Ma Yun, Tian Shuai, Wang Zongbao, Wang Changbo, Chen Xiaowei, Li Wei, Yang Yang, He Shuya
Department of Biochemistry & Biology, University of South China, Hengyang, Hunan 421001, P.R. China.
Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, Institute of Pharmaceutical and Biological Sciences, University of South China, Hengyang, Hunan 421001, P.R. China.
Mol Med Rep. 2016 Aug;14(2):1501-8. doi: 10.3892/mmr.2016.5438. Epub 2016 Jun 23.
Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P‑interacting proteins and determine the biological effect of the interaction. The current study identified CMP‑N‑acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two‑hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β‑galactosidase assay and growth studies with selective media. Furthermore, co‑immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue‑specific regulator of GM1 levels in SH‑SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS.
脆性X智力低下蛋白（FMRP）、脆性X相关蛋白1（FXR1P）和FXR2P是FMR蛋白家族的成员。这些蛋白含有两个KH结构域和一个RGG框，这是RNA结合蛋白的特征。FMRP的缺失会导致脆性X综合征（FXS），这是遗传性智力低下的主要原因。FXR1P在全身表达，对正常肌肉发育很重要，其缺失会导致心脏异常。为了研究FXR1P的功能，进行了一项筛选以鉴定与FXR1P相互作用的蛋白，并确定这种相互作用的生物学效应。当前的研究使用酵母双杂交系统鉴定出CMP-N-乙酰神经氨酸合成酶（CMAS）为一种相互作用蛋白，并使用β-半乳糖苷酶测定法和在选择性培养基上的生长研究在酵母中验证了FXR1P与CMAS之间的相互作用。此外，采用免疫共沉淀法分析FXR1P/CMAS的结合，并进行免疫荧光显微镜检查以检测蛋白的表达和细胞内定位。当前研究的结果表明，FXR1P与CMAS相互作用，并在HEK293T和HeLa细胞的细胞质和细胞核中共定位。因此，构建了一个脆性X相关蛋白1（FXR1）基因过表达载体，以研究FXR1过表达对单唾液酸四己糖神经节苷脂1（GM1）水平的影响。当前研究的结果表明，FXR1P是SH-SY5Y细胞中GM1水平的组织特异性调节因子，但在HEK293T细胞中不是。综上所述，这些结果初步表明FXR1P与CMAS相互作用，并且FXR1P可能通过与CMAS相互作用增强唾液酸的活化，并增加GM1水平以影响神经系统的发育，从而为进一步研究FXS的发病机制提供了证据。
