Characterization of Adaptor proteins and their Posttranslational Modifications involved in ALT (alternative lengthening of telomeres) using Proteomics

端粒酶阴性肿瘤细胞使用被统称为端粒选择性延长（ALT）的机制来抵消复制相关的端粒缩短。人们普遍认为，大多数（如果不是全部的话）ALT机制使用同源重组来维持端粒长度。然而，精确的分子机制尚不清楚。大多数ALT细胞表现出一些特征，包括端粒长度不均一性、高水平的端粒姐妹染色单体交换和称为ALT相关PML小体（apb）的核结构。PML小体是在大多数细胞类型中普遍存在的大分子结构。这些小体参与许多不同的蛋白质-蛋白质和蛋白质-核酸相互作用。然而，PML小体与染色体末端的相互作用是ALT细胞的一个特征，使端粒靠近并促进S/G2期的复制后重组。目前尚不清楚这种时空控制是如何实现的。我们推测，独特的接头蛋白在端粒和PML体之间提供了桥梁，或者组成端粒结合蛋白的特定翻译后修饰是重要的。在本应用程序中，我们建议研究这两种可能性。为了鉴定接头蛋白，我们结合了一种基于pml浸润性疱疹病毒蛋白ICP0的创新方法作为纯化诱饵，并通过最先进的定量蛋白质组学深入表征ALT细胞的端粒- pml复合物。此外，利用高分辨率质谱，我们将建立ALT中端粒相关蛋白翻译后修饰的第一个综合图表。尽管文献中已经描述了几种翻译后修饰，目前尚不清楚这些修饰是否也存在于ALT中，以及它们在ALT中的作用。我们期望本研究的结果将使我们更好地了解ALT的机制。在本提案中，我们将ALT端粒生物学的专业知识（Draskovic和Boussin），一种新的纯化策略（Draskovic），多种胶质瘤干细胞系收集作为模型（Boussin）和基于质谱的蛋白质组学（Butter）的经验结合起来，研究APB的定义及其形成如何受到调节。

Telomerase-negative tumor cells use mechanisms collectively known as Alternative Lengthening of Telomeres (ALT) to counteract the replication-associated telomere shortening. It is generally accepted that most, if not all, ALT mechanisms use homologous recombination to maintain telomere length. However, the precise molecular mechanisms are unknown. Most ALT cells display characteristic features comprising telomere length heterogeneity, high levels of telomere-sister chromatid exchanges and nuclear structures called ALT-associated PML bodies (APBs). PML bodies are macromolecular structures ubiquitously present in most cell types. These bodies are involved in many different protein-protein and protein-nucleic acid interactions. However, the interaction of PML bodies with chromosome extremities is a characteristic feature of ALT cells, bringing telomeres into close proximity and promoting post-replicative recombination in S/G2 phase. It is currently unknown how this spatiotemporal control is achieved. We speculate that unique adaptor proteins provide a bridge between telomeres and the PML body or specific post-translational modifications of constitutive telomere binding proteins are important. In this application, we propose to investigate both possibilities. To identify adaptor proteins, we combine an innovative approach based on the PML-infiltrating Herpes virus protein ICP0 as a bait for purification and in depth characterization by state-of-the-art quantitative proteomics to define the telosome-PML complex in ALT cells. Furthermore, using high-resolution mass spectrometry, we will establish the first comprehensive chart of posttranslational modifications of telomere-associated proteins in ALT. While there have been several posttranslational modifications described in the literature, most of them have only been reported in telomerase-positive cell lines and currently it is unknown whether these modifications are also present in ALT and what their role might be in ALT. We expect that the results of this study will allow us a better understanding of the ALT mechanisms. In this proposal we unite the expertise in ALT telomere biology (Draskovic and Boussin), a new purification strategy (Draskovic), a diverse glioma stem cell line collection as a model (Boussin) and the experience in mass spectrometry based proteomics (Butter) to investigate what defines an APB and how its formation is regulated.