ATR Isomerization in Cellular Responses to UV Damage of DNA

摘要 DNA损伤是人类癌症和许多其他人类疾病的主要原因。作为对.的回应 DNA损伤，细胞激活DNA损伤反应(DDR)途径，如DNA损伤检查点， DNA修复和细胞凋亡。ATR(共济失调毛细血管扩张和RAD3相关)，是 磷脂酰肌醇3-激酶相关蛋白激酶(Pikk)家族是一个主要的DNA损伤检查点 蛋白激酶在DDR中起着关键作用，它通过发出DNA损伤信号，激活检查点， 阻止细胞周期进程，促进DNA修复，恢复DNA完整性。有趣的是，一具身体 来自老鼠模型和人类流行病学研究的证据表明，与ATM不同的是，一种密切相关的 ATR样PIKK家族成员，其缺陷促进癌症发生，ATR抑制抑制 致癌。此外，ATR基因敲除对胚胎是致命的。这些都表明ATR参与了 在调节细胞死亡方面。尽管ATR作为DDR中的一种检查点激酶已被广泛研究 细胞核，对其在细胞质或线粒体中的功能知之甚少。 激活DNA损伤诱导的细胞凋亡。私家侦探S实验室最近的一项发现显示，(A)除了它的 作为核检查点功能的标志，ATR是一种直接作用于线粒体的促生存蛋白 抗紫外线损伤；(B)ATR含有一个类似BH3的结构域，允许ATR发挥像Bcl-2家族蛋白的作用； (C)重要的是，线粒体ATR是受Pin1调控的Pro-ATR顺式异构体，而相比之下， 核ATR是ATR的一种反式异构体；最后(D)ATR的线粒体活性独立于 它的检查点激酶活性和心房颤动。在这个项目中，我们将检验(1)脯氨基的假设 异构化改变ATR的结构，将线粒体特异性活动的ATR功能转化为 促进细胞存活或作为DNA损伤检查点调节器的核功能；(2)抗凋亡活性 线粒体ATR在体内的致癌过程中起着重要的作用，从而抑制 这种活动可减少致癌/致癌，并为癌症预防和 治疗；以及(3)ATR的反式异构体是其DNA损伤检查点活动所必需的，并且后 ATR和/或ATR-ATrip复合体形成的翻译修饰可能在ATR稳定中发挥作用 在原子核中以反式异构体的形式存在。这些假设将在以下具体目标中得到检验。 目的1：确定ATR-Prolyl异构体、ATRH-TBID和ATRH-TbID的结构-功能关系。 Pin1相互作用；目标2：确定Pro异构化在ATR核功能中的作用； 目的：研究ATR异构体在体内的致癌和致癌作用。这个 拟议的研究代表了一项与癌症生物学高度相关的创新努力，并具有 对其他人类疾病的影响，如神经退行性疾病和心血管疾病。

Summary DNA damage is a major cause of human cancers and many other human diseases. In response to DNA damage, cells activate DNA damage response (DDR) pathways such as DNA damage checkpoints, DNA repair, and apoptosis. ATR (ataxia telangiectasia and Rad3-related), a member of the phosphoinositide 3-kinase-related protein kinases (PIKK) family, is a major DNA damage checkpoint protein kinase which plays a critical role in DDR by signaling DNA damage, activating checkpoints, arresting cell cycle progression and facilitating DNA repair to restore DNA integrity. Interestingly, a body of evidence from mouse model and human epidemiologic studies shows that unlike ATM, a closely related ATR-like PIKK family member whose deficiency promotes carcinogenesis, ATR inhibition suppresses carcinogenesis. Moreover, ATR knockout is embryonically lethal. These suggest an involvement of ATR in regulating cell death. Although ATR has been extensively studied as a checkpoint kinase in DDR in the nucleus, little is known about its functions in the cytoplasm or mitochondria, the cellular organelle for activating DNA damage-induced apoptosis. A recent finding from the P.I.’s lab reveals that (a) besides its hallmark nuclear checkpoint functions, ATR is a pro-survival protein functioning directly at mitochondria against UV damage; (b) ATR contains a BH3-like domain that allows ATR to act like a Bcl-2 family protein; (c) importantly, mitochondrial ATR is a prolyl cis-isomeric form of ATR regulated by Pin1 while in contrast, nuclear ATR is a trans-isomeric form of ATR; and finally (d) mitochondria activity of ATR is independent of its checkpoint kinase activity and ATRIP. In this project, we will test the hypotheses that (1) Prolyl isomerization alters the structure of ATR, transforming ATR functions for mitochondria-specific activities to promote cell survival or nuclear functions as a DNA damage checkpoint regulator; (2) Antiapoptotic activity of ATR at mitochondria plays an important role in mediating carcinogenesis in vivo, and thus, suppressing such activity may reduce carcinogenesis/tumorigenesis and provide a strategy for cancer prevention and treatment; and (3) ATR’s trans-isomeric form is required for its DNA damage checkpoint activity, and post- translational modifications of ATR and/or ATR-ATRIP complex formation may play a role in stabilizing ATR in the trans-isomeric form in the nucleus. These hypotheses will be tested in the following specific aims. Aim 1: To define the structure-function relationships of ATR prolyl isomers, and of ATRH-tBid and ATRH- Pin1 interactions; Aim 2: To determine the role of prolyl isomerization in the nuclear functions of ATR; and Aim 3: To determine the in vivo effects of ATR isomers on carcinogenesis and tumorigenesis. The proposed studies represent an innovative effort highly relevant to cancer biology and also having implications in other human diseases such as neurodegenerative and cardiovascular diseases.