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.

一个关键的和充分研究的细胞应激反应途径，未折叠蛋白反应（UPR），保护 生物体对抗几种应激源，包括热、缺氧、饥饿和毒素。帮助修复细胞损伤， 如果压力持续、严重和不可恢复，UPR也可以触发细胞凋亡。因此，适当的监管， 途径是必不可少的，特别是因为它的功能障碍有助于人类病理学，包括自身免疫性疾病， 癌症和神经退行性疾病。 Bracht实验室最近公布了一种从深海中分离出来的线虫Halicebusmephisto的基因组 南非的地表下，超过一公里的地下。这种有机体已经适应了 环境：炎热，缺氧，富含甲烷。因此，生物体表现出自然进化的抗压能力 这通常会导致致命性;我们还发现它的UPR途径是一个发生适应的位点。我们有 证实了RNA干扰（RNAi）通过喂养可用于调节这种生物体的基因表达， 这是对压力恢复力进行分子研究的一个阶段。 目标1。测试ARMET/MANF不仅仅是H中UPR信号的抑制剂的假设。墨菲斯托 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 星形胶质细胞源性神经营养因子（ARMET / MANF），仍然是一个神秘的。虽然它精确的分子功能 证明难以捉摸，我们确定它作为第二个最高上调基因在热应激下在H。墨菲斯托在这一目标下， 我们将分析ARMET / MANF被RNAi敲除后的转录组学变化。 目标2.检验HSF 1在H.墨菲斯托 热休克因子1（HSF 1）是一种特征性的、保守的转录调节因子，在热应激反应中起重要作用。 后生动物然而，我们确定了这种蛋白质通过helitron驱动的基因调控75%的基因的潜力。 扩大其识别范围。这一目标旨在测试HSF 1监管网络的明显重新布线。 目标3.测试热上调蛋白-1（HTUP-1）在热和衣霉素弹性中的作用。 HTUP-1是H. mephisto和其他已知的蛋白质都不一样 匹配，没有可识别的域。我们假设HTUP-1是一种新的调节剂的演变UPR反应， H.墨菲斯托为了研究HTUP-1的功能，我们将通过RNAi对其进行测序，在高温下测量生长表型， 通过qRT-PCR验证敲低，然后进行RNA-seq以检查受影响的途径。 目标4。构建H. mephisto基因在C. elegans线虫as a mechanism机制of heat热resilience弹性. Hsp 70基因是非常好的特点。然而，在H. mephisto我们发现了一个新的Hsp 70家族 基因：Hspa 15;在这里，我们建议评估这些基因是否可以赋予耐热性从头异源 表达于C.优美的因为C.线虫是不耐热的，任何获得的耐热性将很容易检测到， 这种基因背景。