Identification of MANF as a protein interacting with RTN1-C
Identification of MANF as a protein interacting with RTN1-C
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鉴定 MANF 作为与 RTN1-C 相互作用的蛋白质
DOI：
10.1093/abbs/gmu125
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发表时间：
2015-02-01
影响因子：
3.7
通讯作者：
Shen, Yuxian
中科院分区：
文献类型：
--
作者：
Chen, Lijian;Wan, Lijuan;Shen, Yuxian
网状细胞（Reticulons，RTNs）是内质网（Endoplasmic reticulum，ER）相关蛋白家族中的一员，呈网状分布.最近，有证据表明，它们通过特定蛋白的相互作用或调节发挥癌症特异性促凋亡功能。这些证据尤其与RTN 1-C家族成员相关。为了寻找与RTN 1-C相互作用的蛋白质，本研究利用酵母双杂交系统和常规分子生物学技术筛选人胎脑cDNA文库。结果，获得了包括人中脑星形胶质细胞源性神经营养因子（MANF）在内的7个RTN 1-C相互作用蛋白。RTN 1-C与其相互作用蛋白之间的相互作用通过β-半乳糖苷酶测定和选择性培养基中的生长试验来证实。此外，MANF/RTN 1-C相互作用在体外通过谷胱甘肽S-转移酶下拉试验和在体内通过免疫沉淀试验进行验证。免疫荧光检测发现MANF与RTN 1-C共定位于ER。RTN 1-C的敲除减少了MANF在ER中的定位。这些结果为进一步探讨RTN 1-C和MANF在神经退行性疾病和肿瘤中的作用提供了线索。
Reticulons (RTNs) constitute a family of endoplasmic reticulum (ER)-associated proteins with a reticular distribution. Recently, evidence has shown that they exert a cancer-specific proapoptotic function via interaction or modulation of specific proteins. Such evidence is particularly associated with the RTN1-C family members. In order to explore proteins that interact with RTN1-C, the yeast two-hybrid system and regular molecular biological techniques were used to screen the human fetal brain cDNA library. As a result, seven RTN1-C interacting proteins including Homo sapiens mesencephalic astrocyte-derived neurotrophic factor (MANF) were obtained. The interactions between RTN1-C and its interacting proteins were confirmed by beta-galactosidase assay and growth test in selective media. Moreover, the MANF/RTN1-C interaction was verified in vitro by glutathione S-transferase pull-down assay and in vivo by immunoprecipitation assay. By immunofluorescence assay, it was found that MANF co-localized with RTN1-C in the ER. Knockdown of RTN1-C reduced the localization of MANF in the ER. These results provide clues to further explore the function of RTN1-C and MANF in neurodegenerative diseases and cancer.