Stress Granule clearance via Autophagy: Mechanism, Regulation and Consequences

 描述(申请人提供)：应激颗粒(SGS)是在真核生物中保守的非翻译的信使核糖核酸-蛋白质复合体(MRNP)的集合，通常只有在细胞应激期间大量翻译被抑制时才瞬时形成。SGS 隔离mRNPs和信号因子，并通过调节基因表达和细胞信号在细胞应激反应中发挥关键作用。此前，我们确定SGS是通过一种新的选择性自噬途径清除的，我们称之为颗粒吞噬。这一发现很重要，因为它展示了一种清除SGS的根本新方法；以前SGS被认为只能通过非破坏性的方式拆解。因此，吞噬颗粒可能会影响基因在mRNA水平上的表达，并可以解释几种“包涵体”疾病的潜在病理，如肌萎缩侧索硬化症、额颞叶痴呆症和Paget病。这些典型的特征是结构性的SG样聚集和受影响细胞中的自噬缺陷。我们理解中的主要差距包括SGS是如何机械地被吞噬颗粒作用所针对的，这个过程是如何调节的，以及吞噬颗粒作用的生理相关性是什么。我们的长期目标是了解细胞组装、分解或清除mRNP颗粒的方式，这如何影响mRNA调节，以及以异常mRNP颗粒形成为特征的疾病的进展。这项应用的总体目标是识别影响粒细胞吞噬的基因，并确定SG和自噬蛋白之间的蛋白质相互作用，这是粒细胞吞噬活动所必需的。我们还希望确定颗粒吞噬是如何调节的，以及在包涵体疾病中观察到的颗粒吞噬对mRNA稳定性和SG样蛋白聚集体清除的后果是什么。我们的中心假设是选择性自噬受体蛋白与SGS相互作用，并招募自噬机制，特别是在限制SG通过其他方式分解的条件下。使用酵母和细胞系模型系统，以及在SG和自噬领域经过验证的遗传、生化和显微技术，我们将以以下三个目标来验证这一假设：1)哪些基因促进了食肉癖，又是如何促进的？是什么条件导致了食肉现象，这又是如何被调控的？噬粒细胞如何影响信使核糖核酸的衰变和SG样疾病聚集体的清除？这一建议是创新的，因为它将加强我们对一种新的选择性自噬途径的理解，该途径以具有重要生理意义的底物(SGS)为靶标，在疾病中具有含义。这一贡献是重要的，因为更详细地理解吞噬颗粒可能会提出选择性自噬功能的新范式，并阐明与一般细胞生物学相关的mRNA和细胞信号调节的新模式，以及与异常SG聚集特征或引起的疾病相关的新模式。

 DESCRIPTION (provided by applicant): Stress granules (SGs) are assemblies of non-translating mRNA-protein complexes (mRNPs) that are conserved throughout eukaryotes, and which normally form only transiently when bulk translation is inhibited during cellular stress. SGs sequester mRNPs and signaling factors, and play key roles in cellular stress responses by modulating gene expression and cell signaling. Previously, we determined that SGs are cleared via a novel selective autophagic pathway that we termed granulophagy. This finding was important as it demonstrated a fundamentally new way in which SGs are cleared; previously SGs were thought to disassemble only by non-destructive means. Granulophagy thus likely impacts upon gene expression at the mRNA level, and could explain the underlying pathology of several "inclusion body" disorders such as Amyotrophic Lateral Sclerosis, Frontotemporal Lobar Dementia and Paget's disease. These are typically characterized by constitutive SG-like aggregates and autophagy defects in affected cells. Key gaps in our understanding include how are SGs mechanistically targeted by granulophagy, how is this process regulated, and what is the physiological relevance of granulophagy. Our long-term goal is to understand the means by which cells assemble, disassemble or clear mRNP granules, how this affects mRNA regulation, and the progression of diseases characterized by aberrant mRNP granule formation. The overall objectives of this application are to identify genes affecting granulophagy and determine protein interactions between SG and autophagy proteins that are required for granulophagy activity. We also wish to determine how granulophagy is regulated, and what the consequences of granulophagy are on both mRNA stability and clearance of SG-like protein aggregates observed in inclusion body disorders. Our central hypothesis is that selective autophagy receptor proteins interact with SGs and recruit the autophagic machinery, especially under conditions that limit SG disassembly via other means. Using yeast and cell line model systems, and proven genetic, biochemical and microscopy techniques in the SG and autophagy fields, we shall test this hypothesis with the following three aims: 1.) What genes promote granulophagy, and how? 2.) What conditions induce granulophagy, and how is this regulated? 3.) How does granulophagy affect mRNA decay and clearance of SG-like disease aggregates? This proposal is innovative, as it will enhance our understanding of a novel selective autophagic pathway that targets a physiologically important substrate (SGs) with implications in disease. This contribution is important because understanding granulophagy in greater detail may suggest new paradigms of selective autophagic function, and illuminate new modes of mRNA and cell signaling regulation relevant to both general cell biology, and to diseases characterized or caused by aberrant SG accumulation.