Unraveling the role of DJ-1 in cellular RNA dynamics Relevance for neurodegeneration and Parkinson's Disease

揭示 DJ-1 在细胞 RNA 动力学中的作用 与神经退行性变和帕金森病的相关性

• 批准号: 2742591
• 金额: --
• 依托单位: Aston University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2742591
• 关键词: Unraveling; role; DJ; cellular; RNA

IntroductionDJ-1 protein, encoded by the PARK7 (Parkinson disease protein 7) gene, is a conserved protein composed of 189 amino acids, with a crucial role within cells in the cell's protection from oxidative stress, in mitochondrial health and, regulation of autophagy. DJ-1 mutations have been linked to autosomal recessive Parkinson's disease. Indeed, several mutations of DJ-1 have been identified as disease causative in PD patients: L166P, M26I, D149A, E163K and E46D. However, the mechanisms by which DJ-1 initiates neurodegeneration are poorly identified and further studies are needed to elucidate its role in PD pathogenesis.Stress granules (SGs) are membraneless, cytoplasmic aggregates of translationally stalled mRNAs, associated translation initiation factors and multiple RNA-binding proteins (RBPs) that represent the morphological consequence of a RNA triage process induced by various stress stimuli such as heat, UV radiation, viral infections. They have been recognized as precursors of pathological protein aggregates in certain neurodegenerative disorders. The first link between DJ-1 and SG (Stress granules) was the co-localization of the yeast DJ-1 homologs Hsp31 with SG and P-bodies after glucose starvation. Intriguingly, DJ-1 was found to interact with several SGs components in mammalian cells upon induction of hyperosmotic shock and oxidative stress, including eIF4A in an RNA-dependent manner.Remarkably, this study similarly demonstrated an interaction between DJ-1 and several hnRNPs (hnRNPM, hnRNPA2/B1, hnRNPA1, hnRNPV, hnRNP). HnRNPs are considered among the main SGs components, they are RBPs and are intricately linked to RNA metabolism. According to the studies mentioned above, we hypothesize a potential novel role for DJ-1 in SG dynamics and translational control upon stress conditions, which we aim to investigate within this project. Aims and ObjectivesThis research projects aims at investigating the role of DJ-1 in stress granule dynamics to better understand its role in the pathogenesis of Parkinson's Disease.This will be achieved by exploring DJ-1/RNA interactome in different stress conditions, studying the compartmentalization of DJ-1 within stress granules and determining the mRNA populations targeted to SGs in presence or absence of DJ-1. Ultimately, such analyses will further elucidate how DJ-1 loss of function leads to PD, providing important insight into the molecular pathogenesis of this disorder and potentially informing novel therapeutic strategies.MethodologyHuman neuroblastoma SH-SY5Y cells will be exposed to hyperosmotic shock, oxidative stress, or parkinsonian toxins (rotenone, MPP+), and will be lysed in polysome lysis buffer. RNAs interacting with DJ-1 will be purified by co-immunoprecipitation as in Repici et al. (2019), and RNA sequencing will be carried out. Gene ontology and related bioinformatics approaches will be employed to identify functional groups arising from the various stimuli, which will inform the signaling pathways regulated by DJ-1. U-2 OS cells expressing a GFP-G3BP1 fusion protein will be used to isolate SG cores after different types of stress. Immunoblotting will be performed on the SG cores to look for total DJ-1, as well as oxidized forms of DJ-1. This will clarify whether DJ-1 acts as an RNA shuttle protein in the outer shell of SGs or as a core SG component. Mass spectrometry will be used for the analysis of SG components to identify DJ-1 oxidation state and post translational modifications.Wild-type or CRISPR knockout SH-SY5Y cells will be used to study the mRNA populations targeted to SGs in presence or absence of DJ-1.

简介DJ-1 蛋白由 PARK7（帕金森病蛋白 7）基因编码，是一种由 189 个氨基酸组成的保守蛋白，在细胞内保护细胞免受氧化应激、线粒体健康和自噬调节方面发挥着至关重要的作用。 DJ-1 突变与常染色体隐性遗传帕金森病有关。事实上，DJ-1 的几种突变已被确定为 PD 患者的致病因素：L166P、M26I、D149A、E163K 和 E46D。然而，DJ-1 引发神经退行性变的机制尚不清楚，需要进一步研究来阐明其在 PD 发病机制中的作用。应激颗粒 (SG) 是翻译停滞的 mRNA、相关翻译起始因子和多种 RNA 的无膜细胞质聚集体。结合蛋白 (RBP) 代表由各种应激刺激（如热、紫外线辐射、病毒感染）诱导的 RNA 分类过程的形态学结果。它们已被认为是某些神经退行性疾病中病理性蛋白质聚集体的前体。 DJ-1 和 SG（应激颗粒）之间的第一个联系是葡萄糖饥饿后酵母 DJ-1 同源物 Hsp31 与 SG 和 P 体的共定位。有趣的是，在诱导高渗性休克和氧化应激时，发现 DJ-1 与哺乳动物细胞中的几种 SG 成分相互作用，包括以 RNA 依赖性方式的 eIF4A。值得注意的是，这项研究同样证明了 DJ-1 与几种 hnRNP 之间的相互作用。 hnRNPM、hnRNPA2/B1、hnRNPA1、hnRNPV、hnRNP）。 HnRNP 被认为是 SG 的主要成分之一，它们是 RBP，与 RNA 代谢有着错综复杂的联系。根据上述研究，我们假设 DJ-1 在 SG 动力学和应激条件下的翻译控制中具有潜在的新作用，我们的目标是在本项目中进行研究。目的和目标本研究项目旨在研究 DJ-1 在应激颗粒动力学中的作用，以更好地了解其在帕金森病发病机制中的作用。这将通过探索不同应激条件下的 DJ-1/RNA 相互作用组、研究区室化来实现检测应激颗粒内的 DJ-1，并确定在存在或不存在 DJ-1 的情况下针对 SG 的 mRNA 群体。最终，此类分析将进一步阐明 DJ-1 功能丧失如何导致 PD，为了解这种疾病的分子发病机制提供重要见解，并可能为新的治疗策略提供信息。 方法学人神经母细胞瘤 SH-SY5Y 细胞将暴露于高渗性休克、氧化应激，或帕金森毒素（鱼藤酮，MPP+），并将在多核糖体裂解缓冲液中裂解。与 DJ-1 相互作用的 RNA 将通过免疫共沉淀进行纯化，如 Repici 等人所述。 (2019)，并将进行RNA测序。将采用基因本体论和相关生物信息学方法来识别由各种刺激产生的功能组，这将告知 DJ-1 调节的信号通路。表达 GFP-G3BP1 融合蛋白的 U-2 OS 细胞将用于在不同类型的应激后分离 SG 核心。将在 SG 核心上进行免疫印迹以寻找总 DJ-1 以及 DJ-1 的氧化形式。这将澄清 DJ-1 是作为 SG 外壳中的 RNA 穿梭蛋白还是作为核心 SG 成分。质谱法将用于分析 SG 成分，以识别 DJ-1 氧化态和翻译后修饰。野生型或 CRISPR 敲除 SH-SY5Y 细胞将用于研究存在或不存在 DJ 时针对 SG 的 mRNA 群体-1。