AFFINITY PROBES FOR INOSITOL POLYPHOSPHATE RECEPTORS

磷酸肌醇多磷酸（PIPns）是通过磷酸酶和激酶的相互作用生物合成的，并构成细胞通讯中的关键信号分子。已经使用化学合成的亲和探针实现了结合和代谢PIPns的蛋白质的分离、鉴定和表征，所述亲和探针包括光亲和标记物、固体支持物固定化配体、生物素化探针、抗原配体和荧光标记探针。我们建议通过开发用于磷酸肌醇信号通路中细胞内事件的分子传感器来扩展PIPE亲和探针的库和用途。因此，我们将致力于六个方面：（i）用于PIPn结合结构域（PIPnBD）研究的亲和探针的合成;（ii）细胞渗透性PIPn的优化，（iii）开发用于测量PIPn激酶和磷酸酶活性的新的基于荧光和抗体的测定，（iv）使用经典生物化学和体外表达克隆发现新的PIPn结合蛋白，（v）使用肽文库鉴定充当PIPn选择性模拟物的新肽，和（vi）新PIPnBD-PIPn复合物的选择性生物物理学研究。在犹他州大学，我们将专注于亲和探针的制备，分析方法的开发和选定复合物的生物物理研究。在犹他州州立大学，我们将探索抗体和亲和探针在细胞生物学和蛋白质发现中的应用。与其他地方的合作者，新的PIPn结合蛋白将被识别和表征，并将开发独特试剂的用途。

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.