 转录因子的划分对于定义转录因子的机制至关重要 子宫内激素的作用，可能对良好的生殖健康至关重要。