Determining the Genes for Protein S-Palmitoylation

描述(由申请人提供)： 许多蛋白质由于其脂质修饰而集中在特殊的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。最近的数据表明，突触中PSD-95的这种调节对突触的组织和功能至关重要；调节蛋白质脂肪作用的基因突变会导致严重的神经退行性疾病，强调了它们调节对人类健康和疾病的重要性。尽管S-棕榈酰化反应的酶(S-硫酰基转移酶)的性质一直存在争议，但最近来自酵母的数据表明，这种酶是存在的。在脊椎动物系统中的类似研究并没有那么有成效。这项提案中概述的工作旨在通过在培养的单倍体细胞中采用一种新的基因捕获形式来满足这一需求，以实现高通量显微镜格式的全自动读出。该检测系统将在逐个细胞的基础上阐明整个蛋白质S-棕榈酰化途径，每个孔板对数百万个细胞进行成像和单独记录，并与进一步的相关荧光报告读数进行多路复用。这里描述的具体目标是1)构建和表征一种系统，该系统将报告由融合到S棕榈酰化底物(绿色荧光蛋白：SP)上的绿色荧光蛋白的亚细胞定位确定的活细胞的棕榈酰化潜能，并将该系统用作基因陷阱实验的目标群体，该基因陷阱实验旨在识别S-棕榈酰化反应的关键基因。2)通过利用新型机器视觉算法和高通量细胞成像系统，将对S-PATS的搜索扩展到高通量筛选的水平。以S棕榈酰化为概念验证模型通路的这一系统的开发，将为进一步筛选调节报告基因亚细胞分布和浓度的其他基因提供基础。

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. Recent data indicate that such regulation of PSD-95 in the synapse is critical for synaptic organization and function; mutations in genes regulating protein lipidation are known to result in severe neurodegenerative disorders, emphasizing the importance of their regulation to human health and disease. Although characterization of the enzymes for S-palmitoylation (Palmitoyl thio-Acyl Transferases, S- PATs) has been controversial, recent data from yeast indicate that such enzymes exist. Similar studies in vertebrate systems have not been as fruitful. The work outlined in this proposal aims to meet this need by adapting a novel form of gene-trapping in cultured haploid cells for fully automated readout in a high-throughput microscopy format. This assay system will elucidate the entire protein S-palmitoylation pathway on a cell-by-cell basis over millions of cells imaged and individually recorded per well plate and multiplexed against further relevant fluorescent reporter readouts. The specific aims described herein are 1) to construct and characterize a system that will report the palmitoylation potential of living cells as determined by the subcellular localization of a GFP fused to an S-palmitoylation substrate (GFP:SPS) and to use this cellular system as the target population for gene trap experiments designed to identify genes critical for S-palmitoylation 2) to scale the search for S-PATs to the level of a high-throughput screen by utilizing a novel machine-vision algorithm and a high-throughput cellular imaging system. Development of this system using S-palmitoylation as the proof-of-concept model pathway will provide a foundation that can be extended to screens for other genes that regulate the subcellular distribution and concentration of a reporter gene.