黑腹果蝇保幼激素受体Met泛素化修饰位点预测及功能验证
|
摘要
目的预测黑腹果蝇保幼激素(juvenile hormone,JH)受体Methoprene tolerant(Met)的泛素化修饰位点,并验证其功能。方法利用UbPred和CKSAAP_UbSite在线预测工具对Met的泛素化位点进行预测;通过重叠PCR技术将预测的各泛素化修饰位点赖氨酸分别突变为丙氨酸,同时将第46和526位赖氨酸突变为丙氨酸;将突变后的Met插入至表达载体p Ac5. 1/V5-His B中,构建各位点突变的表达质粒;转染果蝇Kc细胞,并经格尔德霉素(geldanamycin,GA)处理,Western blot法检测各位点突变后Met的表达。结果根据UbPred和CKSAAP_UbSite在线网站预测结果,选取7个赖氨酸位点作为Met的潜在泛素化修饰位点,CKSAAP_UbSite及UbPred预测分值最高的分别为第46和526位赖氨酸。构建的Met各位点突变表达质粒转染果蝇Kc细胞后,于相对分子质量约82 000处均可见Met目的蛋白的表达,经GA处理后,Met表达下降。结论成功构建了Met的7个预测泛素化修饰位点的单突变表达质粒及1个双突变表达质粒,这些位点的突变均不足以抑制GA诱导Met的降解。
Objective To predict the ubiquitin sites of juvenile hormone(JH) receptor Methoprene tolerant(Met) in Drosophila melanogaster and analyze their functions. Methods The ubiquitin sites of Met were predicted by UbPred and CKSAAP_UbSite online prediction tools. The lysine in predicted sites was mutated into alanine by over-lapping PCR technique. Meanwhile,the lysine at sites 46 and 526 were mutated into alanine simultaneously. The site mutants of Met were inserted into expression vector pAc5. 1/V5-His Bex,and the constructed recombinant plasmids were transfected to D. melanogaster Kc cells and treated with GA. The expressions of mutated Met at various sites were analyzed by Western blot. Results Seven lysine sites were selected as potential ubiquitin sites of Met according to the prediction result by UbPred and CKSAAP_UbSite websites. The lysine with the highest score predicted by CKSAAP_UbSite was at site 46,while that by UbPred at site 526. Met with a relative molecular mass of about 82 000 was expressed in Kc cells transfected with recombinant plasmids containing various site mutants,of which the expression level decreased after treatment with GA. Conclusion The expression vectors containing a single point mutant of seven predicted ubiquitin sites of Met and that containing the indicated double point mutants were constructed successfully. However,these site mutations were not sufficient to inhibit GA-induced Met degradation.
Objective To predict the ubiquitin sites of juvenile hormone(JH) receptor Methoprene tolerant(Met) in Drosophila melanogaster and analyze their functions. Methods The ubiquitin sites of Met were predicted by UbPred and CKSAAP_UbSite online prediction tools. The lysine in predicted sites was mutated into alanine by over-lapping PCR technique. Meanwhile,the lysine at sites 46 and 526 were mutated into alanine simultaneously. The site mutants of Met were inserted into expression vector pAc5. 1/V5-His Bex,and the constructed recombinant plasmids were transfected to D. melanogaster Kc cells and treated with GA. The expressions of mutated Met at various sites were analyzed by Western blot. Results Seven lysine sites were selected as potential ubiquitin sites of Met according to the prediction result by UbPred and CKSAAP_UbSite websites. The lysine with the highest score predicted by CKSAAP_UbSite was at site 46,while that by UbPred at site 526. Met with a relative molecular mass of about 82 000 was expressed in Kc cells transfected with recombinant plasmids containing various site mutants,of which the expression level decreased after treatment with GA. Conclusion The expression vectors containing a single point mutant of seven predicted ubiquitin sites of Met and that containing the indicated double point mutants were constructed successfully. However,these site mutations were not sufficient to inhibit GA-induced Met degradation.
引文
[1] JINDRA M, BELLES X, SHINODA T. Molecular basis of juvenile hormone signaling[J]. Curr Opin Insect Sci, 2015,11(1):39-46.
[2] JINDRA M,PALLI S R, RIDDIFORD L M. The juvenile hormone signaling pathway in insect development[J]. Annu Rev Entomol,2013,58:181-204. doi:10.1146/annurev-ento-120811-153700.
[3] TIAN L, MA L, GUO E, et al. 20-Hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body[J]. Autophagy, 2013, 9(8):1172-1187.
[4] YAMANAKA N, REWITZ K F, O'CONNOR M B. Ecdysone control of developmental transitions:lessons from Drosophila research[J]. Annu Rev Entomol, 2013, 58(10):497-516.
[5] CHARLES J P, IWEMA T, EPA V C, et al. Ligand-binding properties of a juvenile hormone receptor, Methoprene-tolerant[J]. Proc Natl Acad Sci USA, 2011, 108(52):21128-21133.
[6] KAYUKAWA T, MINAKUCHI C, NAMIKI T, et al. Transcriptional regulation of juvenile hormone-mediated induction of Kruppel homolog 1,a repressor of insect metamorphosis[J].Proc Natl Acad Sci USA, 2012, 109(29):11729-11734.
[7] LI M, MEAD E A, ZHU J. Heterodimer of two bHLH-PAS proteins mediates juvenile hormone-induced gene expression[J]. Proc Natl Acad Sci USA, 2011, 108(2):638-643.
[8] ZHANG Z, XU J, SHENG Z, et al. Steroid receptor coactivator is required for juvenile hormone signal transduction through a bHLH-PAS transcription factor, methoprene tolerant[J]. J Biol Chem, 2011, 286(10):8437-8447.
[9] GUO W, WU Z, SONG J, et al. Juvenile hormone-receptor complex acts on mcm4 and mcm7 to promote polyploidy and vitellogenesis in the migratory locust[J]. PLoS Genet, 2014,10(10):e1004702. doi:10.1371/journal.pgen. 1004702.
[10] ZOU Z, SAHA T T, ROY S, et al. Juvenile hormone and its receptor, methoprene-tolerant, control the dynamics of mosquito gene expression[J]. Proc Natl Acad Sci USA, 2013,110(24):E2173-E2181.
[11] HIRUMA K, KANEKO Y. Hormonal regulation of insect metamorphosis with special reference to juvenile hormone biosynthesis[J]. Curr Top Dev Biol, 2013, 103:73-100.
[12] ABDOU M A, HE Q Y, WEN D, et al. Drosophila Met and Gee are partially redundant in transducing juvenile hormone action[J]. Insect Biochem Mol Biol, 2011, 41(12):938-945.
[13] KONOPOVA B, JINDRA M. Juvenile hormone resistance gene Methoprene-tolerant controls entry into metamorphosis in the beetle Tribolium castaneum[J]. Proc Natl Acad Sci USA,2007, 104(25):10488-10493.
[14] PARHASARATHY R, TAN A, PALLI S R. bHLH-PAS family transcription factor methoprene-tolerant plays a key role in JHaction in preventing the premature development of adult structures during larval-pupal metamorphosis[J]. Mech Dev, 2008,125(7):601-616.
[15] HE Q Y, WEN D, JIA Q Q, et al. Heat shock protein 83(Hsp83)facilitates methoprene-tolerant(Met)nuclear import to modulate juvenile hormone signaling[J]. J Biol Chem,2014,289(40):27874-27885.
[16] HE Q Y, ZHANG Y X, ZHANG X, et al. Nucleoporin Nup-358 facilitates nuclear import of Methoprene-tolerant(Met)in an importin beta-and Hsp83-dependent manner[J]. Insect Biochem Mol Biol, 2017, 81(2):10-18.
[17] ZHANG Y, ZHANG J, LIU C, et al. Neratinib induces ErbB2ubiquitylation and endocytic degradation via HSP90 dissociation in breast cancer cells[J]. Cancer Lett, 2016, 382(2):176-185.
[18] KHATTAR W, FRIED J, XU B, et al. Cksl proteasomal degradation is induced by inhibiting Hsp90-mediated chaperoning in cancer cells[J]. Cancer Chemother Pharmacol, 2015,75(2):411-420.
[19] WILSON T G, FABIAN J. A Drosophila melanogaster mutant resistant to a chemical analog of juvenile hormone[J]. Dev Biol, 1986, 118(1):190-201.
[20] DAIMON T, UCHIBORI M, NAKAO H, et al. Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cyclce[J]. Proc Natl Acad Sci U S A, 2015,112(31):E4226-E4235.
[21] JINDRA M, UHLIROVA M, CHARLES J P, et al. Genetic evidence for function of the bHLH-PAS protein Gee/Met as a juvenile hormone receptor[J]. PLos Genet,2015, 11(7):e1005394. doi:10.1371/journal.pgen. 1005394.
[22] LIU P C, PENG H J, ZHU J S. Juvenile hormone-activated phospholipase C pathway enhances transcriptional activation by the methoprene-tolerant protein[J]. Proc Natl Aad Sci USA, 2015,112(15):E1871-E1879.
[23] LU L,LI D,HE F C. Bioinformatics advances in protein ubiquitination[J]. Heredit, 2013, 35(1):17-26.(in Chinese)卢亮,李栋,贺福初.蛋白质泛素化修饰的生物信息学研究进展[J].遗传,2013, 35(1):17-26.
[24] PICKART C M, COHEN R E. Proteasomes and their kin:proteases in the machine age[J]. Nat Rev Mol Cell Biol, 2004, 5(3):177-187.
[2] JINDRA M,PALLI S R, RIDDIFORD L M. The juvenile hormone signaling pathway in insect development[J]. Annu Rev Entomol,2013,58:181-204. doi:10.1146/annurev-ento-120811-153700.
[3] TIAN L, MA L, GUO E, et al. 20-Hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body[J]. Autophagy, 2013, 9(8):1172-1187.
[4] YAMANAKA N, REWITZ K F, O'CONNOR M B. Ecdysone control of developmental transitions:lessons from Drosophila research[J]. Annu Rev Entomol, 2013, 58(10):497-516.
[5] CHARLES J P, IWEMA T, EPA V C, et al. Ligand-binding properties of a juvenile hormone receptor, Methoprene-tolerant[J]. Proc Natl Acad Sci USA, 2011, 108(52):21128-21133.
[6] KAYUKAWA T, MINAKUCHI C, NAMIKI T, et al. Transcriptional regulation of juvenile hormone-mediated induction of Kruppel homolog 1,a repressor of insect metamorphosis[J].Proc Natl Acad Sci USA, 2012, 109(29):11729-11734.
[7] LI M, MEAD E A, ZHU J. Heterodimer of two bHLH-PAS proteins mediates juvenile hormone-induced gene expression[J]. Proc Natl Acad Sci USA, 2011, 108(2):638-643.
[8] ZHANG Z, XU J, SHENG Z, et al. Steroid receptor coactivator is required for juvenile hormone signal transduction through a bHLH-PAS transcription factor, methoprene tolerant[J]. J Biol Chem, 2011, 286(10):8437-8447.
[9] GUO W, WU Z, SONG J, et al. Juvenile hormone-receptor complex acts on mcm4 and mcm7 to promote polyploidy and vitellogenesis in the migratory locust[J]. PLoS Genet, 2014,10(10):e1004702. doi:10.1371/journal.pgen. 1004702.
[10] ZOU Z, SAHA T T, ROY S, et al. Juvenile hormone and its receptor, methoprene-tolerant, control the dynamics of mosquito gene expression[J]. Proc Natl Acad Sci USA, 2013,110(24):E2173-E2181.
[11] HIRUMA K, KANEKO Y. Hormonal regulation of insect metamorphosis with special reference to juvenile hormone biosynthesis[J]. Curr Top Dev Biol, 2013, 103:73-100.
[12] ABDOU M A, HE Q Y, WEN D, et al. Drosophila Met and Gee are partially redundant in transducing juvenile hormone action[J]. Insect Biochem Mol Biol, 2011, 41(12):938-945.
[13] KONOPOVA B, JINDRA M. Juvenile hormone resistance gene Methoprene-tolerant controls entry into metamorphosis in the beetle Tribolium castaneum[J]. Proc Natl Acad Sci USA,2007, 104(25):10488-10493.
[14] PARHASARATHY R, TAN A, PALLI S R. bHLH-PAS family transcription factor methoprene-tolerant plays a key role in JHaction in preventing the premature development of adult structures during larval-pupal metamorphosis[J]. Mech Dev, 2008,125(7):601-616.
[15] HE Q Y, WEN D, JIA Q Q, et al. Heat shock protein 83(Hsp83)facilitates methoprene-tolerant(Met)nuclear import to modulate juvenile hormone signaling[J]. J Biol Chem,2014,289(40):27874-27885.
[16] HE Q Y, ZHANG Y X, ZHANG X, et al. Nucleoporin Nup-358 facilitates nuclear import of Methoprene-tolerant(Met)in an importin beta-and Hsp83-dependent manner[J]. Insect Biochem Mol Biol, 2017, 81(2):10-18.
[17] ZHANG Y, ZHANG J, LIU C, et al. Neratinib induces ErbB2ubiquitylation and endocytic degradation via HSP90 dissociation in breast cancer cells[J]. Cancer Lett, 2016, 382(2):176-185.
[18] KHATTAR W, FRIED J, XU B, et al. Cksl proteasomal degradation is induced by inhibiting Hsp90-mediated chaperoning in cancer cells[J]. Cancer Chemother Pharmacol, 2015,75(2):411-420.
[19] WILSON T G, FABIAN J. A Drosophila melanogaster mutant resistant to a chemical analog of juvenile hormone[J]. Dev Biol, 1986, 118(1):190-201.
[20] DAIMON T, UCHIBORI M, NAKAO H, et al. Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cyclce[J]. Proc Natl Acad Sci U S A, 2015,112(31):E4226-E4235.
[21] JINDRA M, UHLIROVA M, CHARLES J P, et al. Genetic evidence for function of the bHLH-PAS protein Gee/Met as a juvenile hormone receptor[J]. PLos Genet,2015, 11(7):e1005394. doi:10.1371/journal.pgen. 1005394.
[22] LIU P C, PENG H J, ZHU J S. Juvenile hormone-activated phospholipase C pathway enhances transcriptional activation by the methoprene-tolerant protein[J]. Proc Natl Aad Sci USA, 2015,112(15):E1871-E1879.
[23] LU L,LI D,HE F C. Bioinformatics advances in protein ubiquitination[J]. Heredit, 2013, 35(1):17-26.(in Chinese)卢亮,李栋,贺福初.蛋白质泛素化修饰的生物信息学研究进展[J].遗传,2013, 35(1):17-26.
[24] PICKART C M, COHEN R E. Proteasomes and their kin:proteases in the machine age[J]. Nat Rev Mol Cell Biol, 2004, 5(3):177-187.