中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
8期
677-681
,共5页
王春霞%董鸿铭%赵宇%阎启昌%张劲松
王春霞%董鴻銘%趙宇%閻啟昌%張勁鬆
왕춘하%동홍명%조우%염계창%장경송
视神经蛋白%神经视网膜亮氨酸拉链%蛋白质相互作用%免疫共沉淀%蛋白结合区域
視神經蛋白%神經視網膜亮氨痠拉鏈%蛋白質相互作用%免疫共沉澱%蛋白結閤區域
시신경단백%신경시망막량안산랍련%단백질상호작용%면역공침정%단백결합구역
Optineurin%Neural retina leucine zipper%Protein interaction%Co-immunoprecipitation%Protein binding site
背景 视神经蛋白(OPTN)基因是青光眼和肌萎缩性脊髓侧索硬化的致病基因,在眼部视网膜组织中的表达最多.我们先前的研究分离出与OPTN相互作用的蛋白质,确定其中之一为视网膜色素变性的致病基因——碱性亮氨酸拉链(bZIP)转录因子,即神经视网膜亮氨酸(NRL)拉链,并证实OPTN和NRL两蛋白间存在相互作用. 目的 确定与NRL相互作用所必需的OPTN蛋白结合区域. 方法 构建一系列带FLAG标签的OPTN部分片段缺失质粒,分别与带血凝素标记的NRL(HA-NRL)共转染HeLaS3细胞.用抗HA和抗FLAG标签抗体分别对细胞质和细胞核提取物进行免疫共沉淀和Western blot检测,分析两蛋白质间的相互结合方式,以确定NRL与OPTN蛋白的结合区域.结果 在OPTN的第一、第二和第三缺失区域质粒转染细胞的细胞核组分中均检测出血凝素标记的NRL(HA-NRL)免疫共沉淀条带,但在第四缺失区域质粒中不产生此HA-NRL条带.从所有缺失质粒及全长OPTN质粒转染细胞的细胞质组分中均未观察到NRL条带.结论 本研究进一步证实OPTN和NRL在HeLaS3细胞核内的相互作用,并明确其蛋白结合区域.在共表达NRL和OPTN的HeLaS3细胞中,Flag-OPTN可与HA-NRL相互结合,且OPTN的尾端区域(423-577)是结合NRL所必需的.
揹景 視神經蛋白(OPTN)基因是青光眼和肌萎縮性脊髓側索硬化的緻病基因,在眼部視網膜組織中的錶達最多.我們先前的研究分離齣與OPTN相互作用的蛋白質,確定其中之一為視網膜色素變性的緻病基因——堿性亮氨痠拉鏈(bZIP)轉錄因子,即神經視網膜亮氨痠(NRL)拉鏈,併證實OPTN和NRL兩蛋白間存在相互作用. 目的 確定與NRL相互作用所必需的OPTN蛋白結閤區域. 方法 構建一繫列帶FLAG標籤的OPTN部分片段缺失質粒,分彆與帶血凝素標記的NRL(HA-NRL)共轉染HeLaS3細胞.用抗HA和抗FLAG標籤抗體分彆對細胞質和細胞覈提取物進行免疫共沉澱和Western blot檢測,分析兩蛋白質間的相互結閤方式,以確定NRL與OPTN蛋白的結閤區域.結果 在OPTN的第一、第二和第三缺失區域質粒轉染細胞的細胞覈組分中均檢測齣血凝素標記的NRL(HA-NRL)免疫共沉澱條帶,但在第四缺失區域質粒中不產生此HA-NRL條帶.從所有缺失質粒及全長OPTN質粒轉染細胞的細胞質組分中均未觀察到NRL條帶.結論 本研究進一步證實OPTN和NRL在HeLaS3細胞覈內的相互作用,併明確其蛋白結閤區域.在共錶達NRL和OPTN的HeLaS3細胞中,Flag-OPTN可與HA-NRL相互結閤,且OPTN的尾耑區域(423-577)是結閤NRL所必需的.
배경 시신경단백(OPTN)기인시청광안화기위축성척수측색경화적치병기인,재안부시망막조직중적표체최다.아문선전적연구분리출여OPTN상호작용적단백질,학정기중지일위시망막색소변성적치병기인——감성량안산랍련(bZIP)전록인자,즉신경시망막량안산(NRL)랍련,병증실OPTN화NRL량단백간존재상호작용. 목적 학정여NRL상호작용소필수적OPTN단백결합구역. 방법 구건일계렬대FLAG표첨적OPTN부분편단결실질립,분별여대혈응소표기적NRL(HA-NRL)공전염HeLaS3세포.용항HA화항FLAG표첨항체분별대세포질화세포핵제취물진행면역공침정화Western blot검측,분석량단백질간적상호결합방식,이학정NRL여OPTN단백적결합구역.결과 재OPTN적제일、제이화제삼결실구역질립전염세포적세포핵조분중균검측출혈응소표기적NRL(HA-NRL)면역공침정조대,단재제사결실구역질립중불산생차HA-NRL조대.종소유결실질립급전장OPTN질립전염세포적세포질조분중균미관찰도NRL조대.결론 본연구진일보증실OPTN화NRL재HeLaS3세포핵내적상호작용,병명학기단백결합구역.재공표체NRL화OPTN적HeLaS3세포중,Flag-OPTN가여HA-NRL상호결합,차OPTN적미단구역(423-577)시결합NRL소필수적.
Background Gene encoding optineurin (OPTN) is a causative gene for glaucoma and amyotrophic lateral sclerosis,with a more expression in retina.Our previous study isolated OPTN-interacting proteins and identified that the gene encode the basic leucine zipper (bZIP) transcription factor neural retina leucine (NRL) zipper,a causative gene for retinitis pigmentosa,and further study demonstrated the interaction between OPTN and NRL proteins in nuclei of cultured HeLaS3 cells.Objective This study was to determine the protein binding site of OPTN necessary for NRL binding.Methods A deletion series of OPTN-expression plasmids were constructed and co-expressed with hemagglutinin (HA)-tagged NRL in HeLaS3 cells,respectively.The cytoplasmic and nuclear fractions were used to perform co-immunoprecipitate (CoIP) and Western blot with anti-tag antibodies.Results In the nuclear fractions of cells transfected with the del1 st,del2nd or del3rd plasmid,a band of coimmunoprecipitated HA-labelled NRL (HA-NRL) was detected.However,the del4th plasmid did not produce a band.The NRL band was not found in cytoplasmic fractions from transfected cells with any of the deletion plasmids or with the whole-length OPTN plasmid.Conclusions The protein binding site of OPTN necessary for NRL binding is determined.This result demonstrates the binding of Flag-OPTN and HA-NRL in HeLaS3 cells.A series of partial-deletion OPTN plasmids demonstrated that the tail region (423-577 amino acids) of OPTN was necessary for binding with NRL.
