A Cell-Based HT Imaging Assay for Palmitoylation(RMI)

描述（由申请人提供）：许多蛋白质通过脂质修饰集中在突触和小泡等特定亚细胞区域的质膜上。s -棕榈酰化是一种常见的脂质修饰形式，其独特之处在于它是可逆的和动态的，表明它在信号转导中具有类似于磷酸化的调节作用。这种脂质片段的加入可能会改变受体蛋白的一种或多种特性，如转运到膜上、在膜内重新分配特化微结构域、蛋白质-蛋白质相互作用和酶活性。最近，人们发现了一种能够催化棕榈酰酰基转移酶（palmitoyl acyl transferases, PATs）向蛋白质添加棕榈酸酯的酶的假定特征基元。编码假定的PATs的基因与包括精神分裂症和亨廷顿氏病在内的人类疾病有关，但需要进一步的证据来证明这些基因编码的蛋白质是真正的PATs。不幸的是，研究棕榈酰化的药理学工具几乎不存在。最常用的那些在功效、毒性和作用机制方面的特征很差，这给我们充分表征和理解棕榈酰化相关现象的能力留下了明显的空白。发现和开发此类试剂的关键在于创建和验证一种强大可靠的高通量筛选试验，可用于筛选大型化合物库。我们建议通过使用最先进的高通量成像工作站和新颖的图像分析算法设计和验证基于细胞的荧光高通量成像分析来满足这一需求。我们将利用棕榈酰化的主要特点，即它能够使蛋白质定位到细胞膜上。通过同时定量多种基于蛋白的报告蛋白的亚细胞分布，我们将能够获得有关分子作用机制以及调节棕榈酰化化合物功效的重要信息。

DESCRIPTION (provided by applicant): Many proteins are concentrated on the plasma membrane at specialized subcellular regions like synapses and caveolae by virtue of their lipid modifications. S-palmitoylation, a common form of lipid modification, is unique in that it is reversible and dynamic, suggesting a modulatory role in signal transduction similar to phosphorylation. Addition of this lipid moiety may alter 1 or more properties of the recipient protein such as trafficking to a membrane, redistribution specialized microdomains within a membrane, protein-protein interactions, and enzymatic activity. Very recently a putative signature motif for enzymes capable of catalyzing the addition of paimitate to proteins (palmitoyl acyl transferases or PATs) has been identified. Genes encoding putative PATs have been linked to human diseases including schizophrenia and Huntington's disease but further proof will be required to show that the proteins encoded by these genes are bona fide PATS. Unfortunately, pharmacological tools to study palmitoylation are almost non-existent. Those used most commonly are poorly characterized in terms of their efficacy, toxicity and mechanism of action leaving a conspicuous hole in our ability to characterize and understand fully palmitoylation-related phenomena. The key to discovering and developing such reagents lies in the creation and validation of a robust and reliable high-throughput screening assay that can be used to screen a large compound library. We propose to fill this need by designing and validating a cell-based, fluorescent, high throughput imaging assay using a state-of-the-art high throughput imaging workstation and a novel image analysis algorithm. We will exploit the predominant characteristic of palmitoylation, i. e. its ability to cause a protein to be localized to the cellular plasma membrane. By simultaneously quantifying the subcellular distribution of multiple protein based reporters we will be able to derive important information about the molecular mechanism of action as well as the efficacy of compounds that modulate palmitoylation.