家蚕bHLH转录因子Bmdimm与Bmchip的相互作用

doi:10.3864/j.issn.0578-1752.2016.10.018

Abstract

Abstract: 【Objective】 Bmdimm is a member of the bHLH transcription factors family, which is one of key transcription factors that regulate the expression of fibroin heavy chain gene in the late stage of the fifth instar of Bombyx mori. Bmdimm was predicted to have a potential ubiquitination site of lysine residue K43, and therefore it is presumed that Bmdimm may be modified by ubiquitin and thus degraded. Bmchip belongs to the U-box subfamily of the B. mori ubiquitin ligase E3 family, which is expressed in the posterior silk gland during the fifth instar of B. mori. Studying on the interaction of Bmdimm and Bmchip will promote us to better understand the ubiquitylation of Bmdimm in vivo and its biological significance on the transcriptional regulation of fibroin heavy chain gene. 【Method】 The expression profiles of Bmdimm and Bmchip in the posterior silk gland of B. mori were analyzed based on the B. mori genome microarray database. The CDS sequences of Bmdimm and Bmchip were obtained by PCR cloning and gene synthesis, respectively. Multiple expression vectors were constructed and then transformed into E. coli to express the target proteins to screen the best expression conditions. The fused Bmdimm and Bmchip were purified using Ni²⁺ affinity chromatography, and then digested with protease. The fused tag and target proteins were separated via Ni²⁺ affinity chromatography again. The oligo states of Bmdimm and Bmchip in solution were assessed by gel filtration chromatography. The secondary structures of Bmdimm and Bmchip were studied by circular dichroism spectroscopy. The interaction of Bmdimm and Bmchip in vitro were investigated using Far-western blotting and Pull-down. The affinity of Bmdimm and Bmchip were determined by microscale thermophoresis.【Result】 The B. mori genome microarray expression profiles indicate both Bmdimm and Bmchip were expressed in the posterior silk gland during the fifth instar of B. mori. Multiple expression vectors of Bmdimm and Bmchip were constructed, and it was found that ppSUMO-Bmdimm and pET-32M·3C-Bmchip, 0.3 mmol·L^-1 IPTG and 16℃ were the best expression conditions. Then the recombinant Bmdimm and Bmchip was expressed and purified. Gel filtration analysis suggested both Bmdimm and Bmchip existed mainly as a dimer in solution. Circular dichroism spectroscopy showed both Bmdimm and Bmchip had α-helix structures. Far-western blotting and Pull-down assay suggested Bmdimm could interact with Bmchip in vitro. The dissociation constant K_D of Bmdimm and Bmchip was calculated to be (750±28.6) μmol·L^-1 using microscale thermophoresis.【Conclusion】 The bHLH transcription factor Bmdimm and B. mori ubiquitin ligase Bmchip are expressed in the posterior silk gland during the fifth instar of B. mori. Bmdimm and Bmchip have transient interaction in vitro, implying Bmdimm may be modified by ubiquitin via the interaction with Bmchip and thus down-regulated the transcription of fibroin heavy chain gene.

Key words: Bombyx mori, Bmdimm, Bmchip, interaction, ubiquitylation

ZHAO Peng, WANG Ye-jing, WEI Shu-guang, LIU Li-na, LI Zhen-zhen, ZHAO Ping, HE Hua-wei. Interaction of bHLH Transcription Factor Bmdimm and Bmchip in Bombyx mori[J].Scientia Agricultura Sinica, 2016, 49(10): 2027-2038.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2016.10.018

https://www.chinaagrisci.com/EN/Y2016/V49/I10/2027

References

[1] Kimoto M, Tsubota T, Uchino K, Sezutsu H, Takiya S. LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes, fibH, fibL and fhx, in the silk gland of the silkworm, Bombyx mori. Insect Biochemistry and Molecular Biology, 2015, 56: 29-35.

[2] Kojima K, Kuwana Y, Sezutsu H, Kobayashi I, Uchino K, Tamura T, Tamada Y. A new method for the modification of fibroin heavy chain protein in the transgenic silkworm. Bioscience, Biotechnology and Biochemistry, 2007, 71(12): 2943-2951.

[3] Zhao X M, Liu C, Jiang L J, Li Q Y, Zhou M T, Cheng T C, Mita K, Xia Q Y. A juvenile hormone transcription factor Bmdimm-Fibroin H chain pathway is involved in the synthesis of silk protein in silkworm, Bombyx mori. Journal of Biological Chemistry, 2015, 290(2): 972-986.

[4] Wang Y, Chen K P, Yao Q, Wang W B, Zhu Z. The basic helix-loop-helix transcription factor family in Bombyx mori. Development Genes and Evolution, 2007, 217(10): 715-723.

[5] 阮班军, 代鹏, 王伟, 孙建斌, 张文涛, 颜真, 杨静华. 蛋白质翻译后修饰研究进展. 中国细胞生物学学报, 2014, 36(7): 1027-1037.

Ruan B J, Dai P, Wang W, Sun J B, Zhang W T, Yan Z, Yang J H. Progress on post-translational modification of proteins. Chinese Journal of Cell Biology, 2014, 36(7): 1027-1037. (in Chinese)

[6] 郭芸菲, 王涛, 徐小洁, 宋金洁, 王宣, 王子政, 叶棋浓, 江泽飞. 热休克蛋白70羧基末端相互作用蛋白 (CHIP) 对HER2的降解作用. 细胞与分子免疫学杂志, 2014, 30(6): 611-613.

Guo Y F, Wang T, Xu X J, Song J J, Wang X, Wang Z Z, Ye Q N, Jiang Z F. Degradation of heat shock protein 70 carboxy terminus interacting protein (CHIP) for HER2. Chinese Journal of Cellular and Molecular Immunology, 2014, 30(6): 611-613. (in Chinese)

[7] 张志清, 钱令嘉. 协同伴侣分子CHIP的E3连接酶活性及其生物学意义. 细胞生物学杂志, 2008, 30: 435-439.

Zhang Z Q, Qian L J. E3 ligase activity of CHIP, a cochaperones and its biological significance. Chinese Journal of Cell Biology, 2008, 30: 435-439. (in Chinese)

[8] Huq E, Al-Sady B, Hudson M, Kim C H, Apel M, Quail P H. Phytochrome-interacting factor 1 is a critical bHLH regulator of chlorophyll biosynthesis. Science, 2004, 305(5692): 1937-1941.

[9] Grove C A, De Masi F, Barrasa M I, Newburger D E, Alkema M J, Bulyk M L, Walhout A J M. A multiparameter network reveals extensive divergence between C. elegans bHLH transcription factors. Cell, 2009, 138(2): 314-327.

[10] Wang L H, Baker N E. Proteins and ID proteins: Helix-loop-helix partners in development and disease. Developmental Cell, 2015, 35(3): 269-280.

[11] De Masi F, Grove C A, Vedenko A, Alibes A, Gisselbrecht S S, Serrano L, Bulyk M L, Walhout A J M. Using a structural and logics systems approach to infer bHLH-DNA binding specificity determinants. Nucleic Acids Research, 2011, 39(11): 4553-4563.

[12] Gordan R, Shen N, Dror I, Zhou T, Horton J, Rohs R, Bulyk M L. Genomic regions flanking E-Box binding sites influence DNA binding specificity of bHLH transcription factors through DNA Shape. Cell Reports, 2013, 3(4): 1093-1104.

[13] Wang Y, Chen K, Yao Q, Wang W, Zhu Z. The basic helix- loop-helix transcription factor family in Bombyx mori. Development Genes and Evolution, 2007, 217(10): 715-723.

[14] Hewes R S, Park D, Gauthier S A, Schaefer A M, Taghert P H. The bHLH protein Dimmed controls neuroendocrine cell differentiation in Drosophila. Development, 2003, 130(9): 1771-1781.

[15] Jia D, Sun Y, Konieczny S F. Mist1 regulates pancreatic acinar cell proliferation through p21^CIP1/WAF1. Gastroenterology, 2008, 135(5): 1687-1697.

[16] Pin C L, Rukstalis J M, Johnson C, Konieczny S F. The bHLH transcription factor Mist1 is required to maintain exocrine pancreas cell organization and acinar cell identity. The Journal of Cell Biology, 2001, 155(4): 519-530.

[17] Hamanaka Y, Park D, Yin P, Annangudi S P, Edwards T N, Sweedler J, Meinertzhagen I A, Taghert P H. Transcriptional orchestration of the regulated secretory pathway in neurons by the bHLH protein DIMM. Current Biology, 2010, 20(1): 9-18.

[18] Darosa P A, Wang Z Z, Jiang X M, Pruneda J N, Cong F, Klevit R E, Xu W Q. Allosteric activation of the RNF146 ubiquitin ligase by a poly (ADP-ribosyl) ation signal. Nature, 2015, 517(7533): 223-226.

[19] Hettema E H, Valdez-Taubas J, Pelham H R B. Bsd2 binds the ubiquitin ligase Rsp5 and mediates the ubiquitination of transmembrane proteins. The EMBO Journal, 2004, 23(6): 1279-1288.

[20] Chen Z J. Ubiquitin signalling in the NF-κB pathway. Nature Cell Biology, 2005, 7(8): 758-765.

[21] 洪小琦, 梁敏, 黄芳. 泛素连接酶在神经系统中的作用. 细胞生物学杂志, 2009, 31(3): 325-330.

Hong X Q, Liang M, Huang F. Roles of ubiquitin ligase in the nervous system. Chinese Journal of Cell Biology, 2009, 31(3): 325-330. (in Chinese)

[22] Goo M S, Scudder S L, Patrick G N. Ubiquitin-dependent trafficking and turnover of ionotropic glutamate receptors. Frontiers in Molecular Neuroscience, 2015, 8: Article 60.

[23] 石号, 孙大燕, 李宾, 方合志, 吕建新. 蛋白质量控制研究. 中国细胞生物学学报, 2015, 37(2): 241-248.

Shi H, Sun D Y, Li B, Fang H Z, Lü J X. protein quality control. Chinese Journal of Cell Biology, 2015, 37(2): 241-248. (in Chinese)

[24] Li M Y, Brooks C L, Wu-Baer F, Chen D L, Baer R, Gu W. Mono-versus polyubiquitination: Differential control of p53 fate by Mdm2. Science, 2003, 302(5652): 1972-1975.

[25] Liu Y C. Ubiquitin ligases and the immune response. Annual Review of Immunology, 2004, 22: 81-127.

[26] 何珊, 张令强. 线性泛素化修饰研究进展. 遗传, 2015, 37(9): 911-917.

HE S, ZHANG L q. Research progress in linear ubiquitin modification. Hereditas, 2015, 37(9): 911-917. (in Chinese)

[27] Zou X, Gal L, Blank M. Molecular functions of NEDD4 E3 ubiquitin ligases in cancer. Biochimica et Biophysica Acta (BBA)- Reviews on Cancer, 2015, 1856(1): 91-106.

[28] Murata S, Chiba T, Tanaka K. CHIP: a quality-control E3 ligase collaborating with molecular chaperones. The International Journal of Biochemistry and Cell Biology, 2003, 35(5): 572-578.

[29] Rees I, Lee S, Kim H, Tsai F T F. The E3 ubiquitin ligase CHIP binds the androgen receptor in a phosphorylation-dependent manner. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2006, 1764(6): 1073-1079.

[30] Deng W K, Wang Y B, Liu Z X, Cheng H, Xue Y. HemI: A toolkit for illustrating heatmaps. Plos One, 2014, 9(11): e111988.

[31] Huang P Y, Leu J H, Chen L L. A newly identified protein complex that mediates white spot syndrome virus infection via chitin-binding protein. Journal of General Virology, 2014, 95(8): 1799-1808.

[32] Wang X Q, Corin K, Baaske P, Wienken C J, Moran J W, Duhr S, Braun D, Zhang S G. Peptide surfactants for cell-free production of functional G protein-coupled receptors. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(22): 9049-9054.

[33] Zhou C Z, Confalonieri F, Esnault C, Zivanovic Y, Jacquet M, Janin J, Perasso R, Li Z G, Duguet M. The 62-kb upstream region of Bombyx mori fibroin heavy chain gene is clustered of repetitive elements and candidate matrix association regions. Gene, 2003, 312: 189-195.

[34] Jiang J H, Ballinger C A, Wu Y X, Dai Q, Cyr D M, Hohfeld J, Patterson C. CHIP is a U-box-dependent E3 ubiquitin ligase - Identification of Hsc70 as a target for ubiquitylation. The Journal of Biological Chemistry, 2001, 276(46): 42938-42944.

[35] Stankiewicz M, Nikolay R, Rybin V, Mayer M P. CHIP participates in protein triage decisions by preferentially ubiquitinating Hsp70-bound substrates. The FEBS Journal, 2010, 277(16): 3353-3367.

Related Articles 15

[1]	FAN YanGen,WANG Yu,LIU FuHao,ZHAO XiuXiu,XIANG QinZeng,ZHANG LiXia. Screening and Verification of CsHIPP26.1 Interaction Protein in Tea Plant [J]. Scientia Agricultura Sinica, 2022, 55(8): 1630-1641.
[2]	LI ShiJia,LÜ ZiJing,ZHAO Jin. Identification of R2R3-MYB Subfamily in Chinese Jujube and Their Expression Pattern During the Fruit Development [J]. Scientia Agricultura Sinica, 2022, 55(6): 1199-1212.
[3]	SHI XiaoLong,GUO Pei,REN JingYao,ZHANG He,DONG QiQi,ZHAO XinHua,ZHOU YuFei,ZHANG Zheng,WAN ShuBo,YU HaiQiu. A Salt Stress Tolerance Effect Study in Peanut Based on Peanut//Sorghum Intercropping System [J]. Scientia Agricultura Sinica, 2022, 55(15): 2927-2937.
[4]	WANG Bing,LI HuiMin,CAO HaiQun,WANG GuiRong. Mechanisms and Applications of Plant-Herbivore-Natural Enemy Tritrophic Interactions Mediated by Volatile Organic Compounds [J]. Scientia Agricultura Sinica, 2021, 54(8): 1653-1672.
[5]	ZHAO WeiSong,GUO QingGang,DONG LiHong,WANG PeiPei,SU ZhenHe,ZHANG XiaoYun,LU XiuYun,LI SheZeng,MA Ping. Transcriptome and Proteome Analysis of Bacillus subtilis NCD-2 Response to L-proline from Cotton Root Exudates [J]. Scientia Agricultura Sinica, 2021, 54(21): 4585-4600.
[6]	YANG FengKe,HE BaoLin,DONG Bo,WANG LiMing. Effects of Black Film Mulched Ridge-Furrow Tillage on Soil Water- Fertilizer Environment and Potato Yield and Benefit Under Different Rainfall Year in Semiarid Region [J]. Scientia Agricultura Sinica, 2021, 54(20): 4312-4325.
[7]	YU BaoJun,DENG ZhanZhao,XIN GuoSheng,CAI ZhengYun,GU YaLing,ZHANG Juan. Correlation Analysis of Inosine Monophosphate Specific Deposition Related LNC_003828-gga-miR-107-3P-MINPP1 in Jingyuan Chicken Muscle Tissue [J]. Scientia Agricultura Sinica, 2021, 54(19): 4229-4242.
[8]	ZHANG ZhiXing,MIN XiuMei,SONG Guo,CHEN Hua,XU HaiLong,LIN WenXiong. Identification of 14-3-3 Client Proteins in Rice Grains and Their Response to Exogenous Hormones During the Grain Filling Stage [J]. Scientia Agricultura Sinica, 2021, 54(12): 2523-2537.
[9]	YUAN XinBo,CHENG TingTing,XI XiaoHan,CHEN ZhangYu,WANG RuiHong,KE WeiDong,GUO HongBo. Screening of Polyphenol Oxidase Interaction Proteins from Nelumbo nucifera and Their Verification [J]. Scientia Agricultura Sinica, 2020, 53(18): 3777-3791.
[10]	YUAN GuiBo,MO ShuangRong,QIAN Ying,ZANG DongNan,YANG Fan,JIANG HongLiang,WU Yuan,DING HaiDong. Screening of Interacting Protein of Tomato SIVQ6 by GST Pull-Down [J]. Scientia Agricultura Sinica, 2020, 53(15): 3146-3157.
[11]	ZOU ShuangXia,JIN ChengYan,BAO JianJun,WANG Yue,CHEN WeiHao,WU TianYi,WANG LiHong,LÜ XiaoYang,GAO Wen,WANG BuZhong,ZHU GuoQiang,DAI GuoJun,SHI DongFang,SUN Wei. Differential circRNA Analysis in the Spleen of Hu-sheep Lambs Infected with F17 Escherichia coli [J]. Scientia Agricultura Sinica, 2019, 52(6): 1090-1101.
[12]	CHEN Peng,BAO XiYan,KANG TaoTao,DONG ZhanQi,ZHU Yan,PAN MinHui,LU Cheng. Screening and Identification of Proteins Interacting with Bombyx mori IAP and Their Effects on BmNPV Proliferation [J]. Scientia Agricultura Sinica, 2019, 52(3): 558-567.
[13]	DONG ZhanQi,JIANG YaMing,PAN MinHui. Screening and Identification of Candidate Proteins Interacting with BmHSP60 in the Silkworm (Bombyx mori) [J]. Scientia Agricultura Sinica, 2019, 52(2): 376-384.
[14]	ZHANG Kui, PAN GuangZhao, SU JingJing, TAN Juan, XU Man, LI YuTian, CUI HongJuan. Identification, Expression, Subcelluar Localization, and Function of glial cell missing (gcm) in Silkworm (Bombyx mori) [J]. Scientia Agricultura Sinica, 2018, 51(7): 1401-1411.
[15]	WANG Fei, LI XianYang, HUA XiaoTing, XIA QingYou. Screening and Analysis of Anti-BmNPV Cytokines in Silkworm (Bombyx mori) [J]. Scientia Agricultura Sinica, 2018, 51(4): 789-799.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

Interaction of bHLH Transcription Factor Bmdimm and Bmchip in Bombyx mori

RichHTML

PDF