Protein Overlays for Phosphoinositide Detection

描述（由申请人提供）：磷酸肌醇（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 in cellular蜂窝communication通信.特异性PIPns募集靶信号传导蛋白， 细胞内衔接蛋白连接到内膜位点以激活蛋白 激酶，从而启动信号转导级联，以调节 胞外和内吞作用，蛋白分选，细胞增殖和迁移， 肿瘤发生、凋亡和细胞形状的控制。确定 给定靶蛋白对特定PIPn的选择性已经成为一种新的靶蛋白。 解译下游效应子和新药发现中重要问题 努力寻找可以控制蛋白质-PIPn的潜在疗法 交互. 我们建议开发一个有效的蛋白质覆盖系统的两个组件 其允许确定已知蛋白质的脂质选择性或已知蛋白质的水平。 假定PIPn由脂质激酶和磷酸酶反应产生。这两种方法都使用 同样的原理，即，检测与固定在 硝化纤维素首先，我们将优化脂质印迹方法，我们称之为 PIP-Strips（TM）.“第二，我们将开发高选择性、高亲和力的 利用两个偶联的普列克底物蛋白同源性识别特异性PIPn的蛋白质 (PH)或FYVE域;这种新策略称为Double PH（tm）。的试剂 在第一阶段项目中准备和优化的数据将用于第二阶段， 开发多种高通量筛选方法，例如，快速、自动化 PIPn激酶和磷酸酶的测定以及小分子的筛选 蛋白质-PIPn结合抑制剂 拟定商业应用： 已经设想了开发的产品的一些商业应用 在这份赠款中。PIP-Strips（TM）将主要作为测定脂质结合的工具出售 分离蛋白质的特异性，它们的原始用途。 其他应用包括抗体 克隆蛋白的筛选和内部质量控制。 Lipo-PIP-Strips（TM）也将填充 同样的角色。 双PH试剂将作为单独的产品销售，目标客户为 特异性脂质和集中于一类脂质如3 ′-磷酸化PIPns的试剂盒。

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.