【作者】 王渊；

【导师】 徐涛； 张蓉颖；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2022， 硕士

【摘要】 通过内吞作用,细胞将质膜上的膜蛋白转运至胞内,从而维持细胞膜组分的动态平衡,对细胞信号转导、营养摄取、免疫应答等生命活动的正常运行至关重要。内化后的膜蛋白通常会进入内吞-溶酶体系统中进行分选转运,不同的蛋白走向不同的命运。分选连接蛋白(sorting nexins,SNXs)是一类在内吞体分选过程中发挥调控作用的蛋白家族。SNX27是唯一含有PDZ(PSD95,Dlg1,zo-1)结构域的SNX,通过PDZ结构域与货物蛋白直接结合,并以此调控其内吞后分选转运过程。研究表明SNX27参与调控神经元相关受体的循环转运,SNX27的异常与神经疾病息息相关。但目前对SNX27调控的内吞体分选机制的研究还不够透彻,所以探究SNX27调控的内吞体分选机制有助于更好地理解细胞生命活动运行机制,也为相关疾病的治疗提供新线索和新思路。本研究使用模式生物秀丽隐杆线虫(Caenorhabditis elegans,C.elegans)作为模型,借助共聚焦荧光成像技术探究SNX-27在内吞体分选中发挥作用的机制。首先发现SNX-27直接参与不同特征的CIE途径货物蛋白hTAC和GLUT-1的循环转运。经分析发现与hTAC相比,GLUT-1具有更高的SNX-27依赖性。进一步研究发现GLUT-1的循环转运需要Retromer复合物协助,hTAC则不需要,暗示SNX-27调控不同特征货物蛋白转运存在差异。之后通过共定位分析和RNAi实验发现GLUT-1进入循环路径之前首先到达EEA-1标记的分选内吞体中,表明hTAC与GLUT-1的分选过程发生在早期内吞体的不同微区。最后通过活体动态成像追踪到EEA-1直接参与GLUT-1内吞囊泡向胞内的运输过程。综上所述,本研究发现线虫SNX-27参与调控CIE途径货物蛋白hTAC、GLUT-1的内吞后循环过程,揭示不同特征货物蛋白的分选差异,为进一步探索SNX-27在内吞体分选过程中发挥作用的机制奠定基础。更多还原

【Abstract】 Through endocytosis,cell transport membrane proteins from plasma membrane into cell,thereby maintaining the dynamic balance of plasma membrane components.It is essential for the normal operation of life activities such as cell signaling,nutrient uptake,and immune responses.Internalized proteins usually enter the endolysosomal system for sorting and transport,and different proteins go to different fates.Sorting nexins(SNXs)are a family of proteins that play a regulatory role in endosomal sorting.SNX27 is the only SNX that contains a PDZ(PSD95,Dlg1,zo-1)domain,through which it can directly bind to cargo and thereby regulate its post-endocytosis sorting and transport.Previous studies have shown that SNX27 is involved in the recycling transport of neuron-associated receptors,and that abnormalities of SNX27 are associated with neurological diseases.However,the mechanism of endosomal sorting regulated by SNX27 is still unclear.Therefore,exploring the mechanism of SNX27-regulated endosomal sorting will not only contribute to a better understanding of the mechanism of cell biology,but also provide new clues and ideas for the treatment of related diseases.In this paper,the model organism Caenorhabditis elegans(C.elegans)was used to explore the mechanism of SNX-27 in endosomal sorting by means of confocal fluorescence imaging.First,we found that SNX-27 was directly regulating the recycling transport of CIE cargoes hTAC and GLUT-1.And then,we found that GLUT-1 was more SNX-27 dependent than hTAC.Further studies revealed that Retromer was required for recycling transport of GLUT-1,but not hTAC.This suggested that SNX-27 regulates the transport of different cargoes in different ways.Through colocalization analysis and RNAi experiments,it was found that GLUT-1 reached the EEA-1 labeled sorting endosomes before enter the recycling pathway.It was indicated that the sorting process of hTAC and GLUT-1 occurred in different microdomain.Finally,it was observed by in vivo dynamic imaging that EEA-1 is directly involved in the transport of GLUT-1 endocytic vesicles into the cell.Taken together,this work found that SNX-27 was directly regulating the postendocytosis recycling of CIE pathway cargoes hTAC and GLUT-1,and revealed the sorting differences of different cargoes.It laid the foundation for revealing the mechanism of SNX27-regulated endosomal sorting.更多还原