Protein Overlays for Phosphoinositide Detection

说明书(由申请人提供)：磷脂酰肌醇(PIPns)，例如， 磷脂酰肌醇4，5-二磷酸(PI 4，5)P2是关键的信号分子 在蜂窝通信中。特定的PIPN招募靶向信号蛋白并 细胞内适配蛋白与内膜部位结合激活蛋白 从而启动信号转导级联反应，从而调节 外吞和内吞，蛋白质分选，细胞增殖和迁移， 肿瘤发生、细胞凋亡和细胞形状的控制。确定 给定靶蛋白对特定PIPn的选择性已成为 破译下游效应物和新药开发中的重要问题 努力寻找可能控制蛋白质的潜在疗法-PIPn 互动。 我们建议开发一个有效的蛋白质覆盖系统的两个组成部分 它允许确定已知蛋白质的脂类选择性或 给定的PIPn是由脂质激酶和磷酸酶反应产生的。这两种方法都使用 同样的原理，即检测与固定化PIPns结合的蛋白质 硝化棉。首先，我们将优化脂质印迹方法，我们称之为 “PIP-STRIPS(TM)。”第二，发展高选择性、高亲和力的 利用两个偶联的pleckstrin同源物识别特定PIPn的蛋白质 (Ph)或FYVE域；这种新策略称为双PH(Tm)。试剂盒 在此一期工程中准备和优化的将用于二期至 开发各种高通量筛选方法，如快速、自动化 PIPn激酶和磷酸酶的检测及小分子的筛选 蛋白质-PIPn结合的抑制剂 建议的商业应用： 所开发的产品已经设想了许多商业应用 在这笔赠款中。PIP-STRIPS(TM)将主要作为确定脂质结合的工具销售 分离蛋白的专一性，它们最初的目的。其他应用包括抗体 克隆蛋白的筛选和室内质量控制。Lipo-PIP-Strips(TM)也可以填充 这些相同的角色。双PH试剂将作为单件产品出售，目标是 特定的脂类和专注于一类脂类的试剂盒，如3‘-磷酸化的PIPns。

DESCRIPTION (provided by applicant): Phosphoinositides (PIPns), e.g., phosphatidylinositol 4,5-bisphosphate (PI 4,5) P2) are key signaling molecules in cellular communication. Specific PIPns recruit target signaling proteins and intracellular adapter proteins to inner membrane sites to activate protein kinases and thereby initiate signal transduction cascades in order to regulate exo- and endocytosis, protein sorting, cell proliferation and migration, tumorigenesis, apoptosis, and the control of cell shape. Determining the selectivity of a given target protein for a specific PIPn has become an important issue in deciphering downstream effectors and in new drug discovery efforts for potential therapeutics that could control protein-PIPn interactions. We propose to develop both components of an efficient protein overlay system that allows determining lipid selectivity of a known protein or levels of a given PIPn produced by lipid kinase and phosphatase reactions. Both methods use the same principle, i.e., detection of proteins bound to PIPns immobilized on nitrocellulose. First, we will optimize the lipid blot method, which we call "PIP-Strips (TM)." Second, we will develop highly selective, high-affinity proteins that recognize a specific PIPn using two coupled pleckstrin homology (PH) or FYVE domains; this new strategy is called Double PH(tm). The reagents prepared and optimized in this Phase I project will be used in Phase II to develop a variety of high throughput screening methods, e.g., rapid, automated assays for PIPn kinases and phosphatase and screening for small molecule inhibitors of protein-PIPn binding PROPOSED COMMERCIAL APPLICATION: A number of commercial applications have been envisioned for the products developed in this grant. PIP-Strips(TM) will be primarily sold as tools to determine lipid binding specificity of isolated proteins, their original purpose. Other applications include antibody screening and in house quality control of cloned proteins. Lipo-PIP-Strips(TM) will also fill these same roles. The Double-PH reagents will be sold as individual items targeted to a specific lipid and a kits focussing on a class of lipids such as 3'-phosphorylated PIPns.