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

• 批准号: 7952795
• 负责人: Brent Randall Martin
• 金额: $ 8.08万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7952795
• 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; Hydrolysis; 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; public health relevance; small hairpin RNA; tool; trafficking; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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. 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. Unique chemical tools will be developed to profile the palmitoylation enzymes implicated in the development of metastatic cancer.

描述（由申请人提供）：蛋白质棕榈酰化是一种重要的翻译后修饰，对于在细胞生长和信号传导中起关键作用的许多调节蛋白的运输和定位是必需的。我们最近开发了一种化学蛋白质组学方法，用于通过多个平台代谢掺入和检测棕榈酰化蛋白，包括基于荧光凝胶的检测和基于质谱的鉴定。这种方法在复杂的生物系统中对棕榈酰化蛋白质的分析显示出前所未有的灵敏度，从而鉴定出癌细胞中数百种棕榈酰化蛋白质。这些数据表明，棕榈酰化是一种广泛的翻译后修饰，影响几乎所有的细胞途径的功能。在许多情况下，棕榈酰化被认为是动态调节的，尽管控制这种脂质修饰的机制仍然缺乏表征。为了了解调节动态棕榈酰化的过程，我们将开发一个定量平台，用于棕榈酰化蛋白质的全球比较蛋白质组学分析，包括棕榈酰化的确切位点的鉴定。我们将使用这个平台来询问由棕榈酰转移酶和硫酯酶调节的棕榈酰化蛋白质的群体。一些癌基因需要棕榈酰化来诱导恶性转化，这表明蛋白质棕榈酰硫酯酶可能抑制异常的生长信号传导。通过测定生物正交标记底物的去棕榈酰化，我们已经确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。我们计划通过蛋白质组学鉴定底物，结合细胞转化和致瘤性测定，进一步表征APT1与癌症之间的关系。类似地，几种DHHC棕榈酰酰基转移酶（PAT）已被认为在癌症中发挥重要作用，但其对肿瘤发生的相对贡献的去卷积已被证明具有挑战性。我们建议为PAT创建第一个基于活性的蛋白质组学探针，并表征其在癌症进展不同阶段的活性。我们还将鉴定参与抑制转移的PAT底物。目前，缺乏个别PAT酶的选择性抑制剂。考虑到这一目标，我们将开发一种通用的HTS检测方法来鉴定PAT特异性抑制剂。 公共卫生相关性：蛋白质棕榈酰化是一种重要的翻译后修饰，对于许多在细胞生长和信号传导中起关键作用的调节蛋白的运输和定位是必需的。为了了解调节动态棕榈酰化的过程，我们将开发一个定量平台，用于棕榈酰化蛋白质的全球比较蛋白质组学分析，包括棕榈酰化的确切位点的鉴定。将开发独特的化学工具来描述与转移性癌症发展有关的棕榈酰化酶。