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

PARP1通过可变链长和支化频率的聚腺苷二磷酸核糖(PAR)催化蛋白质的翻译后修饰。多聚(ADP-核糖基化)参与多种细胞功能，如DNA修复、转录和细胞死亡的调节，并参与各种病理生理条件(Rank等人)。核酸研究报告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的开发。本提案的总体目的是揭示PARP1介导的PAR化和单-ADP-核糖化的结构异质性中的生化和细胞功能。这包括目标：i.在PAR代谢过程中作为中间体出现的蛋白质结合的单-ADP-核糖的作用是什么？ii.PAR链长的异质性(短链和长PAR链)的作用是什么？iii.PAR分支率的异质性的作用是什么？在这方面，应该解决以下研究问题：-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？-是否存在针对结构不同的PAR分子的特定读取器，即，是否存在PAR密码？-PAR多样性是否导致蛋白质复合体和酶功能的不同调节？-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PARP1突变体来解决，这将导致单ADP核糖化活性(目标I)，以及PARP1突变体产生改变的链长(目标II)或分支比率(目标III)的PAR。这些突变体及其产生的PAR将使用重组蛋白进行生化分析，并在细胞系统中使用PARP1重组的HAP1细胞，无论是瞬时转染还是CRISPR/Cas工程处理。HAP1细胞是一种非常适合这种用途的人类单倍体癌细胞系。这些分析将以系统和比较的方式进行，涉及几个生化和细胞生物学终点，以破译PARP1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)用新的亲和质谱学方法鉴定PAR结构特异性相互作用，(Iii)通过先进的生物成像技术选择PAR化目标蛋白的时空分布和运输，以及(Vi)在诱导遗传毒性应激时的细胞后果。总之，使用这种方法有望(I)对PARP1的细胞生化有更深入的了解，(Ii)提高我们对其在细胞(病理)生理学中的作用的理解，以及(Iii)从长远来看，帮助量身定做更具体和改进的方法，在癌症治疗中抑制药理学PARP。

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.