【摘要】 目的：探究NLR家族含CARD结构蛋白4(NLRC4)是否存在泛素化修饰，并阐明其分子作用机制。方法：选用高转染效率的HEK293T细胞进行机制初探，应用免疫共沉淀（Co-IP）和镍-NTA亲和沉淀（Ni-NTA pull down）检测NLRC4的泛素化水平；应用亲和纯化技术联合质谱分析筛选与NLRC4相互作用的潜在泛素化调控因子；应用Co-IP、免疫荧光（IF）和邻位连接技术（PLA）验证蛋白的相互作用；通过过表达泛素化调控因子，应用Co-IP实验检测其对NLRC4泛素化水平的影响。结果：NLRC4在HEK293T细胞中存在显著的泛素化修饰；质谱鉴定结果显示E3泛素连接酶自分泌运动因子受体（AMFR）存在于NLRC4纯化的蛋白复合物中；Co-IP、IF及PLA等实验均显示NLRC4与AMFR存在直接相互作用；过表达AMFR可显著增强NLRC4的泛素化水平，泛素化NLRC4占比明显上调（t=5.294,P<0.01）。结论：NLRC4在细胞内存在泛素化修饰，且该过程由E3泛素连接酶AMFR催化。更多还原

【Abstract】 Objective:To investigate whether NLR family CARD domain-containing protein 4(NLRC4)undergoes ubiquitination and elucidate the underlying molecular mechanism. Methods:HEK293T cells with high transfection efficiency were used for initial mechanistic exploration. Ubiquitination levels of NLRC4 were assessed using co-immunoprecipitation(Co-IP) and nickel-NTA affinity precipitation(Ni-NTA pull-down). Affinity purification coupled with mass spectrometry was employed to screen for potential ubiquitination regulators interacting with NLRC4. Protein-protein interactions were validated by Co-IP,immunofluorescence(IF),and proximity ligation assay(PLA). By overexpressing ubiquitination regulatory factors,Co-IP was used to assess their impact on NLRC4 ubiquitination levels. Results:NLRC4 exhibited significant ubiquitination in HEK293T cells. Mass spectrometry analysis identified the E3 ubiquitin ligase autocrine motility factor receptor(AMFR)within the purified NLRC4 protein complex. Co-IP,IF,and PLA consistently demonstrated a direct interaction between NLRC4 and AMFR. Overexpression of AMFR significantly enhanced NLRC4 ubiquitination levels,with the proportion of ubiquitinated NLRC4 markedly increased(t=5.294,P<0.01). Conclusion:NLRC4 undergoes ubiquitination in cells,and this process is catalyzed by the E3 ubiquitin ligase AMFR.更多还原