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

项目总结/摘要 了解细胞如何感知和响应压力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 簇一直被解释为错误折叠蛋白质的聚集体。然而，我们最近证明， （Riback等人，2017），聚（A）结合蛋白（Pab 1），一种普遍保守的应激颗粒标志物，形成 簇通过相分离而没有错误折叠，并且破坏相分离损害细胞生长 在压力下，表明压力引发的聚集的Pab 1是适应性应激反应的一部分。我们 将这种促进健康的压力触发的聚集体称为适应性蛋白质组装体。 认识到压力触发了两类不同的聚集体，适应性蛋白质组装体 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 区分这些不同的基质。Pab 1在生理压力下相分离成水凝胶 条件为了研究Pab 1水凝胶如何被分子伴侣分解，特别是 应激诱导Hsp 104解聚系统-我们开发了一种荧光各向异性测定， Pab 1扩散的定量和动力学测量。这是第一个体外研究系统 适应性蛋白质组装体的解聚。酵母Hsp 104由三个分子伴侣组成， HSP 104/70/40。我们将询问Pab 1解聚与错误折叠聚集体的解聚有何不同。我们 初步数据显示Pab 1水凝胶的解聚比模型的解聚快得多 底物萤火虫荧光素酶。我们还将询问Pab 1的哪些分子特征被分子识别， 监护人Hsp 70优先结合具有延伸的疏水序列的未折叠蛋白，并且 Pab 1的富含脯氨酸的低复杂性结构域中的疏水残基调节Pab 1的相分离。 因此，我们假设，功能改变相分离的Pab 1也将影响解聚。我们将 使用我们最近报道的具有改变的相分离行为的Pab 1突变体来测试这一假设。最后， 基于文献和初步数据，我们有一个强有力的预测，Hsp 70/Ssa 1的磷酸化 在T36处，将增加Hsp 104解聚系统对Pab 1水凝胶的效率。我们将测试 该预测使用荧光各向异性测定。此外，我们采用了进化分析， 方法，以确定其他监管磷酸化位点，我们将测试我们的研究结果生化使用 荧光各向异性测定。这项研究的结果将促进我们了解如何 热休克蛋白104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.