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（SLFN 11）--我们已经证明它可以抑制HIV蛋白的表达，这是由于 发现病毒独特的密码子使用偏好决定了暴露于DNA损伤后的细胞命运。 代理商（DDA）。缺乏SLFN 11的细胞对DDA有抗性，但对其他化疗药物没有抗性。作为 DDAs是最大的一类癌症药物，对它们的耐药性影响了大量的患者群体， 因此，阐明Slfn 11对DDA疗效的分子贡献，并在细胞中恢复Slfn 11是至关重要的。 Slfn11.到目前为止，SLFN 11的缺失导致对DDA的耐药性的事件仍然没有答案。 我们现在表明，SLFN 11抑制ATR翻译响应DDA，以增强细胞杀伤。这 识别抑制翻译是由于ATR中特异性Leu密码子的显著使用。SLFN 11抑制 当Leu（经常）通过TTA或CTT编码时，但当使用其他密码子时，不翻译。我们 证明DDA诱导的、SLFN 11介导的不同tRNA子集（包括tRNA Leu-TAA）的切割 和Leu-AAG。Slfn 11缺陷型细胞中的DDA敏感性可以通过以下方式恢复：1）消除ATR表达; 2） 通过抑制ATR激酶活性;或3）通过使用Gapmers，我们采用了一种新技术， 选择性靶向tRNA Leu-TAA进行降解。我们注意到一种新的密码子特异性调控机制， SLFN 11在DNA损伤反应中的翻译存在，并提供了第一个证据表明， 不同的tRNA允许靶向依赖于这些tRNA的特定蛋白质。我们提供概念验证， 通过Gapmers靶向tRNA是操纵诸如细胞存活或病毒复制的作用的有效方法。 我们的总体目标是提高我们对Slfn 11在肿瘤发生过程中的功能和调节的理解。 细胞和分子水平上的DNA损伤反应。目标1侧重于分析Slfn 11本身， 探索其功能域和调节。我们已经确定了几个抑制性磷酸化位点， 这意味着去磷酸化是SLFN 11激活所必需的，并表明PP 1C γ是SLFN 11激活的关键。 在DNA损伤反应中激活磷酸酶。这些发现需要得到验证和扩大 在其他设置（其他细胞类型和DDA？检查可能的额外（去）磷酸化 网站？确定可能的辅助因子？） 目标2中概述的实验靶向tRNA切割的作用（鉴定切割位点;测试 tRNA转录后修饰的潜在需求;抗切割tRNA Leu-TAA 突变体使细胞具有DDA抗性，这种“突变体”在自然界中存在吗？可能的生物学功能 tRNA衍生的核酸片段？）。 拟议研究的成功完成将支持SLFN 11缺陷癌细胞 可以通过靶向ATR或不同的tRNA对DDA治疗（重新）致敏， II tRNA可能提供一种克服对DDA耐药性的新策略。最后，在我们的艾滋病研究中，我们发现， SLFN 11在逆转录病毒/慢病毒感染期间抑制病毒蛋白的翻译，但不抑制其他病毒。我们 现在认为这种现象的原因是逆转录病毒在整合过程中导致DNA损伤， 从而可能激活SLFN 11。相比之下，例如流感病毒（尽管与HIV密码子偏好相似）不被识别。 受SLFN 11抑制。我们现在假设SLFN 11实际上可能抑制流感或其他“偏见”， 如果SLFN 11通过药理学手段被独立激活，则为病毒。