PINK1与α-synuclein相互作用结构域鉴定

中国生物工程杂志

2011, Vol. 31

Issue (12): 10-14

研究报告

PINK1与α-synuclein相互作用结构域鉴定

付越姣, 段春礼, 龚普盛, 贾焕珍, 张建亮, 赵春礼, 鲁玲玲, 赵焕英, 袁星星, 杨慧

首都医科大学北京神经科学研究所北京市神经再生修复研究重点实验室神经变性病教育部重点实验室北京 100069

Identification Domains of PINK1 Interacting with α-Synuclein

FU Yue-jiao, DUAN Chun-li, GONG Pu-sheng, JIA Huan-zhen, ZHANG ian-liang, ZHAO Chun-li, LU Ling-ling, ZHAO Huan-ying, YUAN Xing-xing, YANG Hui

Beijing Institute for Neuroscience, Capital Medical University, Beijing Center of Neural Regeneration and Repair, Key Laboratory of Neurodegenerative Diseases, Ministry of Education, Beijing 100069, China

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摘要：

目的: 确定PINK1与α-synuclein相互作用的结构域。方法: 将PINK1不同结构域质粒(pcDNA3.1-3xFlag-hPINK1^WT, pcDNA3.1-3xFlag-hPINK1(G309D),pcDNA3.1-3xFlag-hPINK1(ΔN35),pcDNA3.1-3xFlag-hPINK1(ΔC145),pcDNA3.1-3xFlag-hPINK1(156~509), pcDNA3.1-3xFlag-hPINK1(Δ156~581), pcDNA3.1-3xFlag-vector)分别和pCMV-Myc-α-synuclein质粒共转染人胚肾HEK293T细胞,通过免疫共沉淀(Co-IP)技术验证PINK1与α-synuclein相互作用的结构域;同时进行免疫细胞化学染色,利用激光扫描共聚焦显微镜观察两种蛋白的共定位关系。结果: Co-IP实验结果表明PINK1与α-synuclein相互作用的结构域为PINK1的激酶结构域。免疫细胞化学实验也证实α-synuclein只与含激酶结构域的PINK1蛋白在细胞中存在共定位关系。结论: PINK1与α-synuclein相互作用的结构域位于其激酶结构域。

关键词： PINK1; α-synuclein; 激酶结构域; 免疫共沉淀

Abstract:

Objective: To identify domains of PINK1 interacting with α-synuclein. Methods: HEK293T cells were transfected with plasmid encoding human pCMV-Myc-α-synuclein together with human 3xFlag-tagged wild type PINK1 or several deletion mutants(G309D;ΔN35; ΔC145; 156~509; Δ156~581;). After detecting the protein expression by Western blot, the functional domains of hPINK1 interacting with human α-synuclein were detected by co-immunoprecipitation. The co-localization of different domains of hPINK1 and human α-synuclein was detected by immunocytochemistry. Result: Co-IP and immunocytochemistry have proved that the PINK1 kinase domain interacted with α-synuclein. Conclusion: The kinase domain of PINK1 interacting with α-synuclein.

Key words: PINK1 α-synuclein Kinase domain Co-immunoprecipitation

收稿日期: 2011-07-26 出版日期: 2011-12-25

ZTFLH:

Q819

基金资助:

国家重点基础研究发展计划(2011CB504103)、国家自然科学基金(30970940)、北京市教育委员会科技发展计划重点项目(KZ201010025022)、北京市自然基金面上项目(5102012)、北京市高校人才强教资助PHR (200907113)资助项目

通讯作者: 杨慧,电子信箱:huiyang@ccmu.edu.cn E-mail: huiyang@ccmu.edu.cn

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引用本文:

付越姣, 段春礼, 龚普盛, 贾焕珍, 张建亮, 赵春礼, 鲁玲玲, 赵焕英, 袁星星, 杨慧. PINK1与α-synuclein相互作用结构域鉴定[J]. 中国生物工程杂志, 2011, 31(12): 10-14.

FU Yue-jiao, DUAN Chun-li, GONG Pu-sheng, JIA Huan-zhen, ZHANG ian-liang, ZHAO Chun-li, LU Ling-ling, ZHAO Huan-ying, YUAN Xing-xing, YANG Hui. Identification Domains of PINK1 Interacting with α-Synuclein. China Biotechnology, 2011, 31(12): 10-14.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I12/10

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