Unravelling the poly(ADP-ribose) code: How does the structural diversity of poly(ADP-ribose) affect cellular functions during genotoxic stress response

解开聚（ADP-核糖）密码：聚（ADP-核糖）的结构多样性如何影响基因毒性应激反应期间的细胞功能

• 批准号: 386775082
• 负责人: Professor Dr. Aswin Mangerich
• 金额: --
• 依托单位: Lehrstuhl für Ernährungstoxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/386775082?language=en
• 关键词: Unravelling; poly; ADP; ribose; code

PARP1 catalyzes the posttranslational protein modification with poly(ADP-ribose) (PAR) of variable chain length and branching frequency. Poly(ADP-ribosyl)ation (PARylation) is involved in diverse cellular functions, such as DNA repair, transcription, and regulation of cell death, and contributes to various pathophysiological conditions (Rank et al. Nucleic Acids Res. 2016). Importantly, PARP inhibitors are being employed and further developed in clinical cancer therapy.The overall aim of this proposal is to unravel the biochemical and cellular functions in the structural heterogeneity of PARP1-mediated PARylation and mono-ADP-ribosylation. This includes the objectives:I. What is the role of protein-conjugated mono-ADP-ribose that arises as an intermediate during PAR metabolism?II. What is the function of the heterogeneity in PAR chain length (short vs. long PAR chains)?III. What is the function of the heterogeneity in PAR branching ratios?Within this, the following research questions should be addressed:- Do PAR molecules of different quality exhibit different stabilities with regards to their formation and degradation kinetics?- Do specific readers for the structural diverse PAR molecules exist, i.e., does a PAR code exist?- Does PAR diversity lead to differential regulation of protein complexes and enzymatic functions?- How does the structural diversity of PAR translate into specific cellular functions?Experimentally, these questions will be addressed by using PARP1 mutants with modified enzymatic activities, which result in mono-ADP-ribosylation activity (objective i), and PARP1 mutants generating PAR of altered chain lengths (objective ii) or branching ratios (objective iii). These mutants and the PAR produced by them will be analyzed biochemically using recombinant proteins and in cellular systems using PARP1-reconstituted HAP1 cells, either transiently transfected or CRISPR/Cas-engineered. HAP1 cells are a human, haploid cancer cell line ideally suited for this kind of purpose.The analyses will be performed in a systematic and comparative manner concerning several biochemical and cell biological end-points to decipher the different consequences of altered PARP1 activities with a focus on genotoxic stress response. This includes (i) the detailed analysis of the cellular PARylation and NAD+ metabolism, (ii) identification of PAR-structure-specific interactomes by novel affinity mass spectrometry approaches, (iii) spatio-temporal distribution and trafficking of select PARylation target proteins by advanced bioimaging techniques, and (vi) cellular consequences upon induction of genotoxic stress.In conclusion, using this approach it is expected (i) to obtain a deeper insight into the cellular biochemistry of PARP1, (ii) to improve our understanding on its role in cellular (patho-)physiology, and (iii) in the long run, to help tailor more specific and improved approaches for pharmacological PARP inhibition in cancer therapy.

PARP 1催化具有可变链长和分支频率的聚（ADP-核糖）（PAR）的翻译后蛋白质修饰。聚（ADP-核糖基）化（PAR化）参与多种细胞功能，如DNA修复、转录和细胞死亡调控，并导致各种病理生理学状况（Rank et al. Nucleic Acids Res. 2016）。重要的是，PARP抑制剂正在临床癌症治疗中得到应用和进一步开发。该提案的总体目标是阐明PARP 1介导的PARylation和单ADP核糖基化结构异质性中的生化和细胞功能。这包括以下目标： 在PAR代谢过程中作为中间体出现的蛋白结合单ADP-核糖的作用是什么？二. PAR链长度的异质性（短链与长链）的作用是什么？三. PAR分支比的不均匀性的作用是什么？在这方面，应处理以下研究问题： 不同质量的PAR分子在其形成和降解动力学方面是否表现出不同的稳定性？ 是否存在结构多样的PAR分子的特定读取器，即，有PAR代码吗？ PAR多样性是否导致蛋白质复合物和酶功能的差异调节？ PAR的结构多样性如何转化为特定的细胞功能？在实验上，这些问题将通过使用具有修饰的酶活性的PARP 1突变体来解决，所述突变体导致单ADP核糖基化活性（目标i），并且PARP 1突变体产生改变的链长度（目标ii）或分支比（目标iii）的PAR。这些突变体和由它们产生的PAR将使用重组蛋白进行生化分析，并在细胞系统中使用PARP 1重建的HAP 1细胞，无论是瞬时转染还是CRISPR/Cas工程化。HAP 1细胞是一种非常适合这种目的的人类单倍体癌细胞系，将以系统和比较的方式进行分析，涉及几个生化和细胞生物学终点，以解读PARP 1活性改变的不同后果，重点是遗传毒性应激反应。 这包括（i）细胞PAR化和NAD+代谢的详细分析，（ii）通过新的亲和质谱方法鉴定PAR结构特异性相互作用组，（iii）通过先进的生物成像技术选择PAR化靶蛋白的时空分布和运输，以及（vi）诱导遗传毒性应激后的细胞后果。使用这种方法，期望（i）获得对PARP 1的细胞生物化学的更深入的了解，（ii）提高我们对其在细胞（病理）生理学中的作用的理解，和（iii）从长远来看，帮助定制用于癌症治疗中的药理学PARP抑制的更特异和改进的方法。