AFFINITY PROBES FOR INOSITOL POLYPHOSPHATE RECEPTORS

肌醇多磷酸受体的亲和探针

• 批准号: 6130603
• 负责人: GLENN DOWNES PRESTWICH
• 金额: $ 31.2万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 2004-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6130603
• 关键词: affinity labeling; binding proteins; biological signal transduction; chemical synthesis; enzyme activity; expression cloning; inositol phosphates; laboratory mouse; monoclonal antibody; phosphatidylinositols; phosphomonoesterases; protein kinase; protein structure function; receptor; receptor binding

Phosphoinositide polyphosphates (PIPns) are biosynthesized via the interplay of phosphatases and kinases and constitute key signaling molecules in cellular communication. The isolation, identification, and characterization of proteins that bind and metabolize PIPns have been realized using chemically-synthesized affinity probes, including photoaffinity labels, solid-support immobilized ligands, biotinylated probes, antigenic ligands, and fluorescently-labeled probes. We propose to expand the repertoire and uses of PIPE affinity probes by developing molecular sensors for intracellular events in the phosphoinositide signaling pathway. Thus, we will address six areas: (i) synthesis of affinity probes for PIPn-binding domain (PIPnBD) studies; (ii) optimization of cell permeant PIPns, (iii) development of novel fluorescence and antibody- based assays for measuring PIPn kinase and phosphatase activity (iv) discovery of new PIPn-binding proteins using classical biochemistry and in vitro expression cloning, (v) use of peptide libraries to identify novel peptides that act as PIPn selective mimics, and (vi) selected biophysical studies of novel PIPnBD-PIPn complexes. At The University of Utah, we will focus on preparation of affinity probes, development of assays, and biophysical study of selected complexes. At Utah State University, we will explore uses of antibody and affinity probes in cell biology and protein discovery. With collaborators elsewhere, new PIPn binding proteins will be identified and characterized, and uses of unique reagents will be developed.

通过磷酸酶和激酶的相互作用对磷酸肌醇聚磷酸盐（PIPN）进行生物合成，并构成细胞通信中的关键信号分子。使用化学合成的亲和力探针实现了结合和代谢PIPN的蛋白质的隔离，鉴定和表征，包括光疗法标签，固体支持的固定配体，生物素化探针，生物素化的探针，抗原性配体和含cluorescellesscelplyple的探针。我们建议通过在磷酸肌醇信号通路中开发分子传感器来扩大管道亲和力探针的使用。因此，我们将解决六个领域：（i）PIPN结合域（PIPNBD）研究的亲和力探针的合成； （ii）优化细胞渗透性PIPN，（iii）开发新型荧光和基于抗体的测定方法，用于测量PIPN激酶和磷酸酶活性（IV）发现新的PIPN结合蛋白的经典生物化学和使用肽培训（V）选择的PIPN培训（v），从新型PIPNBD-PIPN复合物。在犹他大学，我们将专注于准备亲和力探针的准备，分析的开发以及对选定复合物的生物物理研究。在犹他州立大学，我们将探索细胞生物学和蛋白质发现中抗体和亲和力探针的用途。与其他地方的合作者一起，将确定和表征新的PIPN结合蛋白，并将开发独特的试剂的使用。