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

描述(申请人提供)：许多蛋白质由于其脂质修饰而集中在特定亚细胞区域的质膜上，如突触和小凹。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，提示在信号转导中具有类似于磷酸化的调制作用。这种脂部分的加入可以改变受体蛋白的一种或多种性质，如转运到膜上，重新分布膜内的特殊微域，蛋白质-蛋白质相互作用，以及酶活性。最近，一个假定的特征基序被确定为能够催化蛋白质加成仿酸盐的酶(棕榈酰酰基转移酶或PATS)。编码PATS的基因与包括精神分裂症和亨廷顿病在内的人类疾病有关，但需要进一步的证据才能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。这些最常用的药物在有效性、毒性和作用机制方面的特点很差，这给我们描述和充分理解棕榈酰化相关现象的能力留下了一个明显的漏洞。发现和开发这类试剂的关键在于创建和验证一种强大而可靠的高通量筛选方法，该方法可用于筛选大型化合物文库。我们建议通过使用最先进的高通量成像工作站和新的图像分析算法设计和验证基于细胞的、荧光的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征，即它使蛋白质定位于细胞质膜的能力。通过同时量化多个基于蛋白质的报告的亚细胞分布，我们将能够获得关于调节棕榈酰化的化合物的分子作用机制以及有效性的重要信息。

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.