An analysis of the regulation and functions of a novel family of membraneless organelles in eukaryotic cells

真核细胞新型无膜细胞器家族的调控和功能分析

• 批准号: 9915939
• 负责人: Paul K Herman
• 金额: $ 31.15万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9915939
• 关键词: Amyotrophic Lateral Sclerosis; Area; Biological; Biological Process; Biology; Cell Survival; Cell physiology; Cells; Cellular biology; Complex; Cues; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Granules; Defect; Development; Developmental Biology; Disease; Eukaryota; Eukaryotic Cell; Exhibits; Family; Glean; Knowledge; Link; Maintenance; Malignant Neoplasms; Mediating; Membrane; Messenger RNA; Methods; Molecular; Nature; Neurodegenerative Disorders; Normal Cell; Organelles; Pathology; Phase; Phosphorylation; Phosphotransferases; Physiological; Process; Protein Kinase; Proteins; Quality Control; Regulation; Research; Ribonucleoproteins; Role; Set protein; Signal Transduction; Signaling Protein; Stress; Structure; Type 2 Spinocerebellar Ataxia; Work; cell growth; experimental study; human disease; insight; mutant; novel; novel therapeutics; protein aggregation; protein folding; protein misfolding; proteostasis; recruit; response; segregation; stem; stress granule

The eukaryotic cell is a highly compartmentalized structure that is subdivided into discrete functional areas by the presence of a variety of membrane-enclosed organelles. This segregation of functions is essential for normal cell growth and survival. Interestingly, recent studies have indicated that additional levels of compartmentalization exist within these cells. In particular, a number of cytoplasmic granules that contain distinct sets of proteins and mRNAs have been identified. Two of the best-characterized of these ribonucleoprotein (RNP) structures are the Processing-body (P-body) and stress granule. These granules differ from the more traditional organelles in that they lack a limiting membrane and are rather dynamic in nature. These granules are evolutionarily conserved and have been linked to a number of human diseases, including a variety of cancers and neurodegenerative disorders. However, despite these observations, the physiological functions of these RNP structures remain poorly understood. This lack of understanding represents a critical gap in our current knowledge and attempting to bridge this divide is a primary research focus in our lab. The experiments in this proposal aim to further our understanding of both the biological roles of these RNP granules and the mechanisms that regulate their assembly. The first two aims come at this question of biological function from different directions. In the first, we will assess the physiological consequences of having key signaling proteins associate with the P-body and/or stress granule during conditions of stress. Our focus here is on particular protein kinases and the possibility that this re-localization to RNP granules allows for a rewiring of the signaling networks present in the cell. In the second, we focus on the granule as a whole and ask how cell physiology is altered in mutants that lack these RNP structures. These latter studies focus on a potential role in protein homeostasis that was suggested by recent work from our lab. In particular, we have found that mutants lacking P-bodies exhibit elevated levels of protein misfolding and aggregation. The experiments here aim to determine the underlying mechanisms responsible for these effects and should establish whether these RNP granules have a direct role in the maintenance of protein quality control (PQC). Finally, the third aim examines several key aspects of the regulation and assembly of P-body foci. In particular, the studies will define the molecular mechanism underlying PKA-mediated control of P-body assembly and develop a facile method for the purification of these RNP granules. The three specific aims of the proposal are as follows: (1) determine the physiological consequences of protein kinase recruitment to RNP granules; (2) define the underlying mechanisms responsible for the P-body-mediated effects on PQC; and (3) examine the process and regulation of P-body assembly.

真核细胞是一种高度分隔的结构，可通过 存在多种膜封闭的细胞器。这种功能的隔离对于 正常的细胞生长和生存。有趣的是，最近的研究表明，其他水平 这些细胞内存在分隔。特别是，许多含有的细胞质颗粒 已经确定了不同的蛋白质和mRNA。其中两个最典型的特征 核糖核蛋白（RNP）结构是加工体（P-aboty）和应力颗粒。这些颗粒不同 从更传统的细胞器来看，它们缺乏有限的膜并且本质上是动态的。 这些颗粒在进化上是保守的，并且与许多人类疾病有关，包括 各种癌症和神经退行性疾病。但是，尽管有这些观察，但生理学 这些RNP结构的功能仍然很少了解。缺乏理解代表了一个关键 我们目前的知识和试图弥合这种鸿沟的差距是我们实验室的主要研究重点。 该提案中的实验旨在进一步了解这些生物学作用 RNP颗粒和调节组装的机制。前两个目的是在这个问题 来自不同方向的生物学功能。首先，我们将评估生理后果 在应力条件下，具有关键信号蛋白与P体和/或应力颗粒相关。我们的 这里的重点是特定的蛋白质激酶，并且将这种重新定位到RNP颗粒允许的可能性 单元中存在的信号网络的重新布线。在第二个中，我们专注于整个颗粒和 询问缺乏这些RNP结构的突变体中细胞生理的改变。这些后一个研究的重点是 我们实验室最近的工作提出了蛋白质稳态中的潜在作用。特别是，我们有 发现缺乏P体的突变体表现出蛋白质错误折叠和聚集水平升高。这 这里的实验旨在确定负责这些影响的基本机制，应 确定这些RNP颗粒在维持蛋白质质量控​​制（PQC）中是否具有直接作用。 最后，第三个目的研究了P体焦点的调节和组装的几个关键方面。尤其， 研究将定义PKA介导的P体组装和 开发一种纯化这些RNP颗粒的便利方法。提案的三个具体目标是 如下：（1）确定蛋白激酶募集到RNP颗粒的生理后果； （2） 定义负责P体介导的PQC作用的基本机制； （3）检查 P体组装的过程和调节。