Determining the Genes for Protein S-Palmitoylation

确定蛋白质 S-棕榈酰化的基因

• 批准号: 6918367
• 负责人: David A Zacharias
• 金额: $ 14.55万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-05 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6918367
• 关键词: acyltransferase; biotechnology; cell line; cell membrane; cell morphology; electroporation; fatty acylation; flow cytometry; functional /structural genomics; gene expression; gene mutation; genetic mapping; green fluorescent proteins; high throughput technology; mathematical model; molecular /cellular imaging; palmitates

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.

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。最近的数据表明，突触中PSD-95的这种调节对于突触组织和功能至关重要;已知调节蛋白质脂化的基因突变会导致严重的神经退行性疾病，强调了其调节对人类健康和疾病的重要性。尽管用于S-棕榈酰化的酶（棕榈酰硫代酰基转移酶，S-PAT）的表征一直存在争议，但最近来自酵母的数据表明存在这样的酶。在脊椎动物系统中的类似研究并没有那么富有成效。本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形式的基因捕获来满足这一需求，以高通量显微镜格式进行全自动读出。该测定系统将在每个孔板成像和单独记录的数百万个细胞上逐个细胞地阐明整个蛋白S-棕榈酰化途径，并针对进一步相关的荧光报告物读数进行多路复用。本文所述的具体目的是1）构建和表征将报告活细胞的棕榈酰化潜力的系统，所述棕榈酰化潜力通过与S-棕榈酰化底物融合的GFP（GFP：SPS）的亚细胞定位来确定，并且使用该细胞系统作为基因捕获实验的靶群体，所述基因捕获实验被设计用于鉴定对S-棕榈酰化至关重要的基因2）通过利用新的机器视觉算法和高通量细胞成像系统将S-PAT的搜索扩展到高通量筛选的水平。该系统使用S-棕榈酰化作为概念验证模型途径的开发将提供一个基础，该基础可以扩展到筛选调节亚细胞分布和报告基因浓度的其他基因。