AFFINITY PROBES FOR INOSITOL POLYPHOSPHATE RECEPTORS

磷脂酰肌醇多聚磷酸盐(PIPns)是通过磷酸酶和激酶的相互作用而生物合成的，是细胞通讯中的关键信号分子。结合和代谢PIPns的蛋白质的分离、鉴定和鉴定是利用化学合成的亲和探针实现的，这些亲和探针包括光亲和标记、固相载体固定化配体、生物素化探针、抗原配体和荧光标记探针。我们建议通过开发磷脂酰肌醇信号通路中细胞内事件的分子传感器来扩大管道亲和探针的库和用途。因此，我们将致力于六个方面的研究：(I)PIPn结合结构域(PIPnBD)亲和探针的合成；(Ii)细胞PIPn的优化；(Iii)新型荧光和基于抗体的检测PIPn激酶和磷酸酶活性的方法的发展；(Iv)利用经典生物化学和体外表达克隆发现新的PIPn结合蛋白；(V)使用多肽库来鉴定作为PIPn选择性模拟物的新型多肽；以及(Vi)新型PIPnBD-PIPn复合体的精选生物物理研究。在犹他大学，我们将专注于亲和力探针的制备、分析方法的开发以及选定络合物的生物物理研究。在犹他州立大学，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一起，将识别和表征新的PIPn结合蛋白，并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.