ATR Isomerization in Cellular Responses to UV Damage of DNA

总结 DNA损伤是人类癌症和许多其他人类疾病的主要原因。响应于 DNA损伤，细胞激活DNA损伤反应（DDR）途径，如DNA损伤检查点， DNA修复和凋亡。ATR（共济失调毛细血管扩张和Rad 3相关），一个成员， 磷酸肌醇3-激酶相关蛋白激酶（PIKK）家族，是一个主要的DNA损伤检查点 蛋白激酶通过发出DNA损伤信号，激活检查点， 阻止细胞周期进程并促进DNA修复以恢复DNA完整性。有趣的是，一具尸体 来自小鼠模型和人类流行病学研究的证据表明，与ATM不同， ATR样PIKK家族成员，其缺陷促进致癌作用，ATR抑制抑制 致癌作用此外，ATR敲除是胚胎致死的。这表明ATR参与了 调节细胞死亡虽然ATR作为DDR中的检查点激酶已经被广泛研究，但在 细胞核，很少有人知道它的功能，在细胞质或线粒体，细胞器， 激活DNA损伤诱导的细胞凋亡。私家侦探最近的一项发现实验室发现，（a）除了它的 作为核检查点功能的标志，ATR是直接在线粒体上起作用的促存活蛋白 （B）ATR含有BH 3样结构域，其允许ATR像Bcl-2家族蛋白一样起作用; (c)重要的是，线粒体ATR是受Pin 1调节的ATR的脯氨酰顺式异构体形式，而相反， 核ATR是ATR的反式异构形式;最后（d）ATR的线粒体活性不依赖于 其检查点激酶活性和ATRIP。在本项目中，我们将测试以下假设：（1）脯氨酰 异构化改变了ATR的结构，将ATR功能转化为特异性活性， 促进细胞存活或作为DNA损伤检查点调节剂的核功能;（2）抗凋亡活性 ATR在线粒体中的表达在体内介导癌发生中起重要作用，因此，抑制 这种活性可以减少致癌作用/肿瘤发生并提供癌症预防策略， 治疗;和（3）ATR的反式异构体形式是其DNA损伤检查点活性所必需的，并且治疗后 ATR的翻译修饰和/或ATR-ATRIP复合物的形成可能在稳定ATR中起作用 以反式异构形式存在于原子核中。这些假设将在以下具体目标中得到检验。 目的1：确定ATR脯氨酰异构体、ATRH-tBid和ATRH-tBid的结构-功能关系。 目的2：确定脯氨酰异构化在ATR核功能中的作用; 目的3：研究ATR异构体在体内的致癌作用。的 提出的研究代表了与癌症生物学高度相关的创新努力， 在其他人类疾病如神经退行性疾病和心血管疾病中的应用。

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.