肿瘤坏死因子受体相关因子6泛素化位点突变载体的构建及其功能位泛素化位点的鉴定

doi:10.13481/j.1671-587X.20210303

Abstract

Abstract: Objective

To predict the ubiquitination sites of human tumor necrosis factor receptor-associated factor 6（TRAF6） gene and construct the ubiquitination mutant plasmid， and to explore the effect of mutant plasmid on the relative luciferase activity of nuclear factor kappa-B（NF-κB）and activator protein-1（AP-1） in the human colorectal cancer HCT116 and SW480 cells.

Methods

UbPred， UbiSite， and BDM-PUB softwares were used to predict the ubiquitination sites of TRAF6 gene；the mutation primers were designed by CE Design V1.04 software， and the mutation kits were used for site-directed mutation； the mutated target fragment was amplified by PCR method；the amplified products were digested by Dpnl enzyme to remove the methylated template plasmids；the digested products were recombined under the catalysis of Exnase Ⅱ to obtain the recombinant plasmids；the recombinant plasmids were transformed into the competent cells of DH5α E. coli and the sequence was sequenced. The relative luciferase activities of NF-κB and AP-1 in the colorectal cancer HCT116 and SW480 cells were detected by dual-luciferase reporter gene system.

Results

After DNA sequencing， the ubiquitination mutation site was successfully mutated， and the ubiquitination mutant plasmid was successfully constructed. Compared with TRAF6 wild-type gene strain， the relative luciferase activities of NF-κB and AP-1 in the colorectal cancer HCT116 and SW480 cells were decreased after transfected with 124mut， 319mut， and 331mut plasmids （P<0.05 or P<0.01）， and the relative luciferase activities of NF-κB and AP-1 in the colorectal cancer HCT116 and SW480 cells were the most significantly decreased after transfected with 124mut plasmid（P<0.01）.

Conclusion

The ubiquitination mutant plasmids are successfully constructed.The 124th amino acid of TRAF6 is the most important ubiquitination site， which may affect the activities of NF-κB and AP-1 factors in downstream signaling pathways.

Key words: tumor necrosis factor receptor-associated factor 6, mutant plasmid construction, ubiquitination, luciferase reporter system, nuclear factor kappa-B activator protein

CLC Number:

R735.3

Qin WANG, Chunlin LIN, Zhibin CHENG, Ruofan HE, Penghang LIN, Hui CHEN, Jianxin YE, Guangwei ZHU. Construction of TRAF6 ubiquitination site mutation vectors and identification of its functional ubiquitination sites[J].Journal of Jilin University(Medicine Edition), 2021, 47(3): 551-558.

Figures/Tables 6

Tab. 1

Fig. 1

Fig.2

Fig. 3

Fig. 4

Tab. 2

References

1	SIEGEL R L， MILLER K D， FEDEWA S A， et al. Colorectal cancer statistics， 2017［J］. CA Cancer J Clin， 2017， 67（3）： 177-193.
2	WILSON C， FLIGHT I， ZAJAC I T， et al. Web-based communication strategies designed to improve intention to minimize risk for colorectal cancer： randomized controlled trial［J］. JMIR Cancer， 2018， 4（1）： e2-e9.
3	ZHU G W， CHENG Z B， HUANG Y J， et al. TRAF₆ promotes the progression and growth of colorectal cancer through nuclear shuttle regulation NF-kB/c-Jun signaling pathway［J］. Life Sci， 2019， 235： 116831.
4	HA H， HAN D， CHOI Y. TRAF-mediated TNFR-family signaling［J］. Curr Protoc Immunol， 2009， Chapter 11： Unit11.9D.
5	CAO Z， XIONG J， TAKEUCHI M， et al. TRAF₆ is a signal transducer for interleukin-1［J］. Nature， 1996， 383（6599）： 443-446.
6	ROTHE M， WONG S C， HENZEL W J， et al. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor［J］. Cell， 1994， 78（4）： 681-692.
7	ISHIDA T， MIZUSHIMA S I， AZUMA S， et al. Identification of TRAF6， a novel tumor necrosis factor receptor-associated factor protein that mediates signaling from an amino-terminal domain of the CD40 cytoplasmic region［J］. J Biol Chem， 1996， 271（46）： 28745-28748.
8	李妙，周立军. TRAF6与肿瘤关系的研究进展［J］. 生物技术通报， 2017， 33（6）： 24-31.
9	SUN H， LI X B， FAN L， et al. TRAF₆ is upregulated in colon cancer and promotes proliferation of colon cancer cells［J］. Int J Biochem Cell Biol， 2014， 53： 195-201.
10	DENG L， WANG C， SPENCER E， et al. Activation of the IkappaB kinase complex by TRAF₆ requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain［J］. Cell， 2000， 103（2）： 351-361.
11	SHI J H， SUN S C. Tumor necrosis factor receptor-associated factor regulation of nuclear factor κB and mitogen-activated protein kinase pathways［J］. Front Immunol， 2018， 9： 1849.
12	SHEN H Y， LI L P， YANG S J， et al. Regulatory role of tumor necrosis factor receptor-associated factor 6 in breast cancer by activating the protein kinase B/glycogen synthase kinase 3β signaling pathway［J］. Mol Med Rep， 2017， 16（2）： 2269-2273.
13	MENG Q B， ZHANG W S， XU X L， et al. The effects of TRAF₆ on proliferation， apoptosis and invasion in osteosarcoma are regulated by miR-124［J］. Int J Mol Med， 2018， 41（5）： 2968-2976.
14	华进，程志彬，林春霖，等. 一种高效稳定的磷酸钙转染293T细胞方法的建立及评价［J］. 吉林大学学报（医学版）， 2019， 45（5）： 1177-1181，1198.
15	LIU F，KJUALTE R S.Multitasking with ubiquitin through multivalent interactions［J］.Trends Biochem Sci，2010，35（6）：352-360.
16	HERRMANN J， LERMAN L O， LERMAN A. Ubiquitin and ubiquitin-like proteins in protein regulation［J］. Circ Res， 2007， 100（9）： 1276-1291.
17	SCHWARTZ D C， HOCHSTRASSER M. A superfamily of protein tags： ubiquitin， SUMO and related modifiers［J］.Trends Biochem Sci，2003，28（6）：321-328.
18	MURATANI M， TANSEY W P. How the ubiquitin-proteasome system controls transcription［J］. Nat Rev Mol Cell Biol， 2003， 4（3）： 192-201.
19	MOCCIARO A， RAPE M. Emerging regulatory mechanisms in ubiquitin-dependent cell cycle control［J］. J Cell Sci， 2012， 125（Pt 2）： 255-263.
20	TAN G，QIU M，CHEN L，et al.JS-K，a nitric oxide pro-drug regulates growth and apoptosis through the ubiquitin-proteasome pathway in prostate cancer cells［J］.BMC Cancer，2017，17（1）：367-376.
21	YANG Y， YIN X T， YANG H R， et al. Histone demethylase LSD2 Acts as an E3 ubiquitin ligase and inhibits cancer cell growth through promoting proteasomal degradation of OGT［J］. Mol Cell， 2015， 58（1）： 47-59.
22	FUKUNAGA E， INOUE Y， KOMIYA S， et al. Smurf2 induces ubiquitin-dependent degradation of Smurf1 to prevent migration of breast cancer cells［J］. J Biol Chem， 2008， 283（51）： 35660-35667.
23	MANSOUR M A. Ubiquitination： Friend and foe in cancer［J］. Int J Biochem Cell Biol， 2018， 101： 80-93.
24	陈子琦. 蛋白质泛素化修饰与肿瘤的研究进展［J］. 科学咨询（科技·管理）， 2020（1）： 123-124.
25	HAN C J， YAN P J， HE T， et al. PHLDA1 promotes microglia-mediated neuroinflammation via regulating K63-linked ubiquitination of TRAF₆［J］. Brain Behav Immun， 2020， 88： 640-653.
26	LAMOTHE B，CAMPOS A D，WEBSTER W K，et al.The RING domain and first zinc finger of TRAF₆ coordinate signaling by interleukin-1， lipopolysaccharide， and RANKL［J］. J Biol Chem， 2008， 283（36）： 24871-24880.
27	LOMAGA M A， YEH W C， SAROSI I， et al. TRAF₆ deficiency results in osteopetrosis and defective interleukin-1， CD40， and LPS signaling［J］. Genes Dev， 1999， 13（8）： 1015-1024.
28	SUN L J， DENG L， EA C K， et al. The TRAF₆ ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes［J］. Mol Cell， 2004， 14（3）： 289-301.
29	WU H， LI X M， WANG J R， et al. NUR77 exerts a protective effect against inflammatory bowel disease by negatively regulating the TRAF6/TLR-IL-1R signalling axis［J］. J Pathol， 2016， 238（3）： 457-469.
30	WANG Y Y， TANG Y W， TENG L， et al. Association of beta-arrestin and TRAF₆ negatively regulates Toll-like receptor-interleukin 1 receptor signaling［J］. Nat Immunol， 2006， 7（2）： 139-147.
31	SHAO J H， DING Z R， PENG J H， et al. MiR-146a-5p promotes IL-1β-induced chondrocyte apoptosis through the TRAF6-mediated NF-kB pathway［J］. Inflamm Res， 2020， 69（6）： 619-630.
32	XU C F， CHONG L， YU G， et al. MiR-574-5p alleviates sepsis-induced acute lung injury by regulating TRAF6/NF-κB pathway［J］. Trop J Pharm Res， 2020， 19（4）： 676-682.
33	MENG Q C， LIANG C， HUA J， et al. A miR-146a-5p/TRAF6/NF-kB p65 axis regulates pancreatic cancer chemoresistance： functional validation and clinical significance［J］. Theranostics， 2020， 10（9）： 3967-3979.
34	STARCZYNOWSKT D T W W，LOCKWOOD S，DELEHOUZ E E，al et，TRAF6is an amplified oncogene bridging the RAS and NF-kappa B pathways in human lung cancer［J］.J Clin Invest，2011，121（10）：4095-4105.

Primer	Primer sequence (5'－3')
32mut-F	CGCTGTAACAgAAGATGATAGTGTGGGTGGAACTG
32mut-R	CATCTTcTGTTACAGCGCTACAGGAGCTGGCC
91mut-F	TAGGTTCTGCgAAGCCTGCATCATAAAATCAATAAGG
91mut-R	AGGCTTcGCAGAACCTATGGCCGCATGGCGTT
124mut-F	TGCAgAACGTGAGATTCTTTCTCTGATGGTGA
124mut-R	GAATCTCACGTTcTGCAAAATTGTCTGGAAATAGTTGA
319mut-F	CTGACTGCTgAAATGGAAACTCAGAGTATGTATGTAAGTG
319mut-R	TCCATTTcAGCAGTCAGCTCCCGGATTTGATG
331mut-F	AGTGAGCTCgAACGAACCATTCGAACCCTTGA
331mut-R	GTTCGTTcGAGCTCACTTACATACATACTCTGAGTTTC
371mut-F	AAGAGGAGgAACCTGTTGTGATTCATAGCCCTG
371mut-R	AACAGGTTcCTCCTCTTCTTGACATTTCAAATGC
384mut-F	TTCTACACTGGCgAACCCGGGTACAAACTGTGCA
384mut-R	GGTTcGCCAGTGTAGAATCCAGGGCTATGAAT
469mut-F	GGAACCCAgAAGGTTTTGGCTATGTAACTTTTATGC
469mut-R	AAAACCTTcTGGGTTCCGTGGGATTGTGGGTC

Plasmid	NF-κB		AP-1
Plasmid	HCT116	SW480	HCT116	SW480
pcDNA3.1-3×FLAG-TRAF6	1	1	1	1
pcDNA3.1-3×FLAG-TRAF6 32mut	1.01±0.08	1.04±0.11	1.07±0.05	0.99±0.16
pcDNA3.1-3×FLAG-TRAF6 91mut	0.99±0.09	1.09±0.10	0.88±0.12	0.74±0.11
pcDNA3.1-3×FLAG-TRAF6 124mut	0.34±0.02^**	0.25±0.14^**	0.27±0.01^**	0.33±0.07^**
pcDNA3.1-3×FLAG-TRAF6 319mut	0.63±0.08^**	0.32±0.02^**	0.50±0.07^**	0.44±0.15^**
pcDNA3.1-3×FLAG-TRAF6 331mut	0.68±0.05^*	0.54±0.03^*	0.60±0.04^**	0.54±0.01^*
pcDNA3.1-3×FLAG-TRAF6 371mut	1.15±0.01	0.96±0.16	1.05±0.01	0.99±0.21
pcDNA3.1-3×FLAG-TRAF6 384mut	1.10±0.09	0.98±0.03	1.14±0.10	1.01±0.16
pcDNA3.1-3×FLAG-TRAF6 460mut	0.99±0.09	1.01±0.11	0.99±0.02	0.91±0.10

Construction of TRAF6 ubiquitination site mutation vectors and identification of its functional ubiquitination sites

RichHTML

PDF (PC)

Like

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 6

References

Related Articles 1

Metrics

Comments

Recommended 0