Cellular stress sensing via biomolecular condensation

项目摘要 背景：细胞经历各种各样的环境压力，并且必须能够感知和响应 为了生存而改变。发生的感知过程取决于压力本身，我们有 在S.酿酒酵母，其表现出生物分子缩合， 温度升高。在该组中，一组保守的GTP酶显示出显著的聚集， 任何其他细胞成分的需求，打开了这些蛋白质具有能力的可能性， 来感知热休克此外，这组蛋白质与两种基本的功能反应有关， 在热应激下发生-热休克基因的转录上调和核糖体生物合成的关闭。 对热高度敏感的蛋白质可能作为长期未被识别的传感器上游的大量功能 细胞内的变化，并作为热感测候选人的这一建议。 长期以来被视为恶劣环境条件的有毒后果，最近的研究表明， 生物分子冷凝物的形成是非随机的、适应性的和可逆的。随着这一新兴观点的出现， 如痴呆和ALS，其与非膜结合蛋白聚集体的积累有关 可能是压力感应通路异常激活的结果，这表明我们对压力的理解是错误的。 疾病病理学可能需要重新评估。 具体目的：1：候选物是否足以在体内诱导转录反应？2：什么是 热感应机制3：核糖体上候选缩合的功能相关性是什么 生产？ 研究设计：我将利用一种嗜冷酵母，它在100 ℃时执行热诱导的细胞反应。 温度低于S。啤酒。嗜冷酵母可能含有同源传感器蛋白， 在较低温度下提高灵敏度。我将用它们的基因替换内源性传感器候选基因。 并测定重组S.酿酒酵母来上调热量的产生 特异性转录物和减弱的核糖体生物合成。我将重组这些功能的组成部分 反应，并测试候选传感器的冷凝是否会影响任何一种反应。 候选传感器的浓缩也将在体外使用生物化学和生物物理测定进行研究， 研究它们感知热量的内在能力，以描述感知的机械基础。 培训：本研究将与D.艾伦·德拉蒙德在芝加哥大学， 我的实验技能，首先表征生物分子凝聚的功能相关性， 环境压力传感和进一步扩展到理解生物物理机制。本次培训 将为我未来的职业生涯做好准备，研究环境压力如何塑造细胞行为。

Project Summary Background: Cells experience a wide array of environmental stresses, and must be able to sense and respond to changes in order to survive. The sensing process which occurs depends on the stress itself, and we have identified over 150 heat-sensitive proteins in S. cerevisiae which exhibit biomolecular condensation after temperature increase. Within this group, a conserved set of GTPases displays significant aggregation without the requirement of any other cellular components, opening up the possibility that these proteins have the ability to sense heat shock. Further, this set of proteins is connected to two fundamental functional responses which occur under heat stress -- transcriptional upregulation of heat shock genes and shutoff of ribosome biogenesis. Proteins that are highly sensitive to heat may act as long-unidentified sensors upstream of massive functional changes within the cell, and serve as heat sensing candidates for this proposal. Long viewed as a toxic consequence of harsh environmental conditions, recent work has shown that biomolecular condensate formation is non-random, adaptive, and reversible. With this emerging view, diseases like dementia and ALS which are associated with the accumulation of non-membrane bound protein aggregates might be the result of an aberrant activation of stress sensing pathways, indicating that our understanding of the disease pathology may need to be reevaluated. Specific Aims: 1: Are candidates sufficient to induce the transcriptional response in vivo? 2: What is the mechanism for heat sensing? 3: What is the functional relevance of candidate condensation on ribosome production? Study Design: I will take advantage of a cryophilic yeast which execute their heat-induced cellular responses at lower temperatures than S. cerevisiae. The cryophilic yeast likely contain homologous sensor proteins with increased sensitivity at lower temperatures. I will replace the endogenous sensor candidate genes with their cryophilic homologs and assay the ability of the recombinant S. cerevisiae to upregulate the production of heat- specific transcripts and attenuate ribosome biogenesis. I will reconstitute the components of these functional responses in vitro and test whether the condensation of candidate sensors can affect either response. Condensation of candidate sensors will also be studied in vitro using biochemical and biophysical assays to investigate their intrinsic ability to sense heat to describe the mechanistic underpinnings of sensing. Training: This research will be performed with Dr. D. Allan Drummond at the University of Chicago and will build upon my experimental skills by first characterizing the functional relevance of biomolecular condensation in environmental stress sensing and further expanding into understanding the biophysical mechanism. This training will prepare me for a future career studying how environmental stresses shape cellular behavior.