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.

一种关键的和被充分研究的细胞应激反应途径，未折叠蛋白反应(UPR)，保护 生物体可以抵抗几种应激源，包括高温、缺氧、饥饿和毒素。帮助修复细胞损伤， 如果应激是持续的、严重的和不可恢复的，UPR也可以触发细胞凋亡。因此，对此进行适当的监管 通路是必不可少的，特别是因为它的故障导致了包括自身免疫性疾病在内的人类病理， 癌症和神经退行性疾病。 布拉赫特实验室最近公布了从深海分离的一种线虫Halicephalobus mephisto的基因组 南非的陆地地下，地下超过一公里。这种有机体已经适应了压力 环境：炎热、低氧、富含甲烷。因此，生物体对压力表现出一种自然进化的弹性 这通常会导致致命性；我们还发现其UPR途径是发生适应的位置。我们有 证实了通过进食的RNA干扰(RNAi)可以用来调节这种生物中的基因表达，设置了 为应激恢复的分子研究做准备。 目的1.验证ARMET/MANF不只是H.mephisto中UPR信号的抑制因子的假设。 2003年发现的富含精氨酸的UPR信号基因在早期肿瘤/中脑组织中发生突变 星形胶质细胞源性神经营养因子(ARMET/MANF)，至今仍是个谜。而它的精确的分子功能 事实证明，它难以捉摸，我们确定它是热应激下H.mephisto中第二个高度上调的基因。在这个目标中， 我们将对Armet/MANF被RNAi击倒时的转录变化进行分析。 目的2.验证HSF1在粘虫热弹性中的作用扩大的假说。 热休克因子1(HSF1)是一种特征明确的、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电子驱动调节75%的基因。 扩大其认可网站。这一目标是为了测试HSF1监管网络的这种明显的重新布线。 目的3.检测热上调蛋白-1(HTUP-1)在热和衣霉素抗性中的作用。 HTUP-1是H.mephisto中在高温下表达最高的基因，它不同于任何其他已知的蛋白质--没有BLAST 匹配且没有可识别的域。我们假设HTUP-1是一种新的UPR反应的调节剂 H.梅菲斯托。为了研究HTUP-1的功能，我们将通过RNAi使其失活，测量在高温或高温下的生长表型 衣霉素胁迫，通过qRT-PCR验证基因被敲除，然后进行RNA-SEQ以检测受影响的通路。 目的4.构建线虫耐热性基因的多拷贝阵列。 HSP70基因的特性非常好。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基因赋予耐热性。 在线虫中的表达。由于线虫不耐热，任何获得的耐热性都很容易在 这种遗传背景。