Investigating the molecular basis of evolved stress resilience in a subterrestrial nematode

研究地下线虫进化的应激恢复能力的分子基础

• 批准号: 10438979
• 负责人: John Russell Bracht
• 金额: $ 42.52万
• 依托单位: AMERICAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-07 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10438979
• 关键词: Affect; Antibiotics; Apoptosis; Arginine; Astrocytes; Autoimmune; Autoimmune Diseases; Caenorhabditis elegans; Cellular Stress Response; Development; Disease; Elements; Environment; Family; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genomics; Growth; Heat Stress Disorders; Heat-Shock Proteins 70; Heat-Shock Response; Human Pathology; Hypoxia; Investigation; Laboratories; Life; Link; Malignant Neoplasms; Measures; Methane; Molecular; Mutate; Nematoda; Nerve Degeneration; Neurodegenerative Disorders; Organism; Pathway interactions; Phenotype; Play; Proteins; Publishing; Quantitative Reverse Transcriptase PCR; RNA Interference; Regulation; Role; Signal Transduction; Site; South Africa; South African; Starvation; Stress; Structure; Testing; Therapeutic; Toxin; Tumor stage; Tunicamycin; Work; cell injury; environmental stressor; feeding; gene discovery; gene function; heat-shock factor 1; human disease; inhibitor; knock-down; neoplastic; neurotrophic factor; novel; repaired; resilience; response; stress management; stress resilience; stressor; transcriptome sequencing; transcriptomics

A critical and well-studied cellular stress response pathway, the Unfolded Protein Response (UPR), protects organisms against several stressors including heat, hypoxia, starvation, and toxins. Helping to repair cellular damage, the UPR can also trigger apoptosis if the stress is ongoing, severe, and unrecoverable. Therefore, proper regulation of this pathway is essential, particularly since its malfunction contributes to human pathologies including autoimmune disorders, cancer, and neurodegenerative diseases. The Bracht lab recently published the genome of a nematode, Halicephalobus mephisto, isolated from the deep terrestrial subsurface of South Africa, over a kilometer underground. This organism has adapted to a stressful environment: hot, hypoxic, and rich in methane. Therefore the organism displays a naturally evolved resilience to stresses that would normally cause lethality; we also found that its UPR pathway is a site where adaptation has occurred. We have confirmed that RNA Interference (RNAi) by feeding can be used to modulate gene expression in this organism, setting the stage for a molecular investigation of stress resilience. Aim 1. Test the hypothesis that ARMET/MANF is not just an inhibitor of UPR signaling in H. mephisto. A UPR signaling gene discovered in 2003, Arginine-Rich, Mutated in Early-stage Tumors / Mesencephalic Astrocyte derived Neurotrophic Factor (ARMET / MANF), remains mysterious. While its precise molecular function has proven elusive, we identified it as the second most highly upregulated gene under heat stress in H. mephisto. In this aim, we will perform analysis of the transcriptomic changes when ARMET / MANF is knocked down by RNAi. Aim 2. Test the hypothesis that HSF1 has acquired an expanded role in heat resilience in H. mephisto. Heat-shock factor 1 (HSF1) is a well-characterized, conserved transcriptional regulator of the heat response across metazoa. However, we identified the potential for this protein to regulate 75% of the genes through a helitron-driven expansion of its recognition site. This aim is structured to test this apparent re-wiring of the HSF1 regulatory network. Aim 3. Test the role of HeaT-Upregulated-Protein-1 (HTUP-1) in heat and tunicamycin resilience. HTUP-1 is the most upregulated gene on heat in H. mephisto and it is unlike any other known protein--no blast matches and no recognizable domains. We hypothesize that HTUP-1 is a novel modulator of the evolved UPR response in H. mephisto. To study HTUP-1 function, we will inactivate it by RNAi, measure growth phenotypes under heat or tunicamycin stress, verify knockdown by qRT-PCR, and then perform RNA-seq to examine the pathways affected. Aim 4. Construct multi-copy arrays of H. mephisto genes in C. elegans as a mechanism of heat resilience. Hsp70 genes are extremely well characterized. However, in H. mephisto we uncovered a new family of Hsp70 genes: Hspa15; here we propose to evaluate whether these genes can confer heat tolerance de novo by heterologous expression in C. elegans. Because C. elegans is not thermotolerant, any acquired heat tolerance will be easily detected in this genetic background.

临界且研究良好的细胞应力反应途径，展开的蛋白质反应（UPR）可保护 针对多种压力源的生物，包括热，缺氧，饥饿和毒素。有助于修复细胞损伤， 如果应力持续，严重且无法恢复，UPR也会触发凋亡。因此，对此进行适当的规定 途径是必不可少的，特别是因为它的故障会导致人类病理，包括自身免疫性疾病， 癌症和神经退行性疾病。 Bracht Lab最近发表了线虫的基因组Halicephalobus Mephisto，从深处分离 南非的地下地下地下地下地下地下地下。这种生物已经适应了压力 环境：热，低氧且富含甲烷。因此，生物体表现出对压力的自然发展的弹性 这通常会导致致死性；我们还发现其UPR途径是发生适应性的站点。我们有 确认通过喂养的RNA干扰（RNAi）可用于调节该生物体中的基因表达 分子研究压力弹性的阶段。 AIM 1。检验假设，即装备/MANF不仅是H. Mephisto中UPR信号传导的抑制剂。 2003年发现的UPR信号基因，富含精氨酸，在早期肿瘤 /中脑中突变 星形胶质细胞衍生的神经营养因子（Ambet / Manf）仍然神秘。虽然它的精确分子功能具有 被证明是难以捉摸的，我们将其确定为H. Mephisto热应激下的第二个高度上调的基因。在这个目标中 当RNAi击倒装甲 / MANF时，我们将对转录组变化进行分析。 AIM 2。检验HSF1在H. Mephisto中获得的热弹性作用扩展的假设。 热震因子1（HSF1）是一种良好的，保守的转录调节剂 后生动物。但是，我们确定了该蛋白通过螺旋驱动的75％调节基因的潜力 扩展其识别地点。该目标的结构是测试HSF1调节网络的明显重新开关。 AIM 3。测试热调节蛋白-1（HTUP-1）在热和女霉素弹性中的作用。 HTUP-1是H. Mephisto中热量最上调的基因，它与任何其他已知蛋白质不同 - 没有爆炸 匹配，没有可识别的域。我们假设HTUP-1是在 H. Mephisto。为了研究HTUP-1功能，我们将通过RNAi使其失活，测量在热量下的生长表型 肌霉素应力，验证QRT-PCR的敲低，然后执行RNA-Seq以检查受影响的途径。 AIM 4。在秀丽隐杆线虫中构建H. mephisto基因的多拷贝阵列，作为热弹性的机理。 HSP70基因的特征非常好。但是，在H. Mephisto中，我们发现了HSP70的新家庭 基因：HSPA15;在这里，我们建议评估这些基因是否可以通过异源赋予耐热性。 在秀丽隐杆线虫中的表达。由于秀丽隐杆线虫不是热耐剂，因此在 这种遗传背景。