FBXO22调控凋亡诱导因子的分子机制及其在肿瘤细胞凋亡中的作用

doi:10.3969/j.issn.1674-8115.2021.04.003

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (4): 427-433.doi: 10.3969/j.issn.1674-8115.2021.04.003

FBXO22调控凋亡诱导因子的分子机制及其在肿瘤细胞凋亡中的作用

刘朦迪¹^,²(), 潘漪莲³(), 朱晓娜¹, 杨烁¹, 杨茜¹, 余韵¹()

^1.上海交通大学医学院附属瑞金医院分子医学中心，上海 200025
^2.上海交通大学医学院附属国际和平妇幼保健院生殖遗传科，上海 200030
^3.上海交通大学医学院附属国际和平妇幼保健院产科，上海 200030

收稿日期:2020-07-13 出版日期:2021-04-28 发布日期:2021-05-14
通讯作者: 余韵 E-mail:panda8979@163.com;panyilotus@163.com;yy@shsmu.edu.cn
作者简介:刘朦迪（1989—），女，初级技师，硕士生；电子信箱：panda8979@163.com|潘漪莲（1985—），女，住院医师，硕士；电子信箱：panyilotus@163.com。
基金资助:
国家自然科学基金(81772936);上海交通大学医学院附属国际和平妇幼保健院院级科研基金(GFY5809);上海交通大学医学院高水平地方高校创新团队(SSMU-ZDCX20180800)

Molecular mechanism of apoptosis-inducing factor regulated by FBXO22 and its role in cancer cell apoptosis

Meng-di LIU¹^,²(), Yi-lian PAN³(), Xiao-na ZHU¹, Shuo YANG¹, Qian YANG¹, Yun YU¹()

^1.Molecular Medicine Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^2.Department of Reproductive Genetics, International Peace Maternity & Child Health Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China
^3.Department of Obstetrics, International Peace Maternity & Child Health Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China

Received:2020-07-13 Online:2021-04-28 Published:2021-05-14
Contact: Yun YU E-mail:panda8979@163.com;panyilotus@163.com;yy@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(81772936);Foundation of International Peace Maternity & Child Health Hospital, Shanghai Jiao Tong University School of Medicine(GFY5809);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20180800)

摘要/Abstract

摘要：

目的·挖掘F-box蛋白22（F-box only protein 22，FBXO22）的底物并明确其作用机制，进一步探讨FBXO22在肿瘤细胞凋亡中的作用。方法·利用免疫沉淀联合质谱分析寻找并验证FBXO22的相互作用蛋白凋亡诱导因子（apoptosis-inducing factor，AIF）。采用变性条件下泛素化修饰实验检测FBXO22对AIF泛素化修饰的影响。通过短发夹RNA敲低FBXO22、过表达FBXO22检测AIF的表达水平。利用流式细胞仪检测FBXO22敲低或过表达对结肠癌细胞凋亡的影响。结果·免疫沉淀联合质谱分析显示FBXO22可与AIF结合，变性条件下泛素化修饰实验显示FBXO22可介导AIF的泛素化修饰。敲低FBXO22可上调AIF水平，过表达FBXO22可下调AIF水平。流式细胞术结果显示，敲低FBXO22可促进由甲基硝基亚硝基胍（N-methyl-N'-nitro-N-nitrosoguanidine，MNNG）等诱导的结肠癌细胞凋亡，过表达FBXO22则可抑制MNNG等诱导的结肠癌细胞凋亡。结论·FBXO22通过泛素化修饰降解AIF进而调控由AIF介导的结肠癌细胞凋亡。

关键词: F-box蛋白22, 凋亡诱导因子, 泛素化修饰, 细胞凋亡

Abstract:

Objective·To explore the substrate of F-box only protein 22 (FBXO22) and its mechanism, and further study the role of FBXO22 in cancer cell apoptosis.

Methods·The interaction protein of FBXO22, namely apoptosis-inducing factor (AIF), was searched and identified by immunoprecipitation (IP)-coupled liquid chromatograph mass spectrometer/mass spectrometer (LC-MS/MS) technology. The effect of FBXO22 on ubiquitination of AIF was detected by ubiquitin modification assay under denatured conditions. Short hairpin RNA (shRNA) knockdown FBXO22 and overexpression FBXO22 were used to detect the expression of AIF. The effect of FBXO22 knockdown or overexpression on apoptosis of colon cancer cells was detected by flow cytometry.

Results·IP-coupled LC-MS/MS technology showed that FBXO22 could interact with AIF, and ubiquitin modification assay under denaturing conditions showed that FBXO22 could mediate ubiquitination of AIF. Knockdown of FBXO22 could up-regulate the level of AIF, while overexpression of FBXO22 could down-regulate the level of AIF. The results of flow cytometry showed that knockdown of FBXO22 could promote the apoptosis of colon cancer cells induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), while overexpression of FBXO22 could inhibit the apoptosis of colon cancer cells induced by MNNG.

Conclusion· FBXO22 regulates AIF-mediated apoptosis of colon cancer cells by ubiquitination and degradation of AIF.

Key words: F-box only protein 22 (FBOX22), apoptosis-inducing factor (AIF), ubiquitination modification, cell apoptosis

中图分类号:

R363.1⁺4

刘朦迪, 潘漪莲, 朱晓娜, 杨烁, 杨茜, 余韵. FBXO22调控凋亡诱导因子的分子机制及其在肿瘤细胞凋亡中的作用[J]. 上海交通大学学报(医学版), 2021, 41(4): 427-433.

Meng-di LIU, Yi-lian PAN, Xiao-na ZHU, Shuo YANG, Qian YANG, Yun YU. Molecular mechanism of apoptosis-inducing factor regulated by FBXO22 and its role in cancer cell apoptosis[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(4): 427-433.

图/表 5

图 1 SDS-PAGE及考马斯亮蓝染色检测2组免疫沉淀复合物

Fig 1 Immunoprecipitation complexes of the two groups detected by SDS-PAGE and Coomassie bright blue staining

表 1 质谱鉴定2组差异蛋白的肽段数

Tab 1 Different protein peptide number of the two groups detected by IP/MS technology

Gene	Protein Name	Peptide (FBXO22 group/Control group)
FBXO22	F-box only protein 22	933/1
SKP1	S-phase kinase-associated protein 1	327/0
CUL1	Cullin-1	76/0
RBX1	E3 ubiquitin-protein ligase RBX1	11/0
AIF	Apoptosis-inducing factor 1	9/0

图 2 IP检测FBXO22与AIF的相互作用及其对AIF泛素化水平的影响Note：A. Validation of interaction between exogenous Flag-FBXO22 and endogenous AIF. B/C. Validation of interaction between exogenous Flag-AIF and FBXO22 (B) or FBXO22-ΔF (C). D. Validation of interaction between AIF and SCF complex. E. Effect of FBXO22 or FBXO22-ΔF on ubiquitination of AIF. IP:Flag represents the immune complexes obtained by immunoprecipitation with M2 beads; Input stands for whole cell protein extract.

Fig 2 IP detection of the interaction between FBXO22 and AIF and its effect on the ubiquitination level of AIF

图 3 Western blotting检测FBXO22对AIF水平的影响Note:A. Effect of FBXO22 knockdown on AIF level. B. Effect of FBXO22 or FBXO22-ΔF overexpression on AIF level. C. Expression of AIF in MEF of Fbxo22+/+ and Fbxo22-/- mice. D. Effect of FBXO22 or XIAP knockdown on AIF level in A549 cells. E?G. Effect of FBXO22 knockdown on AIF level in SW620 cells (E), HCT116 cells (F) and PC3 cells (G).

Fig 3 Effect of FBXO22 on AIF level by Western blotting

图 4 FBXO22对AIF介导的肿瘤细胞凋亡的影响Note:A. Apoptosis analysis of SW480 cells (knockdown FBXO22) induced by MNNG and H2O2.B. Apoptosis analysis of SW620 cells (overexpressing FBXO22) induced by MNNG. C. Apoptosis analysis of SW480 cells (overexpressing FBXO22) induced by H2O2. ①P=0.013, ②P=0.004, ③P=0.001, ④P=0.0003, compared with shNC; ⑤P=0.023, ⑥P=0.009, compared with PLVX-ZSGreen-EV.

Fig 4 Effect of FBXO22 on cancer cell apoptosis mediated by AIF

参考文献 27

1	Nandi D, Tahiliani P, Kumar A, et al. The ubiquitin-proteasome system[J]. J Biosci, 2006, 31(1): 137-155.
2	Tang Q, Wu P, Chen H, et al. Pleiotropic roles of the ubiquitin-proteasome system during viral propagation[J]. Life Sci, 2018, 207: 350-354.
3	Kitagawa K, Kitagawa M. The SCF-type E3 ubiquitin ligases as cancer targets[J]. Curr Cancer Drug Targets, 2016, 16(2): 119-129.
4	Jin J, Cardozo T, Lovering RC, et al. Systematic analysis and nomenclature of mammalian F-box proteins[J]. Genes Dev, 2004, 18(21): 2573-2580.
5	Vrba L, Junk DJ, Novak P, et al. p53 induces distinct epigenetic states at its direct target promoters[J]. BMC Genomics, 2008, 9: 486.
6	Johmura Y, Sun J, Kitagawa K, et al. SCF(Fbxo22)-KDM4A targets methylated p53 for degradation and regulates senescence[J]. Nat Commun, 2016, 7: 10574.
7	Tian X, Dai S, Sun J, et al. F-box protein FBXO22 mediates polyubiquitination and degradation of KLF4 to promote hepatocellular carcinoma progression[J]. Oncotarget, 2015, 6(26): 22767-22775.
8	Sun R, Xie HY, Qian JX, et al. FBXO22 possesses both protumorigenic and antimetastatic roles in breast cancer progression[J]. Cancer Res, 2018, 78(18): 5274-5286.
9	Bai J, Wu K, Cao M, et al. SCF FBXO22 targets HDM2 for degradation and modulates breast cancer cell invasion and metastasis[J]. Proceedings of the National Academy of Sciences, 2019, 116(10):201820990.
10	Guo F, Liu J, Han X, et al. FBXO22 suppresses metastasis in human renal cell carcinoma via inhibiting MMP-9-mediated migration and invasion and VEGF-mediated angiogenesis[J]. Int J Biol Sci, 2019, 15(3): 647-656.
11	Lignitto L, LeBoeuf SE, Homer H, et al. Nrf2 activation promotes lung cancer metastasis by inhibiting the degradation of Bach1[J]. Cell, 2019, 178(2): 316-329.e18.
12	Zhu XN, He P, Zhang L, et al. FBXO22 mediates polyubiquitination and inactivation of LKB1 to promote lung cancer cell growth[J]. Cell Death Dis, 2019, 10(7): 486.
13	Ge MK, Zhang N, Xia L, et al. FBXO22 degrades nuclear PTEN to promote tumorigenesis[J]. Nat Commun, 2020, 11(1): 1720.
14	Moubarak RS, Yuste VJ, Artus C, et al. Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis[J]. Mol Cell Biol, 2007, 27(13): 4844-4862.
15	Yang H, Xie Y, Yang D, et al. Oxidative stress-induced apoptosis in granulosa cells involves JNK, p53 and Puma[J]. Oncotarget, 2017, 8(15): 25310-25322.
16	Jang KH, Do YJ, Son D, et al. AIF-independent parthanatos in the pathogenesis of dry age-related macular degeneration[J]. Cell Death Dis, 2017, 8(1): e2526.
17	Joza N, Pospisilik JA, Hangen E, et al. AIF: not just an apoptosis-inducing factor[J]. Ann N Y Acad Sci, 2009, 1171: 2-11.
18	Sevrioukova IF. Apoptosis-inducing factor: structure, function, and redox regulation[J]. Antioxid Redox Signal, 2011, 14(12): 2545-2579.
19	Lewis EM, Wilkinson AS, Davis NY, et al. Nondegradative ubiquitination of apoptosis inducing factor (AIF) by X-linked inhibitor of apoptosis at a residue critical for AIF-mediated chromatin degradation[J]. Biochemistry, 2011, 50(51): 11084-11096.
20	Wilkinson JC, Wilkinson AS, Galbán S, et al. Apoptosis-inducing factor is a target for ubiquitination through interaction with XIAP[J]. Mol Cell Biol, 2008, 28(1): 237-247.
21	Shen SM, Yu Y, Wu YL, et al. Downregulation of ANP32B, a novel substrate of caspase-3, enhances caspase-3 activation and apoptosis induction in myeloid leukemic cells[J]. Carcinogenesis, 2010, 31(3): 419-426.
22	Shen SM, Yu Y, Wu ZX, et al. Apoptosis-inducing factor is a target gene of C/EBPα and participates in adipocyte differentiation[J]. FEBS Lett, 2011, 585(14): 2307-2312.
23	Stambolsky P, Weisz L, Shats I, et al. Regulation of AIF expression by p53[J]. Cell Death Differ, 2006, 13(12): 2140-2149.
24	Pospisilik JA, Knauf C, Joza N, et al. Targeted deletion of AIF decreases mitochondrial oxidative phosphorylation and protects from obesity and diabetes[J]. Cell, 2007, 131(3): 476-491.
25	Vahsen N, Candé C, Brière JJ, et al. AIF deficiency compromises oxidative phosphorylation[J]. EMBO J, 2004, 23(23): 4679-4689.
26	Hangen E, Féraud O, Lachkar S, et al. Interaction between AIF and CHCHD4 regulates respiratory chain biogenesis[J]. Mol Cell, 2015, 58(6): 1001-1014.
27	Shen SM, Guo M, Xiong Z, et al. AIF inhibits tumor metastasis by protecting PTEN from oxidation[J]. EMBO Rep, 2015, 16(11): 1563-1580.

[1]	刘景景, 胡慧洁, 刘子楷, 宋明柯. 钠钙交换体阻滞剂CB-DMB对人胶质母细胞瘤细胞生长的抑制作用[J]. 上海交通大学学报(医学版), 2021, 41(6): 710-716.
[2]	熊强强, 屠俊, 李郡如, 程金科, 左建宏, 陈亚兰. 蛋白质SUMO化修饰的蛋白质组学研究述评[J]. 上海交通大学学报(医学版), 2021, 41(1): 89-94.
[3]	陈巍，韩峥，邹艳丽，黄莎莎，田霞. 溃疡性结肠炎小鼠血清miR-23a-3p和miR-27a-3p的表达水平及其作用的研究[J]. 上海交通大学学报(医学版), 2020, 40(08): 1069-1074.
[4]	阮昕 1，张颖婷 1，韩可琪 1，林龙帅 2，陈晨 1，岳铭 1，王楚翘 1，孙英刚 3，赵庆华 2，贺明 1. SIRT7通过抑制内质网应激蛋白 GRP78减轻脂多糖或 D-氨基半乳糖 /脂多糖诱导的肝细胞凋亡[J]. 上海交通大学学报（医学版）, 2019, 39(8): 812-.
[5]	蒋云凤，程燕咏，孙宇. 长链非编码 RNA Peg13在七氟烷所致的发育期原代神经元凋亡中的作用[J]. 上海交通大学学报（医学版）, 2019, 39(5): 469-.
[6]	金珂，金义超，吕涛，张封臣，张晓华. 丙酮酸乙酯对蛛网膜下腔出血后延迟性脑血管痉挛的治疗作用及机制[J]. 上海交通大学学报（医学版）, 2017, 37(12): 1601-.
[7]	杨晓晓,黄虹婷，刘满华,黄莺 . NF-κB参与调控细胞凋亡[J]. 上海交通大学学报（医学版）, 2017, 37(10): 1446-.
[8]	宋菲，原博 . 脂肪干细胞来源的条件培养基对TGF-β1共同干预下的真皮成纤维细胞致纤维化能力的影响[J]. 上海交通大学学报（医学版）, 2017, 37(05): 588-.
[9]	肖新华，俞淼，吴云昭，吴英理，刘玮 . 腺花素对多发性骨髓瘤细胞的生物学效应及其机制研究[J]. 上海交通大学学报（医学版）, 2017, 37(04): 427-.
[10]	牛伟，周波，吴萍，陈舜杰，蒋更如，张翀 . 家族性高血钾型高血压Cullin3致病突变体neddylation异常的分子机制研究[J]. 上海交通大学学报（医学版）, 2016, 36(10): 1420-.
[11]	张振洲徐冉常青徐颖乐金海燕陈来江宋蓓钟久昌. 重组血管紧张素转换酶2对ApoE基因缺陷小鼠肾脏caspase信号及细胞凋亡的影响[J]. 上海交通大学学报（医学版）, 2016, 36(08): 1115-.
[12]	付宗杰，凌媛，缪婕，杨国源，沈琳辉. 脂联素对骨髓间充质干细胞增殖和凋亡的影响[J]. 上海交通大学学报（医学版）, 2016, 36(05): 631-.
[13]	刘申吒，韩国虎，周月鹏，毛朝明，陈德玉. EGCG协同放射治疗诱导食管鳞癌TE-1细胞凋亡机制的研究[J]. 上海交通大学学报（医学版）, 2016, 36(04): 487-.
[14]	刘畅，陈晓，李诚，等. 神经干细胞移植对大鼠脊髓损伤后Caspase-3和c-fos表达的影响[J]. 上海交通大学学报（医学版）, 2015, 35(3): 343-.
[15]	王新林，耿直. 匹罗卡品致大鼠癫痫持续状态后海马组织中Puma和CytC的表达变化[J]. 上海交通大学学报（医学版）, 2015, 35(2): 193-.

FBXO22调控凋亡诱导因子的分子机制及其在肿瘤细胞凋亡中的作用

Molecular mechanism of apoptosis-inducing factor regulated by FBXO22 and its role in cancer cell apoptosis

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 27

相关文章 15

编辑推荐

Metrics