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: 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
• 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

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.

蛋白质棕榈酰化是贩运和定位所必需的翻译后修改 许多调节蛋白在细胞生长和信号传导中起关键作用。我们最近开发了 用于代谢掺入和检测棕榈酰化蛋白的化学蛋白质学方法 平台，包括基于荧光凝胶的检测和基于质谱的识别。这 方法显示了对复杂生物系统中棕榈酰化蛋白进行分析的前所未有的灵敏度， 导致鉴定癌细胞中数百种棕榈酰化蛋白。这些数据表明 棕榈酰化是一种广泛的翻译后修饰，影响了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，尽管这种机制 控制此脂质修饰的表征仍然很差。为了了解调节的过程 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 棕榈酰化的蛋白质，包括识别棕榈酰化的精确位点。我们将使用此平台来 询问由棕榈酰转移酶和硫酯酶调节的棕榈酰化蛋白质的种群。 几种癌基因需要棕榈酰化来诱导恶性转化，表明蛋白质棕榈酰 硫酯酶可能会抑制异常的生长信号传导。通过测定对生物近似标签的去甲米二酰化 底物，我们已经确定了一种新型的蛋白质硫酯酶，并计划将此测定扩展到其他 未表征的水解酶。我们计划通过 底物的蛋白质组学鉴定以及转化和肿瘤性的细胞测定法。 同样，已经提出了几种DHHC棕榈酰酰基转移酶（PAT）在 癌症，但对肿瘤发生的相对贡献的反卷积已被证明是具有挑战性的。我们建议 为PATS创建第一个基于活动的蛋白质组学探针，并在不同阶段表征其活动 癌症进展。我们还将确定涉及抑制转移的PAT底物。现在， 缺乏单个PAT酶的选择性抑制剂。考虑到这个目标，我们将发展一个一般 HTS分析用于鉴定特异性抑制剂。

项目成果

The next frontier of post-translational modifications.

翻译后修饰的下一个前沿。

• DOI: 10.1002/bip.22370
• 发表时间: 2014
• 期刊: Biopolymers
• 影响因子: 2.9
• 作者: Martin,BrentR

Strategies for profiling native S-nitrosylation.

• DOI: 10.1002/bip.22342
• 发表时间: 2014-02
• 期刊: BIOPOLYMERS
• 影响因子: 2.9
• 作者: Majmudar, Jaimeen D.;Martin, Brent R.
• 通讯作者: Martin, Brent R.

Proteomic analysis of S-acylated proteins in human B cells reveals palmitoylation of the immune regulators CD20 and CD23.

• DOI: 10.1371/journal.pone.0037187
• 发表时间: 2012
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Ivaldi C;Martin BR;Kieffer-Jaquinod S;Chapel A;Levade T;Garin J;Journet A
• 通讯作者: Journet A

Chemical approaches for profiling dynamic palmitoylation.

• DOI: 10.1042/bst20120271
• 发表时间: 2013-02-01
• 期刊: Biochemical Society transactions
• 影响因子: 3.9
• 作者: Martin BR

APT2 Inhibition Restores Scribble Localization and S-Palmitoylation in Snail-Transformed Cells.

• DOI: 10.1016/j.chembiol.2016.12.007
• 发表时间: 2017-01-19
• 期刊: Cell chemical biology
• 影响因子: 8.6
• 作者: Hernandez JL;Davda D;Cheung See Kit M;Majmudar JD;Won SJ;Gang M;Pasupuleti SC;Choi AI;Bartkowiak CM;Martin BR
• 通讯作者: Martin BR