多巴胺通过促进RNF20降解抑制神经细胞中组蛋白H2B泛素化
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摘要
表观遗传修饰参与了药物成瘾的形成过程,而在药物成瘾过程中组蛋白泛素化水平的变化仍未可知。药物成瘾过程中常表现为多巴胺(dopamine, DA)表达量的升高,因此本研究欲探讨多巴胺升高对神经细胞组蛋白泛素化的影响及其机制。Western印迹结果显示,在终浓度0.8 mmol/L的多巴胺作用8 h后,人神经母细胞瘤细胞系SH-SY5Y细胞中环指蛋白20(ring finger protein 20, RNF20)表达量降低(0.29±0.032 vs. 1.0±0.025,P<0.0001),泛素化组蛋白H2B(H2Bub1)表达量下降(0.28±0.032 vs. 1.0±0.017,P<0.0001)。但是RT-PCR结果显示,多巴胺处理SH-SY5Y细胞后,RNF20在mRNA水平的表达无明显变化。在SH-SY5Y细胞中沉默RNF20的表达,H2Bub1在蛋白质水平的表达明显降低(0.20±0.069 vs. 1.0±0.060,P=0.001)。在加入多巴胺的基础上,分别加入蛋白酶体抑制剂MG132、自噬体形成抑制剂3-MA以及空泡型H~+-ATP酶特异性抑制剂Baf-A1等药物来检测RNF20的降解途径,结果发现,加入MG132、3-MA以及Baf-A1后,RNF20表达量均比DA处理组显著上升(1.51±0.095,P=0.0003; 0.89±0.075,P=0.0021; 2.74±0.099,P<0.0001;vs. 0.27±0.044)。上述结果表明,在SH-SY5Y细胞中,RNF20对H2Bub1具有调控作用,多巴胺可通过泛素化及自噬两种途径促进RNF20降解,从而抑制组蛋白H2B泛素化。
Epigenetic modification is involved in the formation of drug addiction, however, it is still unclear whether the level of histone ubiquitination is affected in the process of drug addiction. In the process of drug addiction, the production of dopamine is often increased, but the effect of dopamine on histone ubiquitination remains unknown. This study focused on the effect of dopamine on histone ubiquitination during drug addiction. Western blot showed that after treatment of 0.8 mmol/L dopamine for 8 h, the production of ring finger protein 20(RNF20) in SH-SY5 Y human neuroblastoma cells was decreased(0.29 ± 0.032 vs. 1.0 ± 0.025, P< 0.0001),and the production of ubiquitinated histone H2 B(H2 Bub1) also decreased(0.28 ± 0.032 vs. 1.0 ± 0.017, P< 0.0001). However, RT-PCR results showed that there were no significant differences in mRNA levels of RNF20 expression after SH-SY5 Y cells were treated with dopamine. Silence of RNF20, significantly decreased the production of H2 Bub1 at the protein level in SH-SY5 Y cells(0.2 ± 0.069 vs. 1.0 ± 0.060, P =0.001). Combined with dopamine, proteasome inhibitor MG132, autophagy inhibitor 3-MA, or vacuolar H~+-ATPase specific inhibitor Baf-A1 were added respectively to detect the degradation of RNF20. The results showed that the production of RNF20 increased significantly in groups treated with DA combined with MG132, 3-MA and Baf-A1 as compared with those treated only with DA(1.51 ± 0.095, P=0.0003; 0.89 ± 0.075, P=0.0021; 2.74 ± 0.099, P< 0.0001 vs. 0.27 ± 0.044). These results indicated that dopamine inhibited histone H2 B ubiquitination by degrading RNF20 through ubiquitination and autophagy in SH-SY5 Y cells.
Epigenetic modification is involved in the formation of drug addiction, however, it is still unclear whether the level of histone ubiquitination is affected in the process of drug addiction. In the process of drug addiction, the production of dopamine is often increased, but the effect of dopamine on histone ubiquitination remains unknown. This study focused on the effect of dopamine on histone ubiquitination during drug addiction. Western blot showed that after treatment of 0.8 mmol/L dopamine for 8 h, the production of ring finger protein 20(RNF20) in SH-SY5 Y human neuroblastoma cells was decreased(0.29 ± 0.032 vs. 1.0 ± 0.025, P< 0.0001),and the production of ubiquitinated histone H2 B(H2 Bub1) also decreased(0.28 ± 0.032 vs. 1.0 ± 0.017, P< 0.0001). However, RT-PCR results showed that there were no significant differences in mRNA levels of RNF20 expression after SH-SY5 Y cells were treated with dopamine. Silence of RNF20, significantly decreased the production of H2 Bub1 at the protein level in SH-SY5 Y cells(0.2 ± 0.069 vs. 1.0 ± 0.060, P =0.001). Combined with dopamine, proteasome inhibitor MG132, autophagy inhibitor 3-MA, or vacuolar H~+-ATPase specific inhibitor Baf-A1 were added respectively to detect the degradation of RNF20. The results showed that the production of RNF20 increased significantly in groups treated with DA combined with MG132, 3-MA and Baf-A1 as compared with those treated only with DA(1.51 ± 0.095, P=0.0003; 0.89 ± 0.075, P=0.0021; 2.74 ± 0.099, P< 0.0001 vs. 0.27 ± 0.044). These results indicated that dopamine inhibited histone H2 B ubiquitination by degrading RNF20 through ubiquitination and autophagy in SH-SY5 Y cells.
引文
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[14] Alhamwe BA,Khalaila R,Wolf J,et al.Histone modifications and their role in epigenetics of atopy and allergic diseases [J].Allergy Asthma Clin Immunol,2018,14:39
[15] Eric I C,Danny R.Histones:Annotating chromatin [J].Annu Rev Genet,2009,43(1):559
[16] Sadakierska-Chudy A,Frankowska M,Jastrz?bska J,et al.Cocaine administration and its withdrawal enhance the expression of genes encoding histone-modifying enzymes and histone acetylation in the rat prefrontal cortex [J].Neurotox Res,2017,32(1):141-150
[17] Maze I,Feng J,Wilkinson MB,et al.Cocaine dynamically regulates heterochromatin and repetitive element unsilencing in nucleus accumbens [J].Proc Natl Acad Sci U S A,2011,108(7):3035-3040
[18] Rotllant D,Armario A.Brain pattern of histone H3 phosphorylation after acute amphetamine administration:its relationship to brain c-fos induction is strongly dependent on the particular brain area [J].Neuropharmacology,2012,62(2):1073-1081
[19] 陈科,程汉华,周荣家.自噬与泛素化蛋白降解途径的分子机制及其功能 [J].遗传(Chen K,Cheng HH,Zhou RJ.Molecular mechanisms and functions of autophagy and the ubiquitin-proteasome pathway[J].Hereditas),2012,34(1):5-18
[20] 李翔,白洁.多巴胺在可卡因成瘾中作用 [J].中国老年学(Li X,Bai J.The function of dopamine in cocaine addiction[J].Chin J Gerontol),2015,35(15):4372-4374
[2] Dehnes Y,Shan J,Beuming T,et al.Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation [J].Neurochem Int,2014,73:4-15
[3] Gawin FH.Cocaine addiction:psychology and neurophysiology [J].Science,1991,251(5001):1580-1586
[4] 王翔,魏潇凡,张宏权.泛素化的功能及其意义 [J].中国科学:生命科学(Wang X,Wei XF,Zhang HQ.Role of protein ubiquitination and its functional importance[J].Sci Sin Vitae),2015,45(11):1074-1082
[5] Shaid S,Brandts CH,Serve H,et al.Ubiquitination and selective autophagy [J].Cell Death Differ,2013,20(1):21-30
[6] Carvalho BM,Guadagnini D,Tsukumo DML,et al.Modulation of gut microbiota by antibiotics improves insulin signalling in high-fat fed mice [J].Diabetologia,2012,55(10):2823-2834
[7] Vearrier D,Greenberg MI,Miller SN,et al.Methamphetamine:history,pathophysiology,adverse health effects,current trends,and hazards associated with the clandestine manufacture of methamphetamine [J].Dis Mon,2012,58(2):38-89
[8] Sanchez AB,Kaul M.Neuronal stress and injury caused by hiv-1,cart and drug abuse:Converging contributions to hand [J].Brain Sci,2017,7(3).pii:E 25
[9] Shiloh Y,Shema E,Moyal L,et al.RNF20-RNF40:A ubiquitin-driven link between gene expression and the DNA damage response [J].FEBS Lett,2011,585(18):2795-2802
[10] Kim J,Guermah M,McGinty RK,et al.RAD6-mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells [J].Cell,2009,137(3):459-471
[11] 冯波,王蓉,盛树力.神经退行性疾病研究中拟神经细胞模型:人神经母细胞瘤株sh-sy5y的来源特性及应用 [J].中国临床康复(Feng B,Wang R,Sheng SL.Mimic model of nerve cells in the study of neurodegenerative disease:Origin,characteristics and application of human neuroblastoma cell line SH-SY5Y[J].Chin J Clin Rehabil),2006,10(6):121-123
[12] Nakamura K,Kato A,Kobayashi J,et al.Regulation of homologous recombination by RNF20-dependent H2B ubiquitination [J].Mol Cell,2011,41(5):515-528
[13] Godino A,Jayanthi S,Cadet JL.Epigenetic landscape of amphetamine and methamphetamine addiction in rodents [J].Epigenetics,2015,10(7):574-580
[14] Alhamwe BA,Khalaila R,Wolf J,et al.Histone modifications and their role in epigenetics of atopy and allergic diseases [J].Allergy Asthma Clin Immunol,2018,14:39
[15] Eric I C,Danny R.Histones:Annotating chromatin [J].Annu Rev Genet,2009,43(1):559
[16] Sadakierska-Chudy A,Frankowska M,Jastrz?bska J,et al.Cocaine administration and its withdrawal enhance the expression of genes encoding histone-modifying enzymes and histone acetylation in the rat prefrontal cortex [J].Neurotox Res,2017,32(1):141-150
[17] Maze I,Feng J,Wilkinson MB,et al.Cocaine dynamically regulates heterochromatin and repetitive element unsilencing in nucleus accumbens [J].Proc Natl Acad Sci U S A,2011,108(7):3035-3040
[18] Rotllant D,Armario A.Brain pattern of histone H3 phosphorylation after acute amphetamine administration:its relationship to brain c-fos induction is strongly dependent on the particular brain area [J].Neuropharmacology,2012,62(2):1073-1081
[19] 陈科,程汉华,周荣家.自噬与泛素化蛋白降解途径的分子机制及其功能 [J].遗传(Chen K,Cheng HH,Zhou RJ.Molecular mechanisms and functions of autophagy and the ubiquitin-proteasome pathway[J].Hereditas),2012,34(1):5-18
[20] 李翔,白洁.多巴胺在可卡因成瘾中作用 [J].中国老年学(Li X,Bai J.The function of dopamine in cocaine addiction[J].Chin J Gerontol),2015,35(15):4372-4374
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