Quantitative chemical proteomics of dynamic palmitoylation in cells

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，是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所必需的。我们最近开发了一种化学蛋白质组学方法，用于通过多种平台代谢结合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。这种方法在分析复杂生物系统中的棕榈酰化蛋白方面显示出前所未有的敏感性，从而在癌细胞中鉴定出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，影响几乎所有细胞通路的功能。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不清楚。为了了解动态棕榈酰化的调节过程，我们将开发一个定量平台，用于棕榈酰化蛋白的全球比较蛋白质组学分析，包括鉴定棕榈酰化的确切位点。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，这表明蛋白棕榈酰硫酯酶可能抑制异常生长信号。通过分析生物正交标记底物的去棕榈酰化，我们已经鉴定出一种新的蛋白质硫酯酶，并计划将该试验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致瘤性的细胞测定来进一步表征APT1与癌症之间的关系。类似地，一些DHHC棕榈酰酰基转移酶（PATs）已被认为在癌症中发挥重要作用，但它们对肿瘤发生的相对贡献的反折叠已被证明具有挑战性。我们建议为PATs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。我们还将确定参与抑制转移的PAT底物。目前，缺乏单个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检测方法来识别pat特异性抑制剂。

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.