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划分的结构，它被细分为不同的功能区域，通过 存在各种被膜包裹的细胞器。这种职能分离对于 正常的细胞生长和存活。有趣的是，最近的研究表明，额外的水平 这些细胞内部存在区隔。特别是，含有许多细胞质颗粒的 已经确定了不同的蛋白质和mRNAs组。其中两个最具特点的是 核糖核蛋白(RNP)的结构有加工体(P体)和应力颗粒。这些颗粒不同 与更传统的细胞器不同，因为它们缺乏限制膜，本质上是相当动态的。 这些颗粒在进化上是保守的，并与许多人类疾病有关，包括 各种癌症和神经退行性疾病。然而，尽管有这些观察，生理上的 这些RNP结构的功能仍然知之甚少。这种缺乏理解代表着一个关键的 我们目前知识中的差距并试图弥合这一差距是我们实验室的主要研究重点。 这项建议中的实验旨在加深我们对这些细胞的生物学作用的理解 RNP颗粒和调节其组装的机制。最初的两个目标来自于这个问题 来自不同方向的生物功能。首先，我们将评估 在应激条件下，与P小体和/或应激颗粒相关的关键信号蛋白。我们的 这里的重点是特定的蛋白激酶以及这种重新定位到RNP颗粒的可能性 对细胞中的信令网络进行重新布线。在第二部分中，我们将重点放在颗粒的整体上，并 询问缺乏这些RNP结构的突变体的细胞生理学是如何改变的。这些后面的研究集中在一种 我们实验室最近的工作表明，这在蛋白质动态平衡中具有潜在的作用。特别是，我们有 发现缺乏P-小体的突变体表现出更高水平的蛋白质错误折叠和聚集。这个 这里的实验旨在确定造成这些影响的潜在机制，并应该 确定这些RNP颗粒在维持蛋白质质量控制(PQC)中是否有直接作用。 最后，第三个目标研究了P体灶的调节和组装的几个关键方面。特别是， 这些研究将确定PKA介导的P小体组装和调控的分子机制。 建立一种简便的方法来纯化这些RNP颗粒。该提案的三个具体目标是 具体如下：(1)确定RNP颗粒中蛋白激酶重新聚集的生理后果；(2) 定义P-Body对PQC的影响的潜在机制；以及(3)检查 P形体组装的过程和规则。