中国临床药理学杂志2025，Vol.41Issue(9)：1229-1235,7.DOI:10.13699/j.cnki.1001-6821.2025.09.006
代谢酶PGK1琥珀酰化修饰位点突变载体构建及其在HEK-293T细胞中表达的研究
Research on the construction of metabolizing enzyme PGK1 succinylation modification site mutation vector and its expression in HEK-293T cells
摘要
Abstract
Objective To achieve overexpression of phosphoglycerate kinase 1(PGK1)in HEK-293T cells and screen the key site of its succinylation through constructing PGK1 overexpression vector(PGK1-WT)and four lysine site succinylation mutation vectors.Methods GEPIA2 database was used to analyze the expression of PGK1 mRNA in normal breast tissue(291 cases)and breast cancer tissue(135 cases)and the relationship between PGK1 expression level and the overall survival rate of breast cancer patients.Protein modification proteomics was used to detect the levels of protein succinylation in four triple negative breast cancer(TNBC)cell lines(MDA-MB-436,MDA-MB-468,MDA-MB-231,BT-549)and normal breast epithelial cells(MCF-10A);the succinylated positive and negative mutation vectors of K11,K131,K323,K361 were contructed with point mutation kit.The experiment was divided into NC,shPGK1-496,shPGK1-220,PGK1-WT groups and positive and negative mutant groups of K11,K131,K323 and K361.Real-time fluorescence quantitative polymerase chain reaction was used to detect the level of expression of PGK1 mRNA in NC,shPGK1-496 and shPGK1-220 groups.The expression levels of PGK1 proteins in NC,shPGK1-496 and shPGK1-220 groups,and the expression levels of Flag proteins in NC,PGK1-WT and four sites in positive and negative mutant groups were detected by Western blot.The key site of PGK1 succinylation was detected by co-immunoprecipitation.Results Compared with normal breast tissues,the level of PGK1 mRNA was highly expressed in breast cancer tissue,and the survival time of breast cancer patients with high PGK1 expression was shorter.Four lysine sites of PGK1 in TNBC cells were hypersuccinylated.The relative expression levels of PGK1 mRNA in NC,shPGK1-496 and shPGK1-220 groups were 1.03±0.23,0.15±0.11 and 1.42±0.69,respectively;the relative expression levels of PGK1 protein were 1.08±0.05,0.49±0.05 and 1.28±0.01 respectively;compared with NC group,the above indexes in shPGK1-496 group were statistically different(all P＜0.01).The relative expression levels of Flag protein in NC group,PGK1-WT group and positive and negative mutant groups of K11,K131,K323 site were 0,3.43±0.09,3.28±0.10,2.28±0.11,3.03±0.03,2.79±0.11,3.29±0.09 and 2.98±0.04,respectively;compared with the NC group,PGK1-WT group and the positive and negative mutant groups of K11,K131 and K323 had statistical differences(all P＜0.01).The relative expression levels of Flag protein in NC group,PGK1-WT group and K361 positive and negative mutant group were 0,2.58±0.11,3.04±0.02 and 4.04±0.10,respectively;compared with NC group,PGK1-WT group and K361 positive and negative mutant group had statistical significance(all P＜0.01).The levels of lysine succinylation in IgG group,PGK1-WT group and negative mutant groups of K11,K131,K323,K361 were 0,1.53±0.12,1.30±0.18,1.29±0.09,1.23±0.03 and 0.89±0.16,respectively;compared with PGK1-WT group,the negativ mutant group of K361 had statistical significance(P＜0.01).Conclusion The four lysine sites of PGK1 in TNBC cells are highly succinylated,and K361 site is critical to their succinylation.关键词
磷酸甘油酸激酶1/琥珀酰化/突变体/翻译后修饰Key words
phosphoglycerate kinase 1/succinylation/mutant/post-translational modification分类
医药卫生引用本文复制引用
王瑶瑶,刘立琨,高秀丽,朱文斌,郝经伟,岳丽玲..代谢酶PGK1琥珀酰化修饰位点突变载体构建及其在HEK-293T细胞中表达的研究[J].中国临床药理学杂志,2025,41(9):1229-1235,7.基金项目
国家自然科学基金资助项目(81972491) （81972491）
黑龙江省自然科学基金资助项目(H2022H108) （H2022H108）
黑龙江省博士后面上基金资助项目(LBH-Z22295) （LBH-Z22295）
齐齐哈尔医学院研究生创新基金资助项目(QYYCX2022-17) （QYYCX2022-17）
