Defining and targeting the proteome to kill quiescent cells

项目摘要/摘要 在从微生物到人类的有机体中，绝大多数细胞已经退出了细胞分裂周期， 以非增殖性状态存在。对于这些细胞中的许多细胞来说，这是细胞生长和细胞周期的可逆状态 逮捕被称为静默。静止细胞的特征是低代谢、低基因表达和低细胞 活动。静止细胞存在于从致病微生物到人类的各种临床场景中。 肿瘤。由于这些细胞不活跃地循环，许多针对细胞过程的药物，如DNA 复制和蛋白质合成是无效的。我们研究提案的中心假设是，大- 静止期细胞中蛋白质表达的比例重塑是由信号网络介导的 不同的信号来建立共同的基因表达状态。我们假设这一过程会导致 细胞质的根本性变化导致不同的生物物理性质，这可能有利于 静止细胞的长期存活，可用于静止细胞的治疗靶点。 这项提议的目标是用真核细胞模型--酿酒酵母来检验这一假说。 我们的第一个目标是定义蛋白质组被重塑的动力学，以响应不同的信号 开始静默。稳定同位素标记在细胞培养(SILAC)和质量中的应用 光谱，我们将量化三种不同反应的蛋白质表达的动态变化 导致静止的饥饿信号：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中，我们将量化 TORC1、AMPK、PKA和PHO85途径以及信号整合因子RIM15受损的菌株 和SCH9。在目标2中，我们将定义静止细胞的生物物理属性。使用基因编码 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和 量化细胞质扩散的变化以量化细胞质拥挤。找出促成因素 细胞的生物物理性质的改变，我们将量化细胞器大小和丰富的 核糖体等大分子复合体。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和检验增加分子拥挤的假说中的作用 增强了抗压性。为了确定对静止细胞的有效治疗策略，我们将测试 现有抗真菌药物在杀死静止细胞方面的有效性，确定增强 抗真菌药物在静止细胞中的有效性，并测试模拟遗传效应的佐剂的使用。至 确定这些发现的临床相关性，我们将测试新发现的治疗策略 病原真菌、白色念珠菌和光滑念珠菌。我们的研究将为我们提供一个全面的 了解细胞如何重塑他们的蛋白质组以建立静止期，它对 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.