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

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，在细胞内对氧化应激的保护、线粒体的健康和自噬的调节中起着至关重要的作用。DJ-1基因突变与常染色体隐性遗传性帕金森病有关。事实上，DJ-1的几个突变已被确定为帕金森病患者的致病因素：L166P、M26I、D149A、E163K和E46D。然而，DJ-1启动神经退行性变的机制尚不清楚，其在帕金森病发病机制中的作用还需要进一步的研究。应激颗粒(SGS)是由翻译停滞的mRNAs、相关的翻译启动因子和多个RNA结合蛋白(RBP)组成的无膜细胞质聚集体，代表了各种应激刺激(如热、紫外线辐射、病毒感染)诱导的RNA分流过程的形态结果。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与应激颗粒(SG)之间的第一个联系是葡萄糖饥饿后酵母DJ-1同源基因Hsp31与SG和P-Body的共定位。有趣的是，DJ-1被发现在诱导高渗休克和氧化应激时与哺乳动物细胞中的几个SGS成分相互作用，包括以RNA依赖的方式包括eIF4A。值得注意的是，本研究类似地证明了DJ-1与几个hnRNP(hnRNPM、hnRNPA2/B1、hnRNPA1、hnRNPV、hnRNP)之间的相互作用。HnRNPs被认为是SGS的主要组成部分，它们是限制性商业惯例，与RNA代谢有着复杂的联系。根据上面提到的研究，我们假设DJ-1在SG动力学和应力条件下的平移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。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DJ-1在帕金森病发病机制中的作用。研究DJ-1在不同应激条件下与RNA的相互作用，研究DJ-1在应激颗粒中的区划，以及在存在或不存在DJ-1的情况下，确定针对SGS的信使核糖核酸序列。最终，这些分析将进一步阐明DJ-1功能丧失是如何导致帕金森病的，为这种疾病的分子发病机制提供重要的见解，并可能为新的治疗策略提供信息。方法人类神经母细胞瘤SH-SY5Y细胞将暴露于高渗休克、氧化应激或帕金森毒素(鱼藤酮、MPP+)，并将在多聚体裂解缓冲液中裂解。与DJ-1相互作用的RNA将通过免疫共沉淀得到纯化，如Repici等人所述。(2019年)，并进行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各种刺激产生的功能基团，这将为DJ-1调控的信号通路提供信息。表达GFP-G3BP1融合蛋白的U-2OS细胞将用于分离不同类型应激后的SG核心。将对SG核心进行免疫印迹，以寻找总DJ-1以及氧化形式的DJ-1。这将澄清DJ-1是作为SGS外壳中的RNA穿梭蛋白，还是作为SG的核心成分。将使用质谱仪分析SG组分以确定DJ-1的氧化状态和翻译后修饰。野生型或CRISPR基因敲除的SH-SY5Y细胞将被用于研究在DJ-1存在或不存在的情况下针对SGS的信使核糖核酸群体。

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.