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统称为端粒替代性变长（ALT）的机制来抵消复制相关的端粒缩短。一般认为，大多数（如果不是全部）ALT机制使用同源重组来维持端粒长度。然而，精确的分子机制是未知的。大多数ALT细胞显示特征性特征，包括端粒长度异质性，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小体是普遍存在于大多数细胞类型中的大分子结构。这些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细胞的特征，使端粒紧密靠近并促进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，独特的衔接蛋白提供了端粒和PML体之间的桥梁或组成性端粒结合蛋白的特定翻译后修饰是重要的。在本申请中，我们建议调查这两种可能性。为了鉴定接头蛋白，我们结合联合收割机一种基于PML浸润疱疹病毒蛋白ICP 0作为诱饵的创新方法，通过最先进的定量蛋白质组学进行纯化和深入表征，以确定ALT细胞中的端粒体-PML复合物。此外，使用高分辨率质谱，我们将建立ALT中端粒相关蛋白翻译后修饰的第一个全面图表。虽然文献中描述了几种翻译后修饰，但其中大多数仅在端粒酶阳性细胞系中报道，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。我们希望这项研究的结果将使我们更好地了解ALT的机制。在这项提案中，我们结合了ALT端粒生物学（Draskovic和Boussin），新的纯化策略（Draskovic），作为模型的多样化胶质瘤干细胞系集合（Boussin）和基于质谱的蛋白质组学（Butter）的经验，以研究APB的定义及其形成如何受到调控。

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.