Identification of Podocyte Cargo Proteins by Proteomic Analysis of Clathrin-Coated Vesicles.
Identification of Podocyte Cargo Proteins by Proteomic Analysis of Clathrin-Coated Vesicles.
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DOI：
10.34067/kid.0000212020
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发表时间：
2020-04
期刊：
影响因子：
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通讯作者：
Marwin Groener;Ying Wang;Elizabeth Cross;Xuefei Tian;Karen Ebenezer;E. Baik;C. Pedigo;M. Schiffer;Kazunori Inoue;S. Ishibe
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文献类型：
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作者：
Marwin Groener;Ying Wang;Elizabeth Cross;Xuefei Tian;Karen Ebenezer;E. Baik;C. Pedigo;M. Schiffer;Kazunori Inoue;S. Ishibe
网格蛋白介导的内吞作用（CME）在足细胞健康中起着重要作用。基因消融与CME相关的基因已被证明会导致小鼠严重的蛋白尿和足突消退。然而，关于足细胞中网格蛋白包被囊泡（ccv）的货物知之甚少。本研究的目的是从足细胞中分离ccv，并通过蛋白质组学分析鉴定其货物。方法从Podocin-Cre Rosa-DTR flox小鼠肾中分离肾小球，接种白喉毒素，获得纯原代足细胞培养物。采用差梯度超离心分离ccv，免疫印迹和电镜观察ccv的富集情况。采用液相色谱-质谱法（LC-MS）进行蛋白质组学分析。根据先前发表的文献，评估了比转铁蛋白受体蛋白1丰度更高的蛋白质在CCV货物中的潜力。在足细胞中对鉴定的货物蛋白和ccv进行免疫荧光染色以进一步验证。结果对CME的多种蛋白标记物进行免疫印迹检测，发现CCV部位中有丰富的CME蛋白。通过电镜观察到ccv在其他小囊泡中的富集。蛋白质组学共获得了bb0 1200个重要蛋白。多步骤数据分析显示36个ccv相关蛋白，其中10个代表足细胞中新颖的，高度丰富的货物蛋白。观察货运蛋白和ccv在免疫染色上的共定位。结论足细胞CCV货物蛋白的鉴定有助于阐明内吞运输对足细胞健康和维持肾小球环境的重要性。
Background Clathrin-mediated endocytosis (CME) plays a fundamental role in podocyte health. Genetic ablation of genes implicated in CME has been shown to cause severe proteinuria and foot process effacement in mice. However, little is known about the cargo of clathrin-coated vesicles (CCVs) in podocytes. The goal of this study was to isolate CCVs from podocytes and identify their cargo by proteomic analysis. Methods Glomeruli isolated from Podocin-Cre Rosa-DTR flox mouse kidneys were seeded and treated with diphtheria toxin to obtain pure primary podocyte cultures. CCVs were isolated by differential gradient ultracentrifugation, and enrichment of CCVs was assessed by immunoblotting and electron microscopy (EM). Liquid chromatography-mass spectrometry (LC-MS) was performed for proteomic analysis. Proteins with higher abundance than transferrin receptor protein 1 were evaluated for CCV cargo potential against previously published literature. Immunofluorescence staining of identified cargo proteins and CCVs was performed in podocytes for further verification. Results Immunoblotting for multiple protein markers of CME revealed enrichment in the CCV fraction. Enrichment of CCVs among other small vesicles was observed via EM. Proteomics yielded a total of >1200 significant proteins. Multiple-step data analysis revealed 36 CCV-associated proteins, of which 10 represent novel, highly abundant cargo proteins in podocytes. Colocalization of cargo proteins and CCVs on immunostaining was observed. Conclusions Our identification of podocyte CCV cargo proteins helps to elucidate the importance of endocytic trafficking for podocyte health and maintenance of the glomerular environment.