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

描述（由申请人提供）：许多蛋白质由于其脂质修饰而集中在质膜上的特定亚细胞区域，如突触和小窝。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，表明在信号转导中具有类似于磷酸化的调节作用。添加该脂质部分可以改变受体蛋白质的一种或多种性质，例如向膜的运输、膜内的再分布特化微结构域、蛋白质-蛋白质相互作用和酶活性。最近，已经鉴定了能够催化将棕榈酰添加到蛋白质的酶（棕榈酰酰基转移酶或PAT）的推定的特征基序。编码假定的PAT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。最常用的那些在其功效、毒性和作用机制方面的特征很差，这给我们表征和理解完全棕榈酰化相关现象的能力留下了明显的漏洞。发现和开发此类试剂的关键在于创建和验证可用于筛选大型化合物库的稳健且可靠的高通量筛选测定。我们建议通过设计和验证一种基于细胞的荧光高通量成像检测，使用最先进的高通量成像工作站和一种新的图像分析算法来满足这一需求。我们将利用棕榈酰化的主要特点，即。e.其使蛋白质定位于细胞质膜的能力。通过同时定量基于多个蛋白质的报告基因的亚细胞分布，我们将能够获得关于作用的分子机制以及调节棕榈酰化的化合物的功效的重要信息。

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.