Translational inhibition by Schlafen proteins during the DNA damage response

DNA 损伤反应期间 Schlafen 蛋白的翻译抑制

• 批准号: 10080748
• 负责人: MICHAEL DAVID
• 金额: $ 31.55万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10080748
• 关键词: Alkylating Agents; Biochemical; Biological Process; C-terminal; Cause of Death; Cell Survival; Cell physiology; Cells; Codon Nucleotides; DNA; DNA Damage; DNA Synthesis Inhibitors; Diagnostic; Drug resistance; Elements; Event; Exposure to; Family member; Foundations; Future Generations; Gene Expression; Genes; Genetic Transcription; Goals; HIV; Homologous Gene; Human; Infection; Influenza; Interferons; Knockout Mice; Malignant Neoplasms; Mediating; Modification; Molecular; Mus; N-terminal; Nature; Nucleic Acids; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phosphoric Monoester Hydrolases; Phosphorylation Site; Phosphotransferases; Positioning Attribute; Post-Translational Protein Processing; Production; Protein Biosynthesis; Protein Dephosphorylation; Proteins; RNA Helicase; Regulation; Reporting; Resistance; Retroviridae; Ribonuclease H; Role; Site; Testing; Topoisomerase-II Inhibitor; Transfer RNA; Translational Repression; Translations; Type I DNA Topoisomerases; Viral Proteins; Virus; Virus Replication; Work; base; cancer cell; cancer drug resistance; cell killing; cell type; chemotherapeutic agent; cofactor; effective therapy; experimental study; gene product; genome-wide; improved; in vivo; leucine-tRNA; mammalian genome; mouse genome; mutant; neoplastic cell; new technology; novel; novel strategies; nuclease; patient population; preservation; prognostic value; protein expression; response; targeted treatment; treatment strategy

Recently, human Schlafen 11 (SLFN11) - which we had shown to inhibit HIV protein expression due to the distinct codon-usage bias of the virus – was found to determine cell fate after exposure to DNA-damaging agents (DDAs). Cells lacking SLFN11 are resistant to DDAs, but not to other chemotherapeutic drugs. As DDAs are the largest group of cancer drugs, resistance against them impacts a large patient population and thus it is vital to unravel Slfn11's molecular contribution to the efficacy of DDAs, and to restore it in cells w/o Slfn11. So far, the events by which loss of SLFN11 causes resistance to DDAs remained unanswered. We now show that SLFN11 inhibits ATR translation in response to DDAs to enhance cell killing. This discerning inhibition translation is due to the prominent use of specific Leu codons in ATR. SLFN11 inhibits translation when Leu is (frequently) encoded via TTA or CTT, but not when other codons are employed. We demonstrate DDA-induced, SLFN11-mediated cleavage of a distinct tRNA subset including tRNAs Leu-TAA and Leu-AAG. DDA sensitivity in Slfn11-deficient cells can be restored 1) by abrogation of ATR expression; 2) through inhibition of ATR kinase activity; or 3) through the use of Gapmers, a novel technology we adapted to selectively target tRNA Leu-TAA for degradation. We note a novel mechanism of codon-specific regulation of translation by SLFN11 in the DNA damage response exists and provides the first evidence that modulation of a distinct tRNA allows for targeting specific proteins relying on those tRNAs. We provide proof-of-concept that targeting tRNAs by Gapmers is a valid approach to manipulate actions such as cell survival or viral replication. Our overarching goal is to improve our understanding of the function and regulation of Slfn11 during the DNA damage response on a cellular and molecular level. Aim 1 focuses on the analysis of Slfn11 itself, exploring its functional domains and regulation. We already identified several inhibitory phosphorylation sites in Slfn11 implying that dephosphorylation is required for SLFN11 activation, and show that PP1Cγ is the activating phosphatase during the DNA damage response. These findings need to be verified and expanded upon in additional settings (Other cell types and DDAs? Check for possible additional (de)phosphorylation sites? Identify likely cofactors?) The experiments outlined in Aim 2 target the role of the tRNA cleavage (identify cleavage site(s); test potential requirement for post-transcriptional modifications of tRNAs; do cleavage-resistant tRNA Leu-TAA mutants render cells DDA-resistant, and do such “mutants” exist in nature? Possible biological function for the tRNA-derived nucleic acid fragments?). Successful completion of the proposed studies will support the notion that SLFN11-deficient cancer cells can be (re)sensitized to DDA therapy by targeting ATR or distinct tRNAs, and that suppression of specific type II tRNAs might offer a new strategy to overcome resistance to DDAs. Finally, in our HIV studies we found that SLFN11 inhibited the translation of viral proteins during retro/lenti-viral infections, but not other viruses. We now think that the reason for this phenomenon is that retro-viruses cause DNA damage during integration, thereby likely activating SLFN11. In contrast, e.g. Influenza (despite similarity in codon bias to HIV) was not inhibited by SLFN11. We hypothesize now that SLFN11 might actually inhibit Influenza or other “biased” viruses if SLFN11 is independently activated by pharmacological means.

最近，人类Schlafen 11（SLFN11） - 我们已经证明，这抑制了由于 发现病毒的明显密码子 - 使用偏置 - 发现暴露于DNA破坏DNA后的细胞命运 代理（DDA）。缺乏SLFN11的细胞对DDA具有抗性，但对其他化学治疗药物不抗性。作为 DDA是最大的癌症药物，对它们的抵抗会影响大量的患者人群， 这对于阐明SLFN11对DDA效率的分子贡献至关重要，并在带有OP的细胞中恢复它 SLFN11。到目前为止，SLFN11损失的事件导致对DDA的阻力尚未得到解答。 现在，我们表明SLFN11响应DDAS抑制ATR翻译以增强细胞杀伤。这 辨别抑制翻译是由于ATR中特定的LEU密码子的显着使用。 SLFN11抑制 LEU（经常）通过TTA或CTT编码时的翻译，而不是使用其他密码子进行翻译。我们 演示了DDA诱导的SLFN11介导的裂解，包括trnas leu-taa 和Leu-aag。可以通过废除ATR表达来恢复SLFN11缺陷细胞中的DDA敏感性； 2） 通过抑制ATR激酶活性；或3）通过使用Gapmers，我们适应了一种新颖的技术 有选择地靶向tRNA leu-taa进行降解。我们注意到密码子特异性调节的新型机制 SLFN11在DNA损伤响应中的翻译存在，并提供了第一个证据表明 独特的tRNA允许靶向依靠这些TRNA的特定蛋白质。我们提供概念证明 通过Gapmers靶向TRNA是操纵诸如细胞存活或病毒复制之类的作用的有效方法。 我们的总体目标是提高我们对SLFN11功能和调节的理解 DNA损伤反应在细胞和分子水平上。 AIM 1专注于SLFN11本身的分析， 探索其功能领域和调节。我们已经确定了几个抑制性磷酸化位点 SLFN11意味着SLFN11激活需要去磷酸化，并表明PP1Cγ是 在DNA损伤反应过程中激活磷酸酶。这些发现需要验证和扩展 在其他设置（其他单元类型和DDAS？检查）是否可能其他（DE）磷酸化 网站？确定可能的辅助因子？） AIM 2中概述的实验目标是tRNA裂解的作用（识别裂解位点；测试 对TRNA的转录后修饰的潜在要求；做抗裂解的tRNA leu-taa 突变体使细胞具有抗DDA，并且这种“突变体”是否存在于自然界中？可能的生物学功能 tRNA来源的核酸碎片？）。 成功完成拟议的研究将支持SLFN11缺陷癌细胞的观念 可以通过靶向ATR或不同的TRNA对DDA治疗敏感，并且抑制特定类型 II TRNA可能会提供一种克服对DDA的抵抗力的新策略。最后，在我们的艾滋病毒研究中，我们发现 SLFN11抑制了复古/凸光病毒感染期间病毒蛋白的翻译，但没有其他病毒。我们 现在认为这种现象的原因是，复古病毒在整合过程中造成DNA损伤， 因此可能激活SLFN11。相反，例如流感（尽管密码子偏见与HIV相似）不是 被SLFN11抑制。我们现在假设SLFN11实际上可能抑制流感或其他“偏见” 病毒如果SLFN11通过药物手段独立激活。