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.

未折叠蛋白反应 (UPR) 是一条经过充分研究的重要细胞应激反应途径，可保护 生物体抵抗多种应激源，包括热、缺氧、饥饿和毒素。有助于修复细胞损伤， 如果压力持续、严重且无法恢复，UPR 也可能引发细胞凋亡。因此，适当监管这一 途径至关重要，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 实验室最近发表了从深海中分离出来的线虫 Halicephalobus mephisto 的基因组 南非陆地地下，地下一公里多。这个有机体已经适应了压力 环境：炎热、缺氧、富含甲烷。因此，有机体表现出自然进化的抗应激能力 这通常会导致致命；我们还发现它的UPR途径是发生适应的位点。我们有 证实通过喂食进行的 RNA 干扰 (RNAi) 可用于调节该生物体的基因表达，从而设定 压力恢复的分子研究阶段。 目标 1. 检验 ARMET/MANF 不仅仅是 H. mephisto 中 UPR 信号传导抑制剂的假设。 2003 年发现的 UPR 信号基因，富含精氨酸，在早期肿瘤/中脑肿瘤中发生突变 星形胶质细胞衍生的神经营养因子（ARMET/MANF）仍然是个谜。虽然其精确的分子功能已 事实证明它难以捉摸，我们将其确定为 H. mephisto 热应激下第二个高度上调的基因。为了这个目标， 我们将对 ARMET / MANF 被 RNAi 敲低时的转录组变化进行分析。 目标 2. 检验 HSF1 在 H. mephisto 中在热恢复中发挥更大作用的假设。 热休克因子 1 (HSF1) 是一种特征明确、保守的热反应转录调节因子。 后生动物。然而，我们发现这种蛋白质有可能通过氦电子驱动调节 75% 的基因。 扩大其识别位点。这一目标旨在测试 HSF1 监管网络的明显重新布线。 目标 3. 测试 HeaT-Upregulated-Protein-1 (HTUP-1) 在热和衣霉素恢复力中的作用。 HTUP-1 是 H. mephisto 中受热影响最上调的基因，它与任何其他已知的蛋白质不同——没有爆炸 匹配且没有可识别的域。我们假设 HTUP-1 是进化的 UPR 反应的新型调节剂 H.墨菲斯托。为了研究 HTUP-1 功能，我们将通过 RNAi 使其失活，测量热或热条件下的生长表型 衣霉素应激，通过 qRT-PCR 验证敲除，然后进行 RNA-seq 以检查受影响的途径。 目标 4. 在秀丽隐杆线虫中构建 H. mephisto 基因的多拷贝阵列作为热恢复机制。 Hsp70 基因的特征非常明确。然而，在 H. mephisto 中，我们发现了一个新的 Hsp70 家族 基因：Hspa15；在这里，我们建议评估这些基因是否可以通过异源从头赋予耐热性 在线虫中的表达。由于秀丽隐杆线虫不耐热，因此任何获得的耐热性都可以很容易地在 这个遗传背景。