Quantitative chemical proteomics of dynamic palmitoylation in cells

蛋白质棕榈酰化是蛋白质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起关键作用。我们最近开发了一种 用于通过多重代谢掺入和检测棕榈酰化蛋白质的化学蛋白质组学方法 这些平台包括基于荧光凝胶的检测和基于质谱的鉴定。这 该方法显示了在复杂生物系统中分析棕榈酰化蛋白质的前所未有的灵敏度， 从而鉴定出癌细胞中的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，其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 控制这种脂质修饰仍然很难表征。为了了解调节 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，包括棕榈酰化的确切位点的鉴定。我们将利用这个平台， 询问由棕榈酰转移酶和硫酯酶两者调节的棕榈酰化蛋白质的群体。 几个癌基因需要棕榈酰化诱导恶性转化，提示蛋白棕榈酰 硫酯酶可抑制异常生长信号传导。通过测定生物正交标记的 底物，我们已经确定了一种新的蛋白质硫酯酶，并计划将这种测定方法扩展到其他 未表征的水解酶。我们计划通过以下方式进一步描述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偶联。 类似地，已经提出几种DHHC棕榈酰酰基转移酶（PAT）在以下方面发挥重要作用： 癌症，但其对肿瘤发生的相对贡献的反卷积已被证明具有挑战性。我们提出 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。目前， 缺乏单个PAT酶的选择性抑制剂。基于这一目标，我们将制定一项 用于鉴定PAT特异性抑制剂的HTS测定。

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.