Regulation and function of site-specific protein poly-ADP-ribosylation

项目摘要 蛋白质ADP-核糖基化（ADPr）是一种动态的NAD+依赖性翻译后修饰。哺乳动物 催化ADPr的聚（ADP-核糖）聚合酶（PARP）蛋白靶向几种化学上不同的氨基酸 数百种底物蛋白质上侧链官能团介导大量正交信号 转导途径。增加这种复杂性的是ADP-核糖聚合物形成的可能性， 其中所述PARP 1/2和TNKS 1/2酶从单ADPr位点延长ADP-核糖链。突出 聚ADP-核糖在生理学和疾病中的重要性是：（i）PARP 1/2的扩大的临床应用 治疗DNA修复缺陷型癌症的抑制剂，和（ii）TNKS 1/2在Wnt/b-连环蛋白信号传导中的功能， 发育性疾病包括巨胖症的功能障碍。异常ADPr活性也被报道为 心血管和神经退行性疾病的潜在原因，这些发现激发了强烈的 努力阐明PARP底物谱，确定PARP调节机制，并开发PARP 异构体特异性抑制剂。然而，考虑到ADPr在细胞信号传导中的自由部署及其 拓扑复杂的化学性质，我们对特定的单和多ADPr位点如何影响 蛋白质的功能和引发独特的生物活性已经落后。这项工作旨在填补这一空白。 通过开发新的方法来重建ADPr介导的信号传导事件， 控制生化和细胞环境。我们最近开发了一种化学酶策略来安装 丝氨酸ADPr到肽和蛋白质上，完全控制修饰位点和ADP-核糖链长度。 利用这项技术，我们确定了DNA损伤诱导的染色质的关键分子决定因素 重塑和揭示核小体丝氨酸聚ADPr的专门功能。我们现在处于一个独特的 我们的立场是建立在我们的技术和解决PARP生物学的基本问题。我们将探讨 控制聚ADPr活性的机制，并研究不同的修饰位点和伴随的 聚合物长度编码整个细胞的特定生化输出。这些信息可以引导更多 有效的策略来识别和治疗依赖于功能失调的ADPr活性的疾病。

PROJECT SUMMARY Protein ADP-ribosylation (ADPr) is a dynamic, NAD+-dependent post-translational modification. The mammalian poly(ADP-ribose) polymerase (PARP) proteins that catalyze ADPr target several chemically distinct amino acid side chain functionalities on hundreds of substrate proteins to mediate a multitude of orthogonal signal transduction pathways. Adding to this complexity is the potential for ADP-ribose polymer formation, a process wherein the PARP1/2 and TNKS1/2 enzymes elongate ADP-ribose chains from mono-ADPr sites. Highlighting the importance of poly-ADP-ribose in physiology and disease are: (i) the expanding clinical utility of PARP1/2 inhibitors to treat DNA repair-deficient cancers, and (ii) TNKS1/2 function in Wnt/b-catenin signaling and dysfunction in developmental diseases including Cherubism. Aberrant ADPr activity has also been reported as an underlying cause of cardiovascular and neurogenerative diseases, and these findings have inspired intense efforts to elucidate PARP substrate profiles, determine PARP regulatory mechanisms, and develop PARP isoform-specific inhibitors. However, given the liberal deployment of ADPr in cellular signaling and its topologically complex chemical nature, our understanding of how specific mono- and poly-ADPr sites impact protein function and elicit distinct biological activities has lagged behind. The proposed work aims to fill this knowledge gap by developing novel approaches to reconstitute ADPr-mediated signaling events in highly controlled biochemical and cellular environments. We recently developed a chemoenzymatic strategy to install serine ADPr onto peptides and proteins with full control over modification site and ADP-ribose chain length. Using this technology, we identified critical molecular determinants of DNA damage-induced chromatin remodeling and uncovered specialized functions for nucleosome serine poly-ADPr. We are now in a unique position to build upon our technologies and address fundamental questions in PARP biology. We will explore mechanisms that govern poly-ADPr activity and investigate how different modification sites and accompanying polymer lengths encode for specific biochemical outputs throughout the cell. Such information may guide more effective strategies to identify and treat diseases that rely on dysfunctional ADPr activity.