Function, regulation, and conservation of hypoxia-induced glycolysis condensates

缺氧诱导的糖酵解缩合物的功能、调节和保存

• 批准号: 10552295
• 负责人: John Kim
• 金额: $ 44.02万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552295
• 关键词: 3-Dimensional; Binding; Biochemical; Biogenesis; Biosensor; Caenorhabditis elegans; Cancer cell line; Cell Proliferation; Cell Survival; Cells; Consumption; Cytosol; Enzymes; Exhibits; Generations; Genetic; Glucose; Glycolysis; Glycolysis Induction; Glycolysis Pathway; Human; Hypoxia; In Vitro; Learning; Mediating; Metabolic; Mitochondria; Molecular Analysis; Outcome; Oxygen; Pathway interactions; Phase; Physical condensation; Physiological; Production; Proteins; RNA; RNA Binding; Regulation; Research; Respiration; Ribonucleoproteins; Role; Signal Pathway; Solid Neoplasm; Stress; Structure; System; Testing; Therapeutic; Work; Yeasts; biophysical analysis; biophysical properties; cancer cell; enzyme activity; genome-wide; in vivo; insight; metabolomics; novel; response; tumor hypoxia; tumor microenvironment

PROJECT SUMMARY In low oxygen conditions, such as the hypoxic microenvironment of solid tumors, cells cannot perform mitochondrial respiration and rely solely on glycolysis for ATP generation. Thus, to overcome this deficit in energy production, cells require mechanisms to enhance ATP generation in response to hypoxia. We discovered that in hypoxic yeast cells, the enzymes of the glycolysis pathway, which are diffusely localized in the cytosol under normoxic conditions, organize into non-membrane-bound structures we term Glycolytic (G) bodies. G bodies constitute ribonucleoprotein (RNP) condensates formed by phase separation. G body formation is correlated with increased glucose consumption and cell survival and proliferation under hypoxia. Similar structures have been observed in C. elegans and human cancer cell lines, supporting G body formation as an evolutionarily conserved adaptive response. We hypothesize that G bodies enhance rates of glycolysis by coordinating the multiple steps of the pathway to promote cell survival during hypoxic stress conditions. Using biochemical purification, we have determined the protein and RNA constituents of G bodies and our genome-wide deletion screen identified key signaling pathways that influence G body formation. A major gap in the condensate field is the lack of direct evidence for condensate function. Our preliminary results indicate that G bodies exhibit enhanced glycolytic enzyme activity, thus supporting a functional role for these novel RNP condensates. In this proposal, we will employ a diverse array of experimental strategies to investigate G body activity, physiological impact, biogenesis, biophysical properties, and functional conservation. Mechanisms by which G bodies potentiate glycolytic enzyme activity will be pursued using our purified G body in vitro system and by utilizing novel metabolic biosensors in vivo. We will also determine the global metabolic impact of G bodies through metabolomic approaches and investigate the genetic regulation of G body biogenesis and function mediated by conserved energy-sensing signaling pathways. Molecular and biophysical analysis of G bodies will examine the role of non-canonical RNA binding by glycolysis enzymes in condensate formation via phase separation. Finally, we will take the lessons learned in yeast and apply them to human cancer cell lines and 3D spheroid cultures to study the conservation of G body biophysical properties, biogenesis, and physiological function. Successful outcomes of our research will reveal novel basic principles of RNP condensate regulation and function across species and provide mechanistic insights and potential therapeutic strategies to mitigate hypoxic adaption and cell proliferation in solid tumor microenvironments.

项目摘要 在低氧条件下，例如实体瘤的低氧微环境，细胞无法执行 线粒体呼吸，仅依靠糖酵解来产生ATP。因此，要克服这种能源的赤字 生产，细胞需要机制以响应缺氧而增强ATP的产生。我们在 低氧酵母菌细胞，糖酵解途径的酶，它们散布在细胞质中 正常氧条件，组织成非膜结合结构，我们称糖酵解（G）体。 G身体 构成由相分离形成的核糖核蛋白（RNP）冷凝物。 G身体形成相关 在缺氧下增加葡萄糖消耗和细胞存活和增殖。类似的结构也有 在秀丽隐杆线虫和人类癌细胞系中观察到，支持G身体形成是一种进化 保守的适应性反应。我们假设G身体通过协调来提高糖酵解的速率 在低氧应激条件下促进细胞存活的途径的多个步骤。使用生化 纯化，我们确定了G体的蛋白质和RNA成分以及我们的全基因组缺失 屏幕确定了影响G身体形成的键信号通路。冷凝水领域的主要差距是 缺乏凝聚力功能的直接证据。我们的初步结果表明G身体表现出 增强的糖酵解酶活性，从而支持这些新型RNP凝结物的功能作用。在这个 提案，我们将采用各种各样的实验策略来研究身体活动，生理 影响，生物发生，生物物理特性和功能保护。 G尸体的机制 使用我们纯化的G身体在体外系统中将追求增强的糖酵解酶活性，并通过利用 新型代谢生物​​传感器体内。我们还将通过 代谢组方法并研究了G人体生物发生的遗传调节和由 保守的能量感应信号通路。 G身体的分子和生物物理分析将检查 糖酵解酶通过相分离在冷凝物形成中通过糖酵解酶结合的作用。最后， 我们将在酵母中上学的教训，并将其应用于人类癌细胞系和3D球体培养物中 研究了G人体生物物理特性，生物发生和生理功能的保护。成功的 我们研究的结果将揭示RNP冷凝水调节和功能的新基本原理 物种并提供机械见解和潜在的治疗策略，以减轻低氧适应和 实体瘤微环境中的细胞增殖。