Invest Ophthalmol Vis Sci. 2024 Mar 5;65(3):31.doi: 10.1167/iovs.65.3.31.

中文速读

本研究针对FEVR/Norrie病相关的Norrin二聚体界面突变，在HEK293T、HEK293STF和HeLa细胞中利用Entranster-H4000转染质粒，通过Western blot、Co-IP、免疫荧光、双荧光素酶报告实验和RUSH实验分析突变对Norrin分泌、蛋白组装及Norrin/β-catenin通路激活的影响，发现多数突变导致分泌缺陷和信号减弱。

体外（细胞）质粒DNAHEK293THEK293STFHeLa

本文使用：长核酸转染试剂Entranster™-H4000

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Investigating the Impact of Dimer Interface Mutations on Norrin’s Secretion and Norrin/β-Catenin Pathway Activation

Min Liu^{1 2}, Erkuan Dai³, Mu Yang^{1 2}, Shujin Li^{1 2}, Lin Fan⁴, Yining Liu^{1 2}, Haodong Xiao³, Peiquan Zhao³, Zhenglin Yang^{1 2 4}

Affiliations

¹ Sichuan Provincial Key Laboratory for Human Disease Gene Study, Center for Medical Genetics and Department of Laboratory Medicine, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, China.
² Research Unit for Blindness Prevention, Chinese Academy of Medical Sciences (2019RU026), Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China.
³ Department of Ophthalmology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
⁴ The University of Chinese Academy of Sciences, Beijing, China.

PMID: 38517429
PMCID: PMC10981164
DOI: 10.1167/iovs.65.3.31

Abstract

Purpose: This study aimed to investigate the impact of 21 NDP mutations located at the dimer interface, focusing on their potential effects on protein assembly, secretion efficiency, and activation of the Norrin/β-catenin signaling pathway.

Methods: The expression level, secretion efficiency, and protein assembly of mutations were analyzed using Western blot. The Norrin/β-catenin signaling pathway activation ability after overexpression of mutants or supernatant incubation of mutant proteins was tested in HEK293STF cells. The mutant norrin and wild-type (WT) FZD4 were overexpressed in HeLa cells to observe their co-localization. Immunofluorescence staining was conducted in HeLa cells to analyze the subcellular localization of Norrin and the Retention Using Selective Hook (RUSH) assay was used to dynamically observe the secretion process of WT and mutant Norrin.

Results: Four mutants (A63S, E66K, H68P, and L103Q) exhibited no significant differences from WT in all evaluations. The other 17 mutants presented abnormalities, including inadequate protein assembly, reduced secretion, inability to bind to FZD4 on the cell membrane, and decreased capacity to activate Norrin/β-catenin signaling pathway. The RUSH assay revealed the delay in endoplasmic reticulum (ER) exit and impairment of Golgi transport.

Conclusions: Mutations at the Norrin dimer interface may lead to abnormal protein assembly, inability to bind to FZD4, and decreased secretion, thus contributing to compromised Norrin/β-catenin signaling. Our results shed light on the pathogenic mechanisms behind a significant proportion of NDP gene mutations in familial exudative vitreoretinopathy (FEVR) or Norrie disease.

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