Investigating the disaggregation of stress-induced phase-separated poly(A)-binding protein (Pab1) by its cognate disaggregation system

项目摘要/摘要 了解细胞如何感知和响应应激环境变化是细胞生物学的一个主要目标。 在所有真核生物中保守的细胞应激反应的两个代表性特征是：1) 分子伴侣的上调和2)RNA和蛋白质聚集成应激颗粒。这些 长期以来，簇一直被解释为错误折叠的蛋白质的聚集体。然而，我们最近演示了 (Riback等人，2017)Poly(A)结合蛋白(Pab1)，一种普遍保守的应激颗粒标记，形成 通过相分离而不会错误折叠的簇，并且扰乱相分离会损害细胞生长 在胁迫过程中，表明胁迫触发的Pab1聚集是适应性应激反应的一部分。我们 将这种促进健身的压力触发的聚集体称为适应性蛋白质集合。 认识到压力会触发两种不同类型的聚集体的形成，即适应性蛋白质组合 和错误折叠的蛋白质聚集体，促使我们研究分子伴侣是否以及如何 区分这些不同的底物。在生理压力下Pab1相分离成水凝胶 条件。为了研究Pab1水凝胶是如何通过分子伴侣来分解的--特别是 应激诱导的HSP104解聚系统-我们开发了一种荧光各向异性分析，允许 Pab1扩散的定量和动力学测量。这是第一个用于研究的体外系统。 适应性蛋白质组件的解聚。酵母HSP104由三个分子伴侣组成， HSP104/70/40。我们将询问Pab1解聚与错误折叠的聚集体解聚有何不同。我们的 初步数据表明，PAB1水凝胶的解聚速度比模型的解聚速度快得多 萤火虫荧光素酶底物。我们还将询问Pab1的分子特征是由分子识别的 监护人。HSP70优先将未折叠的蛋白质与延伸的疏水序列结合，并且 Pab1富含脯氨酸的低复杂结构域中的疏水残基调节Pab1的相分离。 因此，我们假设改变Pab1相分离的特征也将影响解聚。我们会 使用我们最近报道的具有改变相分离行为的Pab1突变体来验证这一假设。最后， 基于文献和初步数据，我们有一个强有力的预测，即Hsp70/Ssa1的磷酸化 AT T36将提高HSP104解聚系统对Pab1水凝胶的效率。我们将测试 这一预测使用的是荧光各向异性分析。此外，我们还采用了进化分析 确定其他调节磷酸化位点的方法；我们将使用 荧光各向异性分析。这项研究的结果将促进我们对 HSP104解聚系统分散了不同的应力诱导结构。

Project Summary/Abstract Understanding how cells sense and respond to stressful environmental changes is a major goal in cell biology. The two representative features of the cellular stress response that are conserved in all eukaryotes are 1) upregulation of molecular chaperones and 2) aggregation of RNA and protein into stress granules. These clusters had long been interpreted to be aggregates of misfolded proteins. However, we recently demonstrated (Riback et al., 2017) that poly(A)-binding protein (Pab1), a universally conserved stress-granule marker, forms clusters by phase separating without misfolding, and that disrupting phase separation impairs cellular growth during stress, indicating that stress-triggered aggregation of Pab1 is a part of the adaptive stress response. We refer to such fitness-promoting stress-triggered aggregates as adaptive protein assemblies. The realization that stress triggers formation of two distinct class of aggregates, adaptive protein assemblies and misfolded protein aggregates, motivated us to investigate whether and how molecular chaperones can distinguish these different substrates. Pab1 phase-separates into a hydrogel under physiologically stressful conditions. To investigate how Pab1 hydrogels are disaggregated by molecular chaperones—specifically the stress-induced Hsp104 disaggregation system—we developed a fluorescence anisotropy assay that permits both quantitative and kinetic measurement of Pab1 dispersal. This is the first in vitro system for studying disaggregation of adaptive protein assemblies. The yeast Hsp104 consists of three molecular chaperones, Hsp104/70/40. We will ask how Pab1 disaggregation differs from disaggregation of misfolded aggregates. Our preliminary data show that disaggregation of Pab1 hydrogel is much faster than disaggregation of the model substrate firefly luciferase. We will also ask what molecular features of Pab1 are recognized by molecular chaperones. Hsp70 preferentially binds unfolded proteins with an extended hydrophobic sequence, and hydrophobic residues in the proline-rich low-complexity domain of Pab1 modulate phase-separation of Pab1. We thus hypothesize that features which alter phase-separation of Pab1 will also affect disaggregation. We will test this hypothesis using our recently reported Pab1 mutants with altered phase-separation behavior. Finally, we have a strong prediction based on both literature and preliminary data that phosphorylation of Hsp70/Ssa1 at T36 will increase the efficiency of the Hsp104 disaggregation system toward Pab1 hydrogels. We will test this prediction using the fluorescence anisotropy assay. Additionally, we employ an evolutionary analysis approach to identify other regulatory phosphorylation sites; we will test our findings biochemically using the fluorescence anisotropy assay. The outcome of this research will advance our understanding of how the Hsp104 disaggregation system disperses different stress-induced structures.