Objectives: To investigate the subcellular localization and biological functions of transmembrane protein 240 (TMEM240).
Methods: NCBI BLAST and TMHMM bioinformatics software were used for protein sequence analysis and prediction of transmembrane domain of TMEM240. Brain tissues from male C57BL/6 mice (18-20 days old) were examined for distribution of TMEM240 using in situ hybridization, and qPCR and Western blotting were used to detect TMEM240 expression in different mouse tissues and in cortical neurons at different time points (n=3). In the in vitro experiment, HepG2 and Neuro-2a cells were transfected with plasmids for overexpression of TMEM240, and subcellular localization of TMEM240 was analyzed using cell imaging. In primary cultures of cortical neurons isolated from C57BL/6 mice, TMEM240 expression and its biological functions were investigated using qPCR, Western blotting, and immunofluorescence staining.
Results: Human and mouse TMEM240 proteins share a 97.69% similarity in the protein sequences, and both are transmembrane proteins with two transmembrane domains. TMEM240 mRNA and protein were highly expressed in mouse brain tissues and cortical neurons. In isolated mouse cortical neurons, TMEM240 expression reached the peak level after primary culture for 9 days and distributed in scattered spots within the cells. In HepG2 cells, TMEM240 was characterized as intracellular membrane structures and showed 80% colocalization with peroxisomes. In Neuro-2a cells, TMEM240 overexpression caused significant enhancement of the expressions of Rho GDP dissociation inhibitor β (ARHGDIB) at both the mRNA and protein levels.
Conclusions: TMEM240 is a novel intracellular subcellular structure specifically expressed in neurons with significant potential for targeted cellular function regulation.
目的: 探究跨膜蛋白240（TMEM240）的亚细胞定位及其生物学功能。方法: 用NCBI的BLAST工具对蛋白质序列分析，TMHMM生物信息学软件对TMEM240跨膜结构进行分析；体内实验：取18~20 d的雄性C57BL/6小鼠脑组织，设置TMEM240探针组和对照组（3只/组），采用原位杂交技术验证TMEM240在脑组织中的表达分布；采用qPCR、Western blotting检测小鼠不同组织、不同时间皮层神经元TMEM240的表达情况（3只/组）；体外实验：采用质粒转染完成Tmem240过表达体系的构建及稳定克隆的筛选，采用细胞成像技术对TMEM240的亚细胞定位进行研究，确定TMEM240在细胞内的形态和分布，采用qPCR，Western blotting，免疫荧光检测TMEM240在细胞内表达情况并验证其生物学功能；结果: 人源和鼠源TMEM240蛋白质序列相似性达97.69%，TMEM240为含有两个跨膜结构的跨膜蛋白；体内实验：TMEM240的mRNA和蛋白在脑组织及神经元中特异性高表达（P<0.001），原代神经元培养9 d时TMEM240表达水平最高（P<0.001）；体外实验：TMEM240在细胞内呈点状分布，属于细胞内膜性结构，并与过氧化物酶体具有80%共定位；过表达Tmem240的Neuro-2a细胞Rho GDP解离抑制因子β（ARHGDIB）的mRNA和蛋白表达均升高（P<0.05）。结论: TMEM240是一种定位于细胞内的新型亚细胞结构，在神经元中特异性表达，在靶向细胞功能调控方面具有巨大的潜力。.
Keywords: Rho GDP dissociation inhibitor β; TMEM240; neurons; peroxisomes; subcellular localization.