受体相互作用蛋白激酶1(RIPK1)调控人结肠癌HT-29细胞程序性坏死的分子机制

 目的  研究受体相互作用蛋白激酶1(receptor interacting protein kinase 1,RIPK1)在人结肠HT-29细胞程序性坏死过程中的作用,并探讨E3泛素连接酶三重结构域包含蛋白16 (tripartite domain containing protein 16,Trim16)对其作用的潜在调控机制。方法  用肿瘤坏死因子α(tumor necrosis factor α,TNFα)建立HT-29细胞的程序性坏死模型,采用Annexin V-FITC/PI双染色法检测其对凋亡、坏死细胞数目的影响。分别用Western blot和qRT-PCR检测RIPK1在细胞程序性坏死中的表达。构建稳定表达Flag标记的RIPK1的HT-29细胞株,采用Flag标记的pulldown试验结合质谱检测发现与RIPK1有相互作用的新蛋白。应用Ni-NTA pulldown试验检测筛选出的E3泛素连接酶对RIPK1泛素化的调控。结果  TNFα能够成功诱导HT-29细胞程序性坏死。HT-29细胞在TNFα和半胱天蛋白酶抑制剂z-VAD处理后,表现出RIPK1、RIPK3、混合系列蛋白激酶样结构域(mixed lineage kinase domain-like protein,MLKL)表达水平显著增加,并伴随着炎性因子白介素1α(IL1α)和白介素6(IL6)水平明显升高。Flag标记的pulldown试验结合质谱检测发现一个与RIPK1有相互作用的E3泛素连接酶Trim16,体外实验表明Trim16可以增强RIPK1的泛素化程度,其可能调节RIPK1在程序性坏死过程中的作用。结论  RIPK1在TNFα和z-VAD诱导人结肠癌HT-29细胞的程序性坏死过程中起重要作用,E3泛素连接酶Trim16对此过程可能有重要调控作用。

Molecular mechanism of programmed necrosis process regulated by receptorinteracting protein kinase 1 (RIPK1) in human colon cancer HT-29 cells

Objective  To examine the effects of receptor interacting protein kinase 1 (RIPK1) during programmed necrotic cell death in human colon cancer HT-29 cells and to explore the potential regulating mechanism of E3 ubiquitin ligase,tripartite domain containing protein 16 (Trim 16),during the process.Methods  Tumor necrosis factor α (TNF- α) was used to establish the programmed necrosis model of HT-29 cells.Annexin V-FITC/PI double staining method was used to observe the necrosis inducing effects of TNF α in human colon cancer HT-29 cells.Western blot analysis and qRT-PCR were applied to detect RIPK1 expression level during the programmed necrosis process.We generated a HT-29 stable cell line expressing flag-tagged RIPK1.Combining Flag-tagged pulldown and mass spectrometry detection,we were able to find new proteins interacting with RIPK1.Ni-NTA pulldown assay was used to explore the possible regulation effects of new interacting E3 ubiquitin ligase.Results   TNF α could successfully induce the programmed necrosis of HT-29 cells.HT-29 cells demonstrated significantly increased RIPK1,RIPK3,MLKL expression in response to TNFα plus z-VAD treatment,which was accompanied by drastically increased IL1α and IL6 expression.Flag-tagged pulldown and mass spectrometry discovered a new RIPK1 interacting protein Trim16 which was also an E3 ubiquitin ligase.In vitro experiments indicated that Trim16 could enhance the ubiquitination of RIPK1,which may function as a regulating mechanism during programmed necrosis.Conclusions   We discovered that mechanically RIPK1 had a fundamental role during programmed necrosis of HT-29 cells induced by TNFα plus 2-VAD,in which E3 ligase Trim16 may have a potential major regulating effects.