Establishing the importance of DNA helicases and G-quadruplex homeostasis for the maintenance of proteome integrity with age

确定 DNA 解旋酶和 G 四链体稳态对于维持蛋白质组完整性随年龄增长的重要性

• 批准号: BB/W014890/1
• 负责人: Johnathan Labbadia
• 金额: $ 63.18万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW014890%2F1
• 关键词: Establishing; importance; DNA; helicases; quadruplex

The accumulation of misfolded, mislocalised and aggregated proteins (also known as proteostasis collapse) is a highly conserved driver of age-related tissue dysfunction in worms, flies, mice and humans. Our work in the small nematode worm C. elegans has demonstrated that, contrary to long-held assumptions, age-related proteostasis collapse does not simply arise from the accumulation of molecular damage, but instead, emerges from the transcriptional remodelling of the Proteostasis Network (PN; the complete collection of mechanisms that cooperate to maintain proteome integrity) early in life. Given that similar observations have been made in flies and human brain tissues, maintaining the expression of PN genes with age may be a potent way to maintain proteome integrity and prolong healthy tissue function. However, at present, it remains unclear precisely why the expression of PN genes changes with age and how this could be prevented. One currently unexplored and provocative possibility is that alterations in the structure of the genome itself may underlie changes in the expression of PN genes during adulthood. Non-helical DNA secondary structures known as G-quadruplexes (G4s) are highly prevalent across the genome and have emerged as important regulators of gene expression. While dynamic G4 formation can promote transcription factor binding and chromatin accessibility, the stabilisation of G4s can impede RNA polymerase II and repress transcription. As such, maintaining G4 homeostasis is crucial for normal cell function.G4 formation and stability is regulated by the action of DNA helicases, which bind to and actively resolve DNA secondary structures. We recently discovered that the expression of two prominent G4 resolving DNA helicases, wrn-1/WRN and him-6/BLM, declines prior to the loss of proteostasis capacity during early C. elegans adulthood, and that G4 structures are associated with candidate PN genes known to be down-regulated prior to protoestasis collapse. In addition, we find that reduced WRN-1 and HIM-6 activity leads to the repression of PN genes and accelerates the loss of proteostasis capacity in muscle tissues. These observations raise the intriguing possibility that reduced DNA helicase activity and increased G4 stability may be among the earliest events governing the loss of proteostasis capacity with age, and that by maintaining WRN/BLM activity or G4 homeostasis in aged cells, it may be possible to protect the ageing proteome. Here, we propose to explore this possibility by determining the precise relationship between DNA helicase activity, G4 homeostasis, proteostasis collapse and long-term tissue health. We will use a combination of tissue-specific proteostasis sensors and fluorescent reporters/probes to establish the specific tissues in which compromised WRN-1/HIM-6 activity and impaired G4 homeostasis underlies proteostasis collapse. In addition, we will combine genomics, mass-spectrometry-based proteomics and genetic screening approaches to identify the precise genes and proteins that are functionally relevant to age-related proteostasis collapse and tissue dysfunction downstream of reduced WRN-1/HIM-6 activity and compromised G4 homeostasis. Finally, we will genetically engineer animals to preserve wrn-1 and him-6 expression with age and use small molecule "helicase mimetics" to suppress G4 stabilisation in order to prevent transcriptional remodelling of the PN early in adulthood, maintain proteostasis capacity throughout life and promote healthy tissue function with age. We expect that this work will establish a new relationship between DNA secondary structure and the long-term health of the proteome, thereby acting as a foundation for future studies aimed at developing small molecule "helicase mimetics" that can be directed to specific genomic loci in order to discretely maintain the expression of select PN genes, maintain proteome integrity and promote healthy tissue function.

在蠕虫、果蝇、小鼠和人类中，错误折叠、错误定位和聚集蛋白的积累（也称为蛋白质稳态崩溃）是与年龄相关的组织功能障碍的高度保守驱动因素。我们对小线虫秀丽隐杆线虫的研究表明，与长期以来的假设相反，与年龄相关的蛋白质稳态崩溃并不仅仅来自于分子损伤的积累，而是来自于生命早期蛋白质稳态网络（PN；合作维持蛋白质组完整性的完整机制集合）的转录重塑。鉴于在果蝇和人类脑组织中已经进行了类似的观察，随着年龄的增长维持PN基因的表达可能是维持蛋白质组完整性和延长健康组织功能的有效方法。然而，目前尚不清楚为什么PN基因的表达会随着年龄的增长而变化，以及如何预防这种变化。一种目前未被探索和引起争议的可能性是，基因组本身结构的改变可能是成年期PN基因表达变化的基础。非螺旋DNA二级结构称为g -四重体（G4s）在基因组中非常普遍，并已成为基因表达的重要调节因子。虽然G4的动态形成可以促进转录因子的结合和染色质的可及性，但G4s的稳定可以阻碍RNA聚合酶II并抑制转录。因此，维持G4稳态对正常细胞功能至关重要。G4的形成和稳定性受DNA解旋酶的作用调控，DNA解旋酶结合并主动分解DNA二级结构。我们最近发现，两个重要的G4溶解DNA解旋酶，WRN -1/WRN和他-6/BLM的表达在秀丽隐杆线虫成虫早期蛋白酶平衡能力丧失之前下降，并且G4结构与已知在原平衡崩溃之前下调的候选PN基因有关。此外，我们发现WRN-1和HIM-6活性的降低会导致PN基因的抑制，加速肌肉组织中蛋白酶平衡能力的丧失。这些观察结果提出了一种有趣的可能性，即DNA解螺旋酶活性降低和G4稳定性增加可能是随着年龄增长而导致蛋白质平衡能力丧失的最早事件之一，并且通过维持衰老细胞中WRN/BLM活性或G4稳态，可能有可能保护老化的蛋白质组。在这里，我们建议通过确定DNA解旋酶活性、G4稳态、蛋白质稳态崩溃和长期组织健康之间的精确关系来探索这种可能性。我们将使用组织特异性蛋白质平衡传感器和荧光报告/探针的组合来建立特定组织中，受损的WRN-1/ hm -6活性和受损的G4稳态是蛋白质平衡崩溃的基础。此外，我们将结合基因组学、基于质谱的蛋白质组学和遗传筛选方法，以确定在WRN-1/ hm -6活性降低和G4稳态受损的下游，与年龄相关的蛋白质平衡崩溃和组织功能障碍在功能上相关的精确基因和蛋白质。最后，我们将对动物进行基因工程，使其随年龄增长而保持wrn-1和他-6的表达，并使用小分子“解旋酶模拟物”来抑制G4的稳定，以防止成年早期PN的转录重塑，在整个生命中维持蛋白质平衡能力，并随着年龄的增长促进健康的组织功能。我们期望这项工作将建立DNA二级结构与蛋白质组长期健康之间的新关系，从而为未来旨在开发小分子“解旋酶模拟物”的研究奠定基础，这些模拟物可以定向到特定的基因组位点，以离散地维持选择的PN基因的表达，维持蛋白质组的完整性，促进健康的组织功能。