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

项目摘要 环境温度决定生物学。动物利用它们的热环境来引导它们的 迁移、昼夜节律、生长、摄食和性别决定：现在受到威胁的基本行为 气候变化气候变暖同样对人类健康构成挑战。全球每100例死亡中就有1例是死于 与高温有关的原因，这种死亡率正在上升。除了致命的中暑，更温和的热量影响人类 以更普遍和更普遍的方式来维护健康。<40°C的高温会改变和失调人体生理机能， 细胞水平，特别是免疫细胞。这种亚致死性热休克发生在体温过高和中暑， 以及经常在发烧：一个系统性的热休克，调节免疫系统在感染。然而 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知亚致死热休克的理解。 >40°C的极端热休克的细胞生物学特征很好，但对 亚致死，发热范围温度<40°C。然而，我们知道某些免疫细胞上调热量， 发热时休克蛋白表达。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Hsf 1时，通过滴定其阻遏物（热休克蛋白）发生 Hsp 70）远离Hsf 1。这种滴定是由产生新的热冲击诱导的底物引起的， HSP 70结合。这些基板，即上游的热传感器，在亚致死热休克中是无法识别的。 我们假设生物分子凝聚物是帮助细胞感知亚致死温度的底物 冲击.蛋白质和RNA的浓缩或重组成更大的病灶，是对环境的反应。 从酵母菌到人类的所有物种的刺激物。我们的研究小组最近表明，热诱导冷凝物是 酵母中的Hsp 70底物。我们假设亚致死热休克诱导的冷凝物是热休克蛋白70 人体内的底物，使细胞能够感知和响应这种发热范围的温度。目前还不知道 在这些温度下，人类细胞中有哪些蛋白质凝聚，也不知道这种凝聚物是否可能是Hsp 70。 印刷受体.此外，在任何物种中，我们都缺乏对缩合物和Hsp 70如何在分子水平上的理解。 互动.我们正准备利用生物化学、显微镜和生物化学的补充来解开这一知识。 分子水平的方法。首先，我们将揭示在发烧范围内人类细胞系中的蛋白质凝聚 温度下，使用我们小组建立的沉降-质谱法。二是 使用单分子显微镜直接观察冷凝物和Hsp 70如何在分子尺度上相互作用。 总之，这些目标将帮助我们从根本上阐明细胞如何感知和响应亚致死热休克。

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.