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

DJ-1蛋白是由PARK 7（Parkinson disease protein 7）基因编码的一种保守蛋白，由189个氨基酸组成，在细胞内具有保护细胞免受氧化应激、线粒体健康和调节自噬的关键作用。DJ-1突变与常染色体隐性遗传帕金森病有关。事实上，DJ-1的几种突变已被鉴定为PD患者的致病性：L166 P、M26 I、D149 A、E163 K和E46 D。应激颗粒（stress granules，SG）是一种无膜的细胞质聚集体，聚集了转录停滞的mRNA、相关的翻译起始因子和多种RNA结合蛋白（RNA binding proteins，RBP），它们代表了由各种应激刺激如热，紫外线辐射病毒感染它们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Hsp 31与SG和P体的共定位。有趣的是，DJ-1被发现在高渗休克和氧化应激诱导的哺乳动物细胞中与几种SGs组分相互作用，包括以RNA依赖的方式与eIF 4A相互作用。值得注意的是，这项研究类似地证明了DJ-1与几种hnRNP（hnRNPM，hnRNPA 2/B1，hnRNPA 1，hnRNPV，hnRNP）之间的相互作用。HnRNP被认为是主要的SGs组分之一，它们是RBP并且与RNA代谢错综复杂地联系在一起。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。目的与目的本研究旨在探讨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DJ-1/RNA相互作用组，研究DJ-1在应激颗粒中的区室化，以及确定在存在或不存在DJ-1的情况下靶向SGs的mRNA群体来实现。最终，这样的分析将进一步阐明如何DJ-1的功能丧失导致PD，提供重要的洞察这种疾病的分子发病机制，并可能通知新的therapeutic strategy.MethodologyHuman神经母细胞瘤SH-SY 5 Y细胞将暴露于高渗休克，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裂解。与DJ-1相互作用的RNA将通过共免疫沉淀法纯化，如Repici等人（2019）所述，并将进行RNA测序。基因本体论和相关的生物信息学方法将被用来识别由各种刺激产生的功能组，这将为DJ-1调节的信号通路提供信息。表达GFP-G3 BP 1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。将对SG核心进行免疫印迹，以寻找总DJ-1以及DJ-1的氧化形式。这将阐明DJ-1是否作为SG外壳中的RNA穿梭蛋白或作为核心SG组分。将使用质谱法分析SG组分以鉴定DJ-1氧化态和翻译后修饰。将使用野生型或CRISPR敲除SH-SY 5 Y细胞研究在存在或不存在DJ-1的情况下靶向SG的mRNA群体。

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.