Targeted poly(ADP-ribosyl)ation of proteins (Gezielte Poly(ADP-Ribosyl)ierung von Proteinen)

ADP-核糖化和聚ADP-核糖化是以NAD+为底物，由ADP-核糖基转移酶(ARTS)类聚(ADP-核糖)聚合酶(PARPS)催化的蛋白质可逆的翻译后修饰。聚(ADP-核糖)(PAR)是一种大小可变的线状或多支化聚阴离子，可以与多种蛋白质非共价相互作用。PAR的形成与DNA修复、维持基因组稳定性、转录、端粒调节、细胞分裂、能量代谢、细胞死亡、肿瘤抑制和衰老有关。然而，我们对PAR和ADP-核糖修饰如何调节其目标蛋白的性质和功能的了解很少。为了更好地了解这些修饰及其生物学作用，我们建议合成具有不同位置的单和多(ADP-核糖)的蛋白质。作为第一个靶点，我们选择了组蛋白H1.2，它是体内的单核糖和多核糖，参与染色质的紧凑和基因表达的调节。产生的功能化蛋白质将是主要的兴趣和全面的生化后续研究的基础。我们将通过加入一种新的非天然氨基酸来实现项目目标，这种氨基酸通过终止密码子抑制来实现受保护的烷氧胺功能。这将使在烷氧胺官能团释放后，具有正交反应官能团的蛋白质能够进行特定部位的修饰。随后，新的连接化学将被开发出来，以提供一个强大的工具来连接非自然的氨基酸位置-特别是与ADP-核糖和PAR的还原端。这些定义明确的功能化蛋白将被用来研究组蛋白H1.2的单核糖和多(ADP-核糖)的生物学作用，其PAR修饰被认为是染色质结构的调节。

ADP-ribosylation and poly(ADP-ribosy)lation are reversible posttranslational modifications of proteins catalyzed by ADP-ribosyltransferases (ARTs) like poly(ADP-ribose) polymerases (PARPs) using NAD+ as substrate. Poly(ADP-ribose) (PAR) is a linear or multibranched polyanion of variable size that can interact noncovalently with numerous proteins. PAR formation has been associated with DNA repair, maintenance of genomic stability, transcription, telomere regulation, cell division, energy metabolism, cell death, tumour suppression and ageing. However, our knowledge of how PAR and ADP-ribose modifications modulate the properties and regulate the function of their target proteins is sparse. For a better understanding of these modifications and their biological role we propose the synthesis of proteins with distinct sites of mono- and poly(ADP-ribosy)lation. As first target we chose histone protein H1.2, which is mono- and poly(ADP-ribosy)lated in vivo and involved in the compaction of chromatin and regulation of gene expression. The generated functionalized proteins will be of major interest and the basis for comprehensive biochemical follow-up studies. We will reach the project goal by incorporation of a novel unnatural amino acids that bears a protected alkoxyamine functionality via stop codon suppression. This will enable site-specific modification of proteins with orthogonally reacting functional groups after liberation of the alkoxyamine functionality. Subsequently, new ligation chemistry will be developed to offer a powerful tool to conjugate the unnatural amino acid site-specifically with the reducing end of ADP-ribose and PAR. Such well-defined functionalized proteins will be used to investigate the biological role of mono- and poly(ADP-ribosy)lation of histone H1.2, whose PAR-modification has been suggested as a regulator of chromatin structure.