Defining and targeting the proteome to kill quiescent cells

项目总结/摘要 在从微生物到人类的生物体中，绝大多数细胞已经退出细胞分裂周期， 以非增殖状态存在。对于许多这样的细胞，这是细胞生长和细胞周期的可逆状态 被称为静止期。静止细胞的特征在于低代谢、基因表达和细胞增殖。 活动静止细胞在从病原微生物到人类的各种临床情况中被发现 肿瘤的由于这些细胞不活跃地循环，许多靶向细胞过程如DNA的药物 复制和蛋白质合成是无效的。我们研究计划的核心假设是- 静止细胞中蛋白质表达的规模重塑是由信号网络介导的， 不同的信号以建立共同的基因表达状态。我们假设这个过程导致 细胞质中的根本变化导致可以有益于细胞的独特生物物理特性。 静止细胞的长期存活，并且其可用于静止细胞的治疗靶向。 本提案的目的是使用模型真核细胞酿酒酵母（Saccharomyces cerevisiae）来验证这一假设。 我们的第一个目标是定义蛋白质组响应不同信号而重塑的动力学， 启动静止。使用细胞培养物中氨基酸的稳定同位素标记（SILAC）和质量 光谱，我们将量化蛋白质表达变化的动态响应三个不同的 饥饿信号导致静止的开始：氮、碳和磷。测试的作用 在调节蛋白质组重塑的特定信号通路，我们将量化表达动力学， TORC 1、AMPK、PKA和PHO 85通路以及信号整合子RIM 15受损的菌株 的SCH 9。在目标2中，我们将定义静止细胞的生物物理特性。利用基因编码 多聚体纳米颗粒（GEMS）和微流体成像，我们将研究细胞进入静止期， 量化细胞质扩散的变化以量化细胞质拥挤。找出导致 改变细胞的生物物理特性，我们将量化细胞器大小和丰度的变化。 大分子复合物，如核糖体。我们将使用遗传和化学扰动来测试它们的 在调节静止细胞的性质中的作用，并检验增加分子拥挤的假设， 赋予增加的抗应激性。为了确定对静止细胞有效的治疗策略，我们将测试 现有的抗真菌药物在杀死静止细胞方面的有效性， 抗真菌药在静止细胞中的功效，以及模拟遗传效应的佐剂的使用。到 为了确定这些发现的临床相关性，我们将测试新确定的治疗策略， 致病真菌，白色念珠菌和光滑念珠菌。我们的研究将提供全面的 了解细胞如何重塑其蛋白质组以建立静止，其对细胞的影响， 细胞的生物物理特性，以及对抗静止细胞的治疗策略。

Project Summary/Abstract In organisms spanning microbes to humans, the vast majority of cells have exited the cell division cycle and exist in a non-proliferative state. For many of these cells this is a reversible state of cell growth and cell cycle arrest known as quiescence. Quiescent cells are characterized by low metabolic, gene expression, and cellular activity. Quiescent cells are found in diverse clinical scenarios ranging from pathogenic microbes to human tumors. As these cells are not actively cycling, many drugs that target cellular processes such as DNA replication and protein synthesis are ineffective. The central hypothesis of our research proposal is that large- scale remodeling of protein expression in quiescent cells is mediated by signaling networks that respond to distinct signals to establish a common gene expression state. We postulate that this process leads to fundamental changes in the cytoplasm of the cell resulting in distinct biophysical properties that can benefit the long term survival of quiescent cells, and which can be exploited for therapeutic targeting of quiescent cells. The goal of this proposal is to test this hypothesis using the model eukaryotic cell, Saccharomyces cerevisiae. Our first aim is to define the dynamics with which the proteome is remodeled in response to distinct signals that initiate quiescence. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry, we will quantify the dynamics of protein expression changes in response to three different starvation signals that result in the initiation of quiescence: nitrogen, carbon and phosphorus. To test the role of specific signaling pathways in regulating remodeling of the proteome we will quantify expression dynamics in strains impaired for the TORC1, AMPK, PKA and PHO85 pathways as well as the signal integrators, RIM15 and SCH9. In aim 2 we will define the biophysical properties of quiescent cells. Using genetically encoded multimeric nanoparticles (GEMS) and imaging in microfluidics we will study cells as they enter quiescence and quantify changes in cytoplasmic diffusion to quantify cytoplasmic crowding. To identify factors that contribute to the altered biophysical properties of the cell we will quantify changes in organelle size and the abundance of macromolecular complexes such as the ribosome. We will use genetic and chemical perturbations to test their role in modulating the properties of quiescent cells and test the hypothesis that increased molecular crowding confers increased stress resistance. To identify effective therapeutic strategies for quiescent cells we will test the effectiveness of existing antifungal drugs in killing quiescent cells, identify genetic liabilities that enhance the efficacy of antifungals in quiescent cells and test the use of adjuvants that mimic genetic effects. To determine the clinical relevance of these findings, we will test newly identified therapeutic strategies in the pathogenic fungi, Candida albicans and Candida glabrata. Our study will provide a comprehensive understanding of how cells remodel their proteome to establish quiescence, its consequences for the biophysical properties of the cell, and therapeutic strategies for combating quiescent cells.