Poly ADP Ribosylation

聚 ADP 核糖化
  • 文章类型: Journal Article
    DNA-蛋白质交联(DPC)是抑制DNA相关过程的毒性损伤。翻译后修饰(PTM),包括SUMO化和泛素化,在DPC分辨率中发挥核心作用,但是否也涉及其他PTM仍然难以捉摸。这里,我们确定了通过聚ADP核糖基化(PARylation)协调的DPC修复途径。使用非洲爪狼卵提取物,我们表明,单链DNA空位上的DPCs可以通过不依赖复制的机制被靶向降解。在这个过程中,DPC最初被PARP1PAR化,随后被蛋白酶体泛素化和降解。值得注意的是,PARP1介导的DPC拆分是拆分喜树碱诱导的拓扑异构酶1-DNA裂解复合物(TOP1ccs)所必需的。使用Flp-nick系统,我们进一步揭示了在缺乏PARP1活性的情况下,当遇到DNA复制叉时,TOP1cc样病变持续存在并诱导复制体分解。总之,我们的工作揭示了PARP1介导的DPC修复途径,该途径可能是TOP1毒物和PARP抑制剂之间协同毒性的基础.
    DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors.
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  • 文章类型: Journal Article
    DNA聚合酶theta(Polθ)介导的末端连接(TMEJ)修复DNA双链断裂并赋予对遗传毒性剂的抗性。如何在分子水平上调节Polθ以发挥TMEJ仍然缺乏表征。我们发现Polθ与PARP1以HPFl非依赖性方式相互作用并被PARP1化。PARP1通过依赖PARylation的液体分层将Polθ募集到DNA损伤附近,然而,PARylatedPolθ由于无法结合DNA而无法进行TMEJ。PARG介导的Polθ去PARG激活其DNA结合和末端连接活性。与此一致,PARG对TMEJ至关重要,PARG对DNA损伤的时间募集与TMEJ激活和PARP1和PAR的消散相对应。总之,我们展示了TMEJ调控的两步时空机制。首先,PARP1PARylatePole并促进其在失活状态下募集到DNA损伤位点。PARG随后通过去除Pole上的抑制性PAR标记来激活TMEJ。
    DNA polymerase theta (Polθ)-mediated end-joining (TMEJ) repairs DNA double-strand breaks and confers resistance to genotoxic agents. How Polθ is regulated at the molecular level to exert TMEJ remains poorly characterized. We find that Polθ interacts with and is PARylated by PARP1 in a HPF1-independent manner. PARP1 recruits Polθ to the vicinity of DNA damage via PARylation dependent liquid demixing, however, PARylated Polθ cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Polθ reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. In conclusion, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Polθ and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Polθ.
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  • 文章类型: Journal Article
    聚(ADP-核糖)聚合酶(PARP)是调节细胞活动的关键,例如对DNA损伤和细胞死亡的反应。PARP催化单-或聚(ADP-核糖基)形式的可逆翻译后修饰(PTM)。已知这种类型的修饰形成泛素-ADP-核糖(Ub-ADPR)缀合物,该缀合物取决于E3泛素连接酶(DTX)的Deltex家族的作用。特别是,DTX将泛素添加到ADP-核糖中腺苷核糖的3'-OH中,有效地隔离泛素并阻止泛素依赖性信号传导。先前的工作证明了DTX对无蛋白ADPR泛素化的功能,单-ADP-核糖基化肽,和ADP-核糖基化核酸。然而,DTX介导的聚(ADP-核糖基)泛素化的动力学仍有待定义。在这里,我们表明ADPR泛素化功能在其他PAR结合E3连接酶中未发现,并且在DTX家族成员中保守。重要的是,DTX特异性靶向聚(ADP-核糖)链用于泛素化,可被PARG裂解,聚(ADP-核糖)的初级橡皮擦,留下与泛素缀合的腺苷末端ADPR单元。我们的集体结果证明了多(ADP-核糖基)腺苷末端的DTX特异性泛素化,并表明独特的Ub-ADPR缀合过程是PARP-DTX控制细胞活性的基础。
    Poly(ADP-ribose) polymerases (PARPs) are critical to regulating cellular activities, such as the response to DNA damage and cell death. PARPs catalyze a reversible post-translational modification (PTM) in the form of mono- or poly(ADP-ribosyl)ation. This type of modification is known to form a ubiquitin-ADP-ribose (Ub-ADPR) conjugate that depends on the actions of Deltex family of E3 ubiquitin ligases (DTXs). In particular, DTXs add ubiquitin to the 3\'-OH of adenosine ribose\' in ADP-ribose, which effectively sequesters ubiquitin and impedes ubiquitin-dependent signaling. Previous work demonstrates DTX function for ubiquitination of protein-free ADPR, mono-ADP-ribosylated peptides, and ADP-ribosylated nucleic acids. However, the dynamics of DTX-mediated ubiquitination of poly(ADP-ribosyl)ation remains to be defined. Here we show that the ADPR ubiquitination function is not found in other PAR-binding E3 ligases and is conserved across DTX family members. Importantly, DTXs specifically target poly(ADP-ribose) chains for ubiquitination that can be cleaved by PARG, the primary eraser of poly(ADP-ribose), leaving the adenosine-terminal ADPR unit conjugated to ubiquitin. Our collective results demonstrate the DTXs\' specific ubiquitination of the adenosine terminus of poly(ADP-ribosyl)ation and suggest the unique Ub-ADPR conjugation process as a basis for PARP-DTX control of cellular activities.
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  • 文章类型: Journal Article
    聚(ADP-核糖基)化(PARylation),主要由聚(ADP-核糖)聚合酶(PARP)1催化,是参与DNA复制和修复的关键翻译后修饰。这里,我们报告说,没有时间(TIM),复制体的基本支架,是PARylated的,这与它的蛋白水解有关。TIMPARylation需要通过两个聚(ADP-核糖)结合基序识别自修饰的PARP1,启动TIM的蛋白酶体依赖性降解。表达PARylation难治性TIM突变体或受到PARP抑制的细胞在DNA复制叉上积累TIM,导致复制压力和停滞叉的过度切除。机械上,TIM与复制性解旋酶的异常接合阻碍了RAD51的加载和反向叉的保护。因此,缺陷性TIM降解使BRCA2缺陷细胞对复制损伤过敏。我们的研究将TIM定义为PARP1的底物,并阐明了如何通过PARylation控制复制体重塑与停滞的叉保护相关联。因此,我们提出了一种PARP抑制机制,该机制会影响由TIM转换缺陷引起的DNA复制叉不稳定性。
    Poly(ADP-ribosyl)ation (PARylation), catalyzed mainly by poly(ADP-ribose) polymerase (PARP)1, is a key posttranslational modification involved in DNA replication and repair. Here, we report that TIMELESS (TIM), an essential scaffold of the replisome, is PARylated, which is linked to its proteolysis. TIM PARylation requires recognition of auto-modified PARP1 via two poly(ADP-ribose)-binding motifs, which primes TIM for proteasome-dependent degradation. Cells expressing the PARylation-refractory TIM mutant or under PARP inhibition accumulate TIM at DNA replication forks, causing replication stress and hyper-resection of stalled forks. Mechanistically, aberrant engagement of TIM with the replicative helicase impedes RAD51 loading and protection of reversed forks. Accordingly, defective TIM degradation hypersensitizes BRCA2-deficient cells to replication damage. Our study defines TIM as a substrate of PARP1 and elucidates how the control of replisome remodeling by PARylation is linked to stalled fork protection. Therefore, we propose a mechanism of PARP inhibition that impinges on the DNA replication fork instability caused by defective TIM turnover.
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  • 文章类型: Journal Article
    ADP-核糖基转移酶PARP1和PARP2通过检测DNA损伤并诱导聚ADP-核糖基化依赖性染色质松弛和修复蛋白的募集在DNA修复机制中起主要作用。催化PARP抑制剂用作抗癌药物,尤其是在由致敏突变引起的肿瘤的情况下。最近,一项研究表明,组蛋白PAR化因子(HPF1)与PARP1/2形成联合活性位点。HPF1与PARP1/2的相互作用改变了从天冬氨酸/谷氨酸到丝氨酸的修饰位点,这已被证明是DNA损伤背景下的关键ADP核糖基化事件。因此,破坏PARP1/2-HPF1相互作用可能是药物开发阻断PARP1/2活性的替代策略.在这项研究中,我们描述了一种基于FRET的高通量筛选试验,以筛选抗PARP-HPF1相互作用的抑制剂库.我们优化了FRET信号的条件,并通过多种方式竞争FRET对验证了相互作用。该测定是稳健的并且易于自动化。有效的筛选显示了该测定的强大性能,我们发现了两种化合物二甲基丙烯紫草素和碱宁,对PARP1/2-HPF1相互作用具有μM抑制效力。该测定将有助于发现针对HPF1-PARP1/2复合物的抑制剂并开发潜在的新的有效抗癌剂。
    ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents.
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  • 文章类型: Journal Article
    聚(ADP-核糖基化)(PARylation)是由ADP-核基转移酶(ART)的子集介导的翻译后修饰。尽管基于PARylation抑制的疗法被认为是对抗癌症和肌病等衰弱疾病的途径,这种修饰在细胞分化等生理过程中的作用尚不清楚。这里,我们证明了Tankyrase1(TNKS1),PARylating艺术,在肌生成中起主要作用,已知驱动肌肉纤维形成和再生的重要过程。尽管所有真正的PARP都在肌肉细胞中表达,使用siRNA介导的敲减或药理学抑制的实验表明,TNKS1是负责在肌生成过程中催化PARylation的酶。通过这次活动,TNKS1控制编码生肌调节因子如核磷蛋白(NPM)和肌原蛋白的mRNA的周转。TNKS1通过靶向RNA结合蛋白如人抗原R(HuR)来介导这些作用。HuR具有保守的TNKS结合基序(TBM),其突变不仅阻止了HuR与TNKS1及其PARA化的关联,但也阻止了HuR调节NPM和肌原蛋白mRNA的周转以及促进肌生成。因此,我们的数据揭示了TNKS1作为RBP介导的转录后事件的关键调节因子的新作用,这些转录后事件是重要过程所必需的,如肌肉发生.
    Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.
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  • 文章类型: Journal Article
    染色质重塑剂ALC1被PARP1/PARP2及其辅因子HPFl沉积的DNA损伤诱导的聚(ADP-核糖)激活。ALC1已经成为癌症药物的靶点,但它是如何被招募到ADP-核糖基化核小体来影响它们在DNA断裂附近的定位的,目前尚不清楚。在这里,我们发现PARP1/HPF1优先在最接近DNA断裂的组蛋白H2B尾巴上启动ADP-核糖基化。为了剖析这种不对称的后果,我们仅在一侧产生具有确定的ADP-核糖基化H2B尾的核小体。与这种不对称核小体结合的ALC1的低温电子显微镜结构表明在一侧优先接合。使用单分子FRET,我们证明了这种不对称募集导致从最接近ADP-核糖基化位点的DNA接头的定向滑动。我们的数据表明,ALC1将核小体从DNA断裂中滑出,使其更易于修复因子的机制。
    The chromatin remodeler ALC1 is activated by DNA damage-induced poly(ADP-ribose) deposited by PARP1/PARP2 and their co-factor HPF1. ALC1 has emerged as a cancer drug target, but how it is recruited to ADP-ribosylated nucleosomes to affect their positioning near DNA breaks is unknown. Here we find that PARP1/HPF1 preferentially initiates ADP-ribosylation on the histone H2B tail closest to the DNA break. To dissect the consequences of such asymmetry, we generate nucleosomes with a defined ADP-ribosylated H2B tail on one side only. The cryo-electron microscopy structure of ALC1 bound to such an asymmetric nucleosome indicates preferential engagement on one side. Using single-molecule FRET, we demonstrate that this asymmetric recruitment gives rise to directed sliding away from the DNA linker closest to the ADP-ribosylation site. Our data suggest a mechanism by which ALC1 slides nucleosomes away from a DNA break to render it more accessible to repair factors.
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  • 文章类型: Journal Article
    细胞内ATP-核糖基转移酶PARP1和PARP2有助于DNA碱基切除修复(BER)和DNA去甲基化,并参与早期哺乳动物发育的表观遗传编程。最近,蛋白质组学分析确定BER蛋白被PARP共价聚ADP核糖基化。这种翻译后修饰在BER过程中的作用是未知的。这里,我们显示PARP1可感知在TET-TDG介导的活性DNA去甲基化过程中产生的AP位点和SSB,并将PAR共价连接到每个参与的BER蛋白上。共价PARylation从DNA中分离出BER蛋白,这加速了修复过程的完成。始终如一,抑制mESC中的PARylation导致减少的基因座特异性TET-TDG靶向DNA去甲基化,减少随机DNA损伤的一般修复。我们的发现建立了共价蛋白PARylation在协调与动态DNA甲基化相关的分子过程中的关键功能。
    The intracellular ATP-ribosyltransferases PARP1 and PARP2, contribute to DNA base excision repair (BER) and DNA demethylation and have been implicated in epigenetic programming in early mammalian development. Recently, proteomic analyses identified BER proteins to be covalently poly-ADP-ribosylated by PARPs. The role of this posttranslational modification in the BER process is unknown. Here, we show that PARP1 senses AP-sites and SSBs generated during TET-TDG mediated active DNA demethylation and covalently attaches PAR to each BER protein engaged. Covalent PARylation dissociates BER proteins from DNA, which accelerates the completion of the repair process. Consistently, inhibition of PARylation in mESC resulted both in reduced locus-specific TET-TDG-targeted DNA demethylation, and in reduced general repair of random DNA damage. Our findings establish a critical function of covalent protein PARylation in coordinating molecular processes associated with dynamic DNA methylation.
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  • 文章类型: Review
    Tankyrases,聚(ADP-核糖)聚合酶家族中的通用蛋白质组,对翻译后聚(ADP-核糖基)至关重要,影响各种细胞功能并导致疾病,尤其是癌症。因此,tankyrase已成为抗癌药物开发的重要靶标。药物发现中的新兴方法旨在破坏tankyrases与其结合伴侣之间的相互作用,它取决于伴侣蛋白内的tankyrase结合基序(TBM)和tankyrase内的锚蛋白重复簇结构域。我们的研究解决了识别和排名TBM的挑战。我们对现有文献进行了全面回顾,将TBM分为三个不同的组,每个人都有自己的评分系统。为了促进这一进程,我们介绍TBMHunter-anaccessible,基于Web的工具。这个用户友好的平台提供了一种免费且有效的方法来筛选和评估任何给定蛋白质中的潜在TBM。TBMHunter可以同时处理单个蛋白质或蛋白质列表。值得注意的是,我们的结果表明,TBMHunter不仅可以识别已知的TBM,而且还可以发现新的TBM。总之,我们的研究提供了一个全面的角度对TBM,并提出了一个易于使用,精确,以及用于识别和评估任何蛋白质中潜在TBM的免费工具,从而加强专注于tankyrase的研究和药物开发工作。
    Tankyrases, a versatile protein group within the poly(ADP-ribose) polymerase family, are essential for post-translational poly(ADP-ribosyl)ation, influencing various cellular functions and contributing to diseases, particularly cancer. Consequently, tankyrases have become important targets for anti-cancer drug development. Emerging approaches in drug discovery aim to disrupt interactions between tankyrases and their binding partners, which hinge on tankyrase-binding motifs (TBMs) within partner proteins and ankyrin repeat cluster domains within tankyrases. Our study addresses the challenge of identifying and ranking TBMs. We have conducted a comprehensive review of the existing literature, classifying TBMs into three distinct groups, each with its own scoring system. To facilitate this process, we introduce TBM Hunter-an accessible, web-based tool. This user-friendly platform provides a cost-free and efficient means to screen and assess potential TBMs within any given protein. TBM Hunter can handle individual proteins or lists of proteins simultaneously. Notably, our results demonstrate that TBM Hunter not only identifies known TBMs but also uncovers novel ones. In summary, our study offers an all-encompassing perspective on TBMs and presents an easy-to-use, precise, and free tool for identifying and evaluating potential TBMs in any protein, thereby enhancing research and drug development efforts focused on tankyrases.
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  • 文章类型: Journal Article
    PARP1/2抑制剂(PARPi)是临床上用于治疗具有BRCA缺陷的癌症的有效药物。PARPi在BRCA缺陷型癌症之外的成功和适用性有限,它们的作用因抗性机制而减弱。组蛋白PARylation因子(HPF1)的最新发现及其通过与PARP1形成共有的活性位点在PARylation反应中的作用提高了可以鉴定靶向PARP1-HPF1复合物的新型抑制剂的可能性。在本文中,我们描述了一种简单且具有成本效益的高通量筛选(HTS)方法,旨在发现PARP1-HPF1复合物的抑制剂。HTS验证后,我们首先应用此方法筛选了一个小型的PARP集中的化合物库,然后使用机器人自动化对我们的方法进行了10,000种化合物的试验筛选,并验证了>100种命中。这项工作首次证明了发现PARP1-HPF1复合物的有效抑制剂的能力,这可能有效用作为探针,以更好地了解DNA损伤反应和作为治疗癌症。
    PARP1/2 inhibitors (PARPi) are effective clinically used drugs for the treatment of cancers with BRCA deficiencies. PARPi have had limited success and applicability beyond BRCA deficient cancers, and their effect is diminished by resistance mechanisms. The recent discovery of Histone PARylation Factor (HPF1) and the role it plays in the PARylation reaction by forming a shared active site with PARP1 raises the possibility that novel inhibitors that target the PARP1-HPF1 complex can be identified. Herein we describe a simple and cost-effective high-throughput screening (HTS) method aimed at discovering inhibitors of the PARP1-HPF1 complex. Upon HTS validation, we first applied this method to screen a small PARP-focused library of compounds and then scale up our approach using robotic automation to conduct a pilot screen of 10,000 compounds and validating >100 hits. This work demonstrates for the first time the capacity to discover potent inhibitors of the PARP1-HPF1 complex, which may have utility as probes to better understand the DNA damage response and as therapeutics for cancer.
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