Investigating Biomolecular Condensates and Heat Shock Proteins in Cellular Responses to Sublethal Heat Shock and Fever

项目摘要 环境温度决定了生物学。动物利用他们的热环境来引导他们的 迁徙、昼夜节律、生长、摄食和性别决定：现在受到威胁的基本行为 气候的变化。变暖的气温同样对人类健康构成了挑战。全球约每100人中就有1人死于 死于与高温相关的原因，而且这种死亡率正在上升。除了致命的中暑外，更温和的高温影响人类 以更为普遍和普遍的方式传播健康信息。40摄氏度的高温改变和失调了人体的生理机能 细胞水平，特别是在免疫细胞中。这种亚致命性的热休克会在体温过高和中暑时发生， 以及经常发烧：感染期间调节免疫系统的系统性热休克。还没有 即使在众所周知的发烧背景下，我们也缺乏对人类细胞如何感觉亚致死热休克的理解。 极端高温冲击&40°C的细胞生物学特征已经得到很好的描述，但人们对其了解的还远远不够 然而，我们确实知道，某些免疫细胞会上调体温 对发烧作出反应的休克蛋白表达。热休克蛋白的诱导，或热休克反应， 在真核生物中，当热激活转录因子HSF1时，通过其抑制物(热休克蛋白)的滴定 HSP70)远离HSF1。这种滴定是由于产生了新的、热休克诱导的底物 HSP70结合。这些底物，即上游的热感应器，在亚致命的热休克中是无法识别的。 我们假设生物分子凝聚体就是这些帮助细胞感受亚致命热量的底物。 令人震惊。缩合，或蛋白质和RNA重组成更大的焦点，发生对环境的反应 从酵母到人类的各种刺激。我们的小组最近表明，热诱导凝析油是 酵母菌中的HSP70底物。我们假设热休克引起的亚致死冷凝物是Hsp70 人类体内的底物，使细胞能够感知并对这种发烧范围的温度做出反应。目前尚不清楚 在这种温度下，哪些蛋白质在人类细胞中凝聚，或者这种凝聚物是否可能是热休克蛋白70 底物。此外，在任何物种中，我们都缺乏对冷凝物和热休克蛋白70如何在分子水平上的理解 互动。我们已经准备好利用生化、显微和 分子水平的方法。首先，我们将发现在发热范围内人类细胞系中的蛋白质凝聚。 温度，采用本课题组建立的沉淀-质谱法。第二，我们将 使用单分子显微镜，直接观察冷凝物和Hsp70如何在分子水平上相互作用。 总之，这些目标将帮助我们从根本上阐明细胞如何感知和反应亚致死热休克。

Project Summary Environmental temperature dictates biology. Animals use their thermal environments to guide their migration, circadian rhythms, growth, feeding, and sex determination: essential behaviors now threatened by changing climates. Warming temperatures likewise challenge human health. About 1 in 100 deaths globally stem from heat-related causes, and such mortality is rising. Beyond lethal heatstroke, more mild heat affects human health in far more common and pervasive ways. Heat <40°C alters and dysregulates human physiology down to the cellular level, particularly in immune cells. Such sublethal heat shocks occur in hyperthermia and heat illness, as well as frequently in fever: a systemic heat shock which regulates the immune system during infection. Yet even in the well-known context of fever, we lack understanding of how human cells sense sublethal heat shock. The cell biology of extreme heat shock >40°C is well-characterized, but far less is understood about sublethal, fever-range temperatures <40°C. However, we do know that certain immune cells upregulate heat shock protein expression in response to fever. The induction of heat shock proteins, or the heat shock response, occurs in eukaryotes when heat activates transcription factor Hsf1, via titration of its repressor (heat shock protein Hsp70) away from Hsf1. This titration is caused by the generation of new, heat shock-induced substrates for Hsp70 to bind. These substrates, i.e. the upstream sensors of heat, are unidentified in sublethal heat shock. We hypothesize biomolecular condensates are these substrates which help cells sense sublethal heat shock. Condensation, or reorganization of proteins and RNA into larger foci, occurs in response to environmental stimuli across species from yeast to humans. Our group showed recently that heat-induced condensates are Hsp70 substrates in yeast. We hypothesize that sublethal heat shock-induced condensates are Hsp70 substrates in humans, enabling cells to sense and respond to such fever-range temperatures. It is not known what proteins condense in human cells at these temperatures, nor if such condensates might be Hsp70 substrates. Moreover, in any species, we lack molecular-scale understanding of how condensates and Hsp70 interact. We are poised to unlock exactly this knowledge using a complement of biochemical, microscopic, and molecular-level approaches. First, we will uncover protein condensation in human cell lines at fever-range temperature, using the established sedimentation-mass spectrometry method of our group. Second, we will observe directly how condensates and Hsp70 interact at the molecular scale, using single-molecule microscopy. Together, these aims will help us elucidate fundamentally how cells sense and respond to sublethal heat shock.