【作者】 杨超；

【导师】 史岸冰；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2024， 博士

【摘要】 背景:内吞体是存在于真核细胞细胞质中的膜区室,负责将细胞外物质、质膜蛋白和脂质等内吞进入细胞的货物进行分类、分选,继而将这些货物循环回到质膜、高尔基体或转运至溶酶体降解。内吞体在各种生理过程中都发挥着关键作用,比如营养物质的摄取、受体调控的信号传导、膜成分再循环和免疫应答等。内吞体属性及功能是由内吞体膜上的特定生物大分子决定的,其中包括各种磷脂酰肌醇和作为分子开关的Rab小GTP酶(Rab GTPases,Rabs),它们通过募集下游的各种效应蛋白质来协同调控内吞体的多类型功能。RAB-10/Rab10定位于分选内吞体,参与调控囊泡循环运输,相关研究表明RAB-10缺失会导致磷脂酰肌醇-4,5-二磷酸(phosphatidylinositol 4,5-bisphosphate,PI(4,5)P2)在内吞体上大量累积。PI(4,5)P2是一种在细胞质膜中富集的磷脂酰肌醇,多领域广泛研究提示其在质膜上的合成和分解代谢与天然免疫反应活化密切相关,然而学界对于内吞体膜上的PI(4,5)P2的功能意义仍待深入剖析。目前普遍认为,PI(4,5)P2在内吞体上的主要功能是通过招募与其高亲和的功能蛋白来调节囊泡循环运输,而内吞体PI(4,5)P2代谢与动物天然免疫反应之间的相关性尚不明确。目的:以秀丽隐杆线虫作为天然免疫反应研究模型,探究RAB-10调控的内吞体磷脂酰肌醇代谢与天然免疫反应之间的关系。方法:通过脂质组学实验来检测突变线虫的脂质代谢差异,遗传学实验构建不同基因型背景的线虫株系,分子遗传学实验构建转基因线虫株系,细胞生物学实验检测细胞层面蛋白变化,计算机科学实验预测了蛋白相互作用的氨基酸位点,生物化学实验检测蛋白质表达量变化和蛋白质相互作用,分子生物学实验构建质粒载体。结果:病原菌感染存活实验结果表明,RAB-10缺失突变的线虫rab-10(ok1494)在被铜绿假单胞菌(pseudomonas aeruginosa,P.aeruginosa)侵染以后存活率显著低于野生型动物。脂质组学和细胞学实验结果表明,rab-10(ok1494)突变线虫的免疫反应降低是由于PI(4,5)P2在内吞体上累积,导致其水解产物二酰甘油(diacylglycerol,DAG)在肠细胞中的水平下降,进而降低了线虫PMK-1/p38 MAPK天然免疫通路的应答。遗传筛选和细胞实验结果表明,UNC-16/JIP3作为RAB-10的效应蛋白因子,介导磷脂酶C(phospholipase C,PLC)EGL-8的内吞体募集,进而通过水解PI(4,5)P2产生DAG来激活PMK-1/p38 MAPK天然免疫通路。蛋白相互作用实验结果表明,UNC-16的LZII结构域二聚化是其与活性RAB-10相互作用及招募EGL-8到内吞体的先决条件。蛋白实验表明,线虫被病原菌侵染以后,肠细胞中RAB-10活性显著增加。蛋白生化实验、细胞实验和实时定量PCR实验表明,RAB-10的活性增加是其活化辅助因子LET-413/Erbin表达水平增加所致,而LET-413转录水平由核激素受体NHR-25/NR5A1/2所调控。结论:秀丽隐杆线虫在被病原体感染时,肠上皮细胞中的NHR-25很可能参与识别病原体感染信号,随后入核促进LET-413表达,高水平的LET-413提升了RAB-10的活性水平,活性态RAB-10进而定位到内吞体,通过其效应因子UNC-16募集EGL-8到内吞体膜上,促进PI(4,5)P2水解并提升信号分子DAG水平,随后p38 MAPK天然免疫信号通路被激活,促进线虫对病原体入侵的抵御能力。更多还原

【Abstract】 Background:The endosome is a membrane-bound compartment present in the cytoplasm of eukaryotic cells,responsible for sorting and segregating endocytosed extracellular materials,membrane proteins,and lipids for recycling back to the plasma membrane,Golgi apparatus,or transport to lysosomes for degradation.Endosomes play pivotal roles in various physiological processes such as nutrient uptake,regulation of receptor-mediated signaling,membrane component recycling,and immune responses.The properties and functions of endosomes are determined by specific biomolecules present on the endosomal membrane,including various phosphoinositides and Rab small GTPases,which act as molecular switches by recruiting downstream effector proteins to coordinate the diverse functions.RAB-10/Rab10 localizes to sorting endosomes and is involved in regulating endocytic recycling.Studies indicate that loss of RAB-10 leads to a substantial accumulation of phosphatidylinositol 4,5-bisphosphate(PI(4,5)P2)on endosomes.PI(4,5)P2,an abundant phosphoinositide on the plasma membrane,has been extensively studied,suggesting its close association with the activation of innate immune responses through its synthesis and breakdown on the plasma membrane.However,the functional significance of PI(4,5)P2 on endosomal membranes remains to be further elucidated.It is generally believed that the primary role of PI(4,5)P2 on endosomes is to regulate vesicle trafficking by recruiting functional or structural proteins,while the relationship between endosomal PI(4,5)P2 metabolism and innate immune responses remains unclear.Objective:By utilizing Caenorhabditis elegans as a model,we aim to elucidate the interplay between RAB-10-mediated endosomal phosphoinositide metabolism and innate immune responses.Methods:Lipidomics experiments were conducted to assess variations in lipid metabolism in mutant nematodes.Genetic experiments were used to establish nematode strains with different genotypes.Molecular genetic experiments were performed to create transgenic nematode strains,while cell biology experiments were employed to investigate protein alterations at the cellular level.Computer science experiments were carried out to predict amino acid sites for protein interactions,and biochemical experiments were conducted to measure changes in protein expression levels and interactions.Additionally,molecular biology experiments were conducted to construct plasmid vectors.Results:Experimental results from infection survival experiments indicate that the survival rate of the RAB-10 deficient mutant,rab-10(ok1494),significantly decreases upon invasion by Pseudomonas aeruginosa.Lipidomics and cellular experiments suggest that the reduced immune response in the rab-10(ok1494)mutant worms is attributed to the accumulation of PI(4,5)P2 in endosomes,leading to a decrease in the levels of its hydrolysis product,diacylglycerol(DAG),in intestinal cells,subsequently attenuating the response of the PMK-1/p38 MAPK innate immune pathway.Genetic screening and cellular experiments reveal that UNC-16/JIP3,as an effector protein of RAB-10,mediates the recruitment of phospholipase C(PLC)EGL-8 to endosomes,thereby activating the PMK-1/p38 MAPK innate immune pathway through the hydrolysis of PI(4,5)P2 to DAG.Protein interaction experiments demonstrate that dimerization of UNC-16’s LZII domain is a prerequisite for its interaction with active RAB-10 and recruitment of EGL-8 to endosomes.Protein assays indicate a significant increase in RAB-10 activity in intestinal cells following pathogen invasion.Biochemical,cellular,and real-time quantitative PCR experiments suggest that the upregulation of RAB-10activity is attributed to an increase in the expression of its activating co-factor LET-413/Erbin,with the transcriptional level of LET-413 being regulated by the nuclear hormone receptor NHR-25/NR5A1/2.Conclusion:When infected by pathogens,the nematode C.elegans likely engages the nuclear hormone receptor NHR-25 in recognizing signals of pathogen invasion in intestinal epithelial cells.Subsequently,NHR-25 translocates into the nucleus to promote LET-413expression.Elevated levels of LET-413 enhance the activity of RAB-10,which then localizes to endosomes.Through its effector UNC-16,active RAB-10 recruits EGL-8 to the endosomal membrane,facilitating hydrolysis of PI(4,5)P2 and elevating DAG levels.This cascade activates the p38 MAPK innate immune signaling pathway,enhancing the worm’s defense against pathogen invasion.更多还原