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

蛋白质的细胞系综的稳态（蛋白质稳态）是适应性的基本要求。蛋白质稳态的突然或进行性下降可能是病理性的，但也常在衰老中观察到。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种类积累的联系。衰老和疾病中的蛋白质稳态下降取决于影响蛋白质稳态的机制的细胞网络内的事件。此外，个体的衰老轨迹可能取决于多个遗传变异对该网络的综合影响。蛋白质组范围的测量正在迅速改变我们如何研究蛋白质稳定等复杂性状的范式。在这里，我们建议应用现代质谱法为基础的方法，能够检测蛋白质组范围内的蛋白质功能状态的变化，研究的鲁棒性的模式生物酿酒酵母蛋白毒性应力引起的热。最近开发的基于有限蛋白水解的结构蛋白质组学方法（“LiP-MS”）的应用将允许我们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鲁棒性作为网络。我们的目的是确定的遗传基础的鲁棒性，以热应力的基础上，一组遗传上不同的酵母菌株。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鲁棒性的个体遗传决定因素与调控模块的功能状态联系起来。通过归纳法，我们将绘制热胁迫鲁棒性的机制网络图，而机制假说将从网络信息和分子数据中推导出来。除了调查耐热性状的个体间差异外，我们还将确定先前的胁迫经历的影响。酵母菌经轻度胁迫预处理后可获得耐热性。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导致的变化重叠。此外，我们还将研究热应激记忆建立的遗传条件。总之，本研究将采用面向网络的方法，结合蛋白质组学方法，阐明细胞蛋白质稳态失效和防御的基本过程。深入研究从蛋白质组范围内的测量推导出的机制假说将使用详细的分子生物学实验进行。因此，我们的方法来识别相互关联的决定因素的压力鲁棒性的能力将得到证明。

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.