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Quantitative chemical proteomics of dynamic palmitoylation in cells

细胞中动态棕榈酰化的定量化学蛋白质组学

基本信息

批准号：
8516469
负责人：
Brent Randall Martin
金额：
$ 28.55万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-20 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8516469
关键词：
Adenocarcinoma Cell Affinity Chromatography Azides Biological Assay Biotin Cancer cell line Cell Line Cell physiology Cells Cellular Assay Chemicals Colon Adenocarcinoma Complement Coupled Data Detection Development Disseminated Malignant Neoplasm Enzymes Event Family Gel Goals Growth HRAS gene Human Hydrolase Individual Label Libraries Light Light Cell Link Lipids Malignant - descriptor Malignant Neoplasms Mass Spectrum Analysis Metabolic Metastasis Suppression Methods Mind Modification Mus Neoplasm Metastasis Oncogenes Palmitic Acylation Site Participant Pathology Pathway interactions Peptides Play Population Post-Translational Protein Processing Process Protein Dynamics Proteins Proteome Proteomics Relative (related person)Research Research Design Resolution Role Series Serine Hydrolase Signal Transduction Site Staging Stearic Acids Testing Transferase Triazoles Tumor Suppressor Proteins Tumorigenicity base cancer cell cancer proteomics cell growth comparative complex biological systems genetic regulatory protein high throughput screening inhibitor/antagonist instrument migration novel palmitoylation small hairpin RNA tool trafficking tumor progression tumorigenesis

项目摘要

Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of numerous regulatory proteins that play key roles in cell growth and signaling. We have recently developed a chemo-proteomic method for metabolic incorporation and detection of palmitoylated proteins by multiple platforms, including fluorescent gel-based detection and mass spectrometry-based identification. This approach shows unprecedented sensitivity for profiling palmitoylated proteins in complex biological systems, leading to the identification of hundreds of palmitoylated proteins in cancer cells. These data indicate that palmitoylation is a widespread post-translational modification that influences the function of nearly all cellular pathways. In many cases, palmitoylation is thought to be dynamically regulated, although the mechanisms that control this lipid modification remain poorly characterized. In order to understand the processes regulating dynamic palmitoylation, we will develop a quantitative platform for global comparative proteomic analysis of palmitoylated proteins, including identification of exact sites of palmitoylation. We will use this platform to interrogate the population of palmitoylated proteins regulated by both palmitoyl transferases and thioesterases. Several oncogenes require palmitoylation to induce malignant transformation, suggesting protein palmitoyl thioesterases may repress aberrant growth signaling. By assaying de-palmitoylation of bio-orthogonally labeled substrates, we have identified a novel protein thioesterase, and plan to expand this assay to other uncharacterized hydrolases. We plan to further characterize the relationship between APT1 and cancer by proteomic identification of substrates coupled with cellular assays of transformation and tumorigenicity. Similarly, several DHHC palmitoyl acyl transferases (PATs) have been suggested to play important roles in cancer, yet deconvolution of their relative contributions to tumorigenesis has proven challenging. We propose to create the first activity-based proteomics probe for PATs and characterize their activity at different stages of cancer progression. We will also identify PAT substrates involved in suppressing metastasis. Currently, selective inhibitors of individual PAT enzymes are lacking. With this goal in mind, we will develop a general HTS assay for identifying PAT-specific inhibitors.

蛋白质棕榈酰化是转运和定位所必需的重要翻译后修饰许多调节蛋白在细胞生长和信号传导中发挥关键作用。我们最近开发了一个通过多种方法代谢掺入和检测棕榈酰化蛋白质的化学蛋白质组学方法平台，包括基于荧光凝胶的检测和基于质谱的识别。这该方法在分析复杂生物系统中的棕榈酰化蛋白质方面表现出前所未有的灵敏度，导致癌细胞中数百种棕榈酰化蛋白的鉴定。这些数据表明棕榈酰化是一种广泛的翻译后修饰，影响几乎所有细胞的功能途径。在许多情况下，棕榈酰化被认为是动态调节的，尽管其机制控制这种脂质修饰的特征仍不清楚。为了了解调节过程动态棕榈酰化，我们将开发一个定量平台，用于全球比较蛋白质组学分析棕榈酰化蛋白质，包括棕榈酰化确切位点的鉴定。我们将利用这个平台询问由棕榈酰转移酶和硫酯酶调节的棕榈酰化蛋白质群体。几种癌基因需要棕榈酰化才能诱导恶性转化，表明棕榈酰蛋白硫酯酶可以抑制异常的生长信号传导。通过测定生物正交标记的去棕榈酰化底物，我们已经鉴定了一种新的蛋白质硫酯酶，并计划将该测定扩展到其他未表征的水解酶。我们计划通过以下方式进一步表征 APT1 与癌症之间的关系：底物的蛋白质组学鉴定与转化和致瘤性的细胞测定相结合。同样，几种 DHHC 棕榈酰酰基转移酶 (PAT) 被认为在癌症，但事实证明，解卷积它们对肿瘤发生的相对贡献具有挑战性。我们建议创建第一个基于活性的 PAT 蛋白质组学探针，并表征其在不同阶段的活性癌症进展。我们还将鉴定参与抑制转移的 PAT 底物。现在，缺乏单个 PAT 酶的选择性抑制剂。考虑到这一目标，我们将制定一个总体用于鉴定 PAT 特异性抑制剂的 HTS 测定。