中国科学院生物物理研究所
国家 “十一五”传染病重大专项资助项目(2008ZX10002-008)
INSTITUTE of BIOPHYSICS,CHINESE ACADEMY OF SCIENCE
This work was supported by a grant from National Major Science and Technology Special Project of China (2008ZX10002-008)
选取功能未知的泛素连接酶RNF149作为研究对象.该分子同GRAIL(gene related to anergy in lymphocytes)具有较高的同源性,属于Ⅰ型跨膜蛋白.激光共聚焦显微镜的观察结果显示,RNF149是一个定位在溶酶体上的蛋白质,它与CD9在细胞内有共定位.免疫共沉淀实验证明RNF149与CD9有相互作用.RNF149通过泛素分子的第48位赖氨酸多泛素化CD9.在HeLa细胞中转染数量一定的CD9质粒和数量梯度增加的RNF149质粒24 h后,蛋白质印迹检测外源RNF149和CD9的表达,结果表明,随外源RNF149表达量梯度增高,外源CD9的表达量梯度降低.在HEK293T细胞内以shRNA敲低内源的RNF149,并检测内源CD9的变化,发现RNF149被敲低后内源CD9的量增多.上述结果提示,CD9很有可能是RNF149的底物,被RNF149通过泛素化降解.此外,RNF149被敲低的HEK293T细胞增殖受到抑制,这可能与其内源CD9的量增多有关,提示RNF149可能是一种细胞增殖的调控因子.
Our research selected the RNF149, the novel ubiquitin ligase which owned the high identity to GRAIL and belonged to the typeⅠ transmembrane protein, as our object. By the confocal laser scanning microscope, it was demonstrated that RNF149 is located at lysosome and the RNF149 is co-located with CD9. The interactions between RNF149 and CD9 were demonstrated by immune co-precipitation.RNF149 polyubiquitinates CD9 via ubiquitin Lys-48. The HeLa cells were co-transfected with the same quantity of the CD9 plasmids and the gradient increase quantity of the RNF149 plasmids. We found that the exogenous quantity of CD9 was decreasing with the increased expression of the exogenous RNF149. In HEK293T cells, the knocking down RNF149 by shRNA led to the increase of the endogenous CD9. All these evidence suggested that CD9 maybe regulated by RNF149. In addition, the knocking down RNF149 by shRNA led to the inhibition of the cell proliferation in HEK293T cells. This phenomenon suggested that the RNF149 possibly could be considered as the regulatory factor of the cell proliferation.
李燕,阮林浩,索塔林,王 鹏,唐 捷. RNF149通过泛素化介导的CD9降解调控细胞增殖[J].生物化学与生物物理进展,2013,40(12):1230-1238
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