Structural and genetic variability of the proteostasis network in Saccharomyces cerevisiae.

蛋白质细胞整体的动态平衡(蛋白质平衡)是健身的基本要求。蛋白稳定性的突然或进行性下降可以是病理性的，但在衰老过程中也很常见。我们还没有确定这种恶化的确切原因，也不了解它与疾病中有毒蛋白质物种积累的联系。蛋白质稳定性在衰老和疾病中的下降取决于影响蛋白质稳定性的细胞网络中的事件。此外，个体的衰老轨迹可能依赖于这个网络上多个遗传变异的综合影响。蛋白质组范围的测量正在迅速改变我们如何研究复杂特征的范式，如蛋白质平衡。在这里，我们建议应用基于现代质谱学的方法，能够在蛋白质组范围内检测蛋白质功能状态的变化，以研究模式生物酿酒酵母对热引起的蛋白毒性胁迫的稳健性。最近发展起来的基于有限蛋白分解的结构蛋白质组学方法(“LIP-MS”)的应用将使我们能够通过同时监测多个细胞过程来研究蛋白质稳定机制和热应激稳健性。我们的目标是基于一组遗传差异的酵母菌株来确定耐热应激的遗传基础。表型测量将与个体遗传、转录和蛋白质组组成的信息相结合。这些数据将使我们能够将热应激稳健性的个体遗传决定因素与调节模块的功能状态联系起来。通过归纳法，我们将绘制热应激稳健性的机制网络，而机制假说将从网络信息和分子数据中推导出来。除了调查耐热性性状的个体间差异外，我们还将确定先前应激经验的影响。酵母经过温和的应激处理后，可以获得耐热性。我们将评估由于热应激稳健性的个体间决定因素的经验而引起的变化的重叠。此外，我们将研究建立热应激记忆的遗传要求。综上所述，本研究将采用面向网络的方法结合包罗万象的蛋白质组学方法来阐明细胞蛋白平衡失败和防御的基本过程。将使用详细的分子生物学实验对从蛋白质组范围的测量得出的机制假说进行深入研究。因此，我们的方法识别压力稳健性的相互关联的决定因素的能力将得到证明。

Homeostasis of the cellular ensemble of proteins (proteostasis) is a basic requirement for fitness. Sudden or progressive decline of proteostasis can be pathologic but is also commonly observed in aging. We have not identified the exact causes of this deterioration nor do we understand its link to the accumulation of toxic protein species in disease. Proteostasis decline in aging and disease depends on events within the cellular network of mechanisms that affect proteostasis. Moreover, individual aging trajectories likely depend on combined effects of multiple genetic variants on this network.Proteome-wide measurements are rapidly changing the paradigms of how we study complex traits such as proteostasis. Here, we propose to apply modern mass spectrometry-based methods that are able to detect protein functional state changes on a proteome-wide scale to study the robustness of the model organism Saccharomyces cerevisiae to proteotoxic stress caused by heat. Application of a recently developed structural proteomics method based on limited proteolysis (“LiP-MS”) will allow us to study proteostasis mechanisms and heat stress robustness as a network by simultaneous monitoring of multiple cellular processes. We aim to detemine the genetic foundation of robustness to heat stress based on a panel of genetically different yeast strains. Phenotypic measurements will be combined with information on the individual genetic, transcriptional and proteomic make-up. These data will allow us to relate individual genetic determinants of heat stress robustness to the functional state of regulatory modules. By means of induction, we will chart the network of mechanisms that underlies heat stress robustness, whereas mechanistic hypotheses will be deduced from network information and molecular data.In addition to the investigation of inter-individual differences in thermotolerance traits, we will also determine the influence of prior stress experience. Yeast can acquire thermotolerance following mild stress pre-treatment. We will assess the overlap of changes due to experience with inter-individual determinants of heat stress robustness. Furthermore, we will study the genetic requirements for the establishment of heat stress memory.In sum, this study will take a network-oriented approach combined with encompassing proteomics methods to elucidate basic processes underlying failure and defense of cellular proteostasis. In-depth investigation of mechanistic hypotheses deduced from proteome-wide measurements will be carried out using detailed molecular biology experiments. Thereby, the ability of our approach to identify inter-connected determinants of stress robustness will be demonstrated.