家蚕热休克蛋白HSP90相互作用蛋白的筛选与鉴定

doi:10.3864/j.issn.0578-1752.2022.06.016

摘要/Abstract

摘要：

【目的】HSP90是热休克蛋白家族的成员之一,在昆虫的抗逆性和变态发育中发挥重要作用,已有研究表明HSP90能够促进家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus,BmNPV）的增殖,但作用机制尚不清楚。本研究通过鉴定BmHSP90的相互作用蛋白,为其促进BmNPV增殖的作用机制解析提供参考。【方法】构建连接在pIZ/V5-His的BmHSP90^HA真核过表达载体,在家蚕BmN-SWU1细胞中转染48 h后,感染BmNPV继续培养48 h后收集蛋白,保留总蛋白后将这些蛋白均分两管,进行免疫共沉淀,分别用抗HA抗体和IgG抗体钓取相互作用蛋白,对蛋白胶进行硝酸银染色后,获取差异条带并做质谱分析,将质谱结果与信息分析结合进行候选互作蛋白筛选,并克隆鉴定互作蛋白。通过免疫荧光验证HSP90与互作蛋白的共定位情况,并进一步采用免疫共沉淀试验确定其是否存在相互作用关系。【结果】硝酸银染色结果显示,试验组与对照组在90、70和60 kD附近处存在差异条带,并验证了90 kD处的差异条带为诱饵蛋白;将另外两条差异带进行质谱分析,共鉴定到7个候选相互作用蛋白,通过分析选择其中两个候选蛋白作后续研究,分别为Tubulin-specific chaperone E（Tbce）和Golgin subfamily A member 5（Golga5）。BmTbce的最大开放阅读框长度为1 728 bp,编码576个氨基酸,BmGolga5的最大开放阅读框长度为1 854 bp,编码618个氨基酸;同源比对和系统进化树显示BmTbce的微管结合结构域（cytoskeleton-associated protein-glycine-rich,CAP-Gly）位于N端且在不同物种之间保守性较高,BmGolga5的跨膜区（transmembrane domain,TMD）位于C端,也较为保守;荧光共定位显示BmHSP90与BmTbce和BmGolga5在细胞质中发生共定位,并进一步通过免疫共沉淀证明BmHSP90^HA和BmTbce^Flag、BmHSP90^HA和BmGolga5^Flag具有相互作用关系。【结论】经过筛选与鉴定,在BmNPV感染家蚕细胞的过程中,与家蚕热休克蛋白HSP90发生相互作用的蛋白为BmTbce和BmGolga5。

关键词: 家蚕, 家蚕核型多角体病毒, HSP90, Tbce, Golga5

Abstract:

【Objective】HSP90 is a member of the heat shock protein family and plays an important role in insect resistance and metamorphosis. Studies have shown that HSP90 can promote the proliferation of Bombyx mori nucleopolyhedrovirus (BmNPV), but the mechanism of action is unclear. The objective of this study is to identify the interacting proteins of BmHSP90, and to provide a reference for the analysis of its mechanism of promoting BmNPV proliferation.【Method】The BmHSP90 ^HA eukaryotic overexpression vector linked to pIZ/V5-His was constructed. After transfection in BmN-SWU1 cells for 48 h, BmNPV was infected and cultured for 48 h to collect the proteins. After the total protein was retained, the proteins were divided into two tubes and co-immunoprecipitation was performed. The interacting protein was caught with anti-HA antibody and IgG antibody, respectively, after staining the protein gel with silver nitrate, the different bands were obtained and mass spectrometry analysis was performed. The mass spectrometry results were combined with the information analysis to screen candidate interacting proteins, and then the interacting proteins were cloned and identified. The co-localization of HSP90 and the interacting protein was verified by immunofluorescence, and the co-immunoprecipitation experiment was further used to determine whether they had an interaction relationship.【Result】The results of silver nitrate staining showed that the experimental group and the control group had different bands near 90, 70 and 60 kD, and verified that the different bands at 90 kD were bait proteins. The other two different bands were analyzed by mass spectrometry, and a total of 7 candidate interacting proteins were identified. Two of the candidate proteins were selected for follow-up study through analysis, namely Tubulin-specific chaperone E (Tbce) and Golgin subfamily A member 5 (Golga5). The maximum open reading frame length of the BmTbce is 1 728 bp, which encodes 576 amino acids, and the maximum open reading frame length of the BmGolga5 is 1 854 bp, which encodes 618 amino acids. The homology alignment and phylogenetic tree showed that the microtubule binding domain of BmTbce (cytoskeleton-associated protein-glycine-rich, CAP-Gly) was located at the N-terminus and was highly conserved among different species. The transmembrane domain (TMD) of BmGolga5 was located at the C-terminus and was also conservative. The fluorescence co-localization showed that BmHSP90 co-localized with BmTbce and BmGolga5 in the cytoplasm, and it was further proved by co-immunoprecipitation that BmHSP90 ^HA and BmTbce^Flag, BmHSP90^HA and BmGolga5^Flag had an interaction relationship.【Conclusion】After screening and identification, in the process of BmNPV infection of B. mori cells, the proteins that interact with the B. mori heat shock protein HSP90 are BmTbce and BmGolga5.

Key words: Bombyx mori, BmNPV, HSP90, Tbce, Golga5

龙艳碧,吴云飞,张倩,陈鹏,潘敏慧. 家蚕热休克蛋白HSP90相互作用蛋白的筛选与鉴定[J]. 中国农业科学, 2022, 55(6): 1253-1262.

LONG YanBi,WU YunFei,ZHANG Qian,CHEN Peng,PAN MinHui. Screening and Identification of HSP90 Interacting Proteins in Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2022, 55(6): 1253-1262.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.06.016

https://www.chinaagrisci.com/CN/Y2022/V55/I6/1253

图/表 9

表1

图1

表2

图2

图3

图4

图5

图6

图7

参考文献 38

[1]	RITOSSA F. A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia, 1962,18:571-573. doi: 10.1007/BF02172188
[2]	TISSIÉRES A, MITCHELL H K, TRACY U M. Protein synthesis in salivary glands of Drosophila melanogaster: Relation to chromosome puffs. Journal of Molecular Biology, 1974,84(3):389-398. doi: 10.1016/0022-2836(74)90447-1
[3]	LANDAIS I, POMMET J M, MITA K, NOHATA J, GIMENEZ S, FOURNIER P, DEVAUCHELLE G, DUONOR-CERUTTI M, OGLIASTRO M. Characterization of the cDNA encoding the 90 kDa heat-shock protein in the Lepidoptera Bombyx mori and Spodoptera frugiperda. Gene, 2001,271(2):223-231. doi: 10.1016/S0378-1119(01)00523-6
[4]	RICHTER K, HASLBECK M, BUCHNER J. The heat shock response: Life on the verge of death. Molecular Cell, 2010,40(2):253-266. doi: 10.1016/j.molcel.2010.10.006
[5]	MOGK A, BUKAU B. Role of sHsps in organizing cytosolic protein aggregation and disaggregation. Cell Stress and Chaperones, 2017,22(4):493-502. doi: 10.1007/s12192-017-0762-4
[6]	BAR-LAVAN Y, SHEMESH N, BEN-ZVI A. Chaperone families and interactions in metazoa. Essays in Biochemistry, 2016,60(2):237-253. doi: 10.1042/EBC20160004
[7]	DALIDOWSKA I, GAZI O, SULEJCZAK D, PRZYBYLSKI M, BIEGANOWSKI P. Heat shock protein 90 chaperones E1A early protein of adenovirus 5 and is essential for replication of the virus. International Journal of Molecular Sciences, 2021,22(4):2020. doi: 10.3390/ijms22042020
[8]	ZHANG Y, ZHANG Y A, TU J. Hsp90 is required for snakehead vesiculovirus replication via stabilization the viral L protein. Journal of Virology, 2021,95(16):e00594-21.
[9]	JING L, BUCHNER J. Structure, function and regulation of the Hsp90 machinery. Biomedical Journal, 2013,36(3):106-117. doi: 10.4103/2319-4170.113230
[10]	WU P, SHANG Q, HUANG H, ZHANG S, ZHONG J, HOU Q, GUO X. Quantitative proteomics analysis provides insight into the biological role of Hsp90 in BmNPV infection in Bombyx mori. Journal of Proteomics, 2019,203:103379. doi: 10.1016/j.jprot.2019.103379
[11]	TSOU Y L, LIN Y W, CHANG H W, LIN H Y, SHAO H Y, YU S L, LIU C C, CHITRA E, SIA C, CHOW Y H. Heat shock protein 90: Role in enterovirus 71 entry and assembly and potential target for therapy. PLoS ONE, 2013,8(10):e77133. doi: 10.1371/journal.pone.0077133
[12]	IYER K, CHAND K, MITRA A, TRIVEDI J, MITRA D. Diversity in heat shock protein families: Functional implications in virus infection with a comprehensive insight of their role in the HIV-1 life cycle. Cell Stress and Chaperones, 2021,26(5):743-768. doi: 10.1007/s12192-021-01223-3
[13]	MIYATA Y, YAHARA I. p53-independent association between SV40 large T antigen and the major cytosolic heat shock protein, HSP90. Oncogene, 2000,19(11):1477-1484. doi: 10.1038/sj.onc.1203475
[14]	MOMOSE F, NAITO T, YANO K, SUGIMOTO S, MORIKAWA Y, NAGATA K. Identification of Hsp90 as a stimulatory host factor involved in influenza virus RNA synthesis. The Journal of Biological Chemistry, 2002,277(47):45306-45314. doi: 10.1074/jbc.M206822200
[15]	WYLER E, MOSBAUER K, FRANKE V, DIAG A, GOTTULA L T, ARSIE R, KLIRONOMOS F, KOPPSTEIN D, HONZKE K, AYOUB S, et al. Transcriptomic profiling of SARS-CoV-2 infected human cell lines identifies HSP90 as target for COVID-19 therapy. iScience, 2021,24(3):102151. doi: 10.1016/j.isci.2021.102151
[16]	张凤霞. 棉铃虫Hsp90、胸腺素和丝氨酸蛋白酶抑制因子在发育中的表达模式与激素调控[D]. 济南: 山东大学, 2010.
	ZHANG F X. Expression patterns and hormonal regulation of Hsp90, thymosin and serine protease inhibitors in the development of Helicoverpa armigera[D]. Ji’nan: Shandong University, 2010. (in Chinese)
[17]	CHEN B, WAGNER A. Hsp90 is important for fecundity, longevity, and buffering of cryptic deleterious variation in wild fly populations. BMC Evolutionary Biology, 2012,12:25. doi: 10.1186/1471-2148-12-25
[18]	MCCLELLAN A J, XIA Y, DEUTSCHBAUER A M, DAVIS R W, GERSTEIN M, FRYDMAN J. Diverse cellular functions of the Hsp90 molecular chaperone uncovered using systems approaches. Cell, 2007,131(1):121-135. doi: 10.1016/j.cell.2007.07.036
[19]	SHANG Q, WU P, HUANG H L, ZHANG S L, TANG X D, GUO X J. Inhibition of heat shock protein 90 suppresses Bombyx mori nucleopolyhedrovirus replication in B. mori. Insect Molecular Biology, 2020,29(2):205-213. doi: 10.1111/imb.v29.2
[20]	PAN M H, CAI X J, LIU M, LV J, TANG H, TAN J, LU C. Establishment and characterization of an ovarian cell line of the silkworm, Bombyx mori. Tissue and Cell, 2010,42(1):42-46. doi: 10.1016/j.tice.2009.07.002
[21]	BASCOM R A, SRINIVASAN S, NUSSBAUM R L. Identification and characterization of golgin-84, a novel Golgi integral membrane protein with a cytoplasmic coiled-coil domain. The Journal of Biological Chemistry, 1999,274(5):2953-2962. doi: 10.1074/jbc.274.5.2953
[22]	SATOH A, WANG Y, MALSAM J, BEARD M B, WARREN G. Golgin-84 is a rab1 binding partner involved in Golgi structure. Traffic, 2003,4(3):153-161. doi: 10.1034/j.1600-0854.2003.00103.x
[23]	DIAO A, RAHMAN D, PAPPIN D J, LUCOCQ J, LOWE M. The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation. The Journal of Cell Biology, 2003,160(2):201-212. doi: 10.1083/jcb.200207045
[24]	PINOT M, GOUD B, MANNEVILLE J B. Physical aspects of COPI vesicle formation. Molecular Membrane Biology, 2010,27(8):428-442. doi: 10.3109/09687688.2010.510485
[25]	SOHDA M, MISUMI Y, YAMAMOTO A, NAKAMURA N, OGATA S, SAKISAKA S, HIROSE S, IKEHARA Y, ODA K. Interaction of Golgin-84 with the COG complex mediates the intra-Golgi retrograde transport. Traffic, 2010,11(12):1552-1566. doi: 10.1111/tra.2010.11.issue-12
[26]	TONGMUANG N, YASAMUT U, SONGPRAKHON P, DECHTAWEWAT T, MALAKAR S, NOISAKRAN S, YENCHITSOMANUS P T, LIMJINDAPORN T. Coat protein complex I facilitates dengue virus production. Virus Research, 2018,250:13-20. doi: 10.1016/j.virusres.2018.03.021
[27]	LIMJINDAPORN T, WONGWIWAT W, NOISAKRAN S, SRISAWAT C, NETSAWANG J, PUTTIKHUNT C, KASINRERK W, AVIRUTNAN P, THIEMMECA S, SRIBURI R, SITTISOMBUT N, MALASIT P, YENCHITSOMANUS P T. Interaction of dengue virus envelope protein with endoplasmic reticulum-resident chaperones facilitates dengue virus production. Biochemical and Biophysical Research Communications, 2009,379(2):196-200. doi: 10.1016/j.bbrc.2008.12.070
[28]	HOWE C, GARSTKA M, AL-BALUSHI M, GHANEM E, ANTONIOU A N, FRITZSCHE S, JANKEVICIUS G, KONTOULI N, SCHNEEWEISS C, WILLIAMS A, ELLIOTT T, SPRINGER S. Calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules. The EMBO Journal, 2009,28(23):3730-3744. doi: 10.1038/emboj.2009.296
[29]	WU Y, DING Y, ZHENG X, LIAO K. The molecular chaperone Hsp90 maintains Golgi organization and vesicular trafficking by regulating microtubule stability. Journal of Molecular Cell Biology, 2020,12(6):448-461. doi: 10.1093/jmcb/mjz093
[30]	高囡囡, 鲍岚. 微管蛋白的翻译后修饰及功能研究. 生命科学, 2015,27(3):363-373.
	GAO N N, BAO L. Post-translational modification and function of tubulin. Chinese Bulletin of Life Sciences, 2015,27(3):363-373. (in Chinese)
[31]	TIAN G, HUANG Y, ROMMELAERE H, VANDEKERCKHOVE J, AMPE C, COWAN N J. Pathway leading to correctly folded β-tubulin. Cell, 1996,86(2):287-296. doi: 10.1016/S0092-8674(00)80100-2
[32]	JIN S, PAN L, LIU Z, WANG Q, XU Z, ZHANG Y Q. Drosophila tubulin-specific chaperone E functions at neuromuscular synapses and is required for microtubule network formation. Development, 2009,136(9):1571-1581. doi: 10.1242/dev.029983
[33]	METIVIER M, GALLAUD E, THOMAS A, PASCAL A, GAGNE J P, POIRIER G G, CHRETIEN D, GIBEAUX R, RICHARD-PARPAILLON L, BENAUD C, GIET R. Drosophila tubulin-specific chaperone E recruits tubulin around chromatin to promote mitotic spindle assembly. Current Biology, 2021,31(4):684-695. doi: 10.1016/j.cub.2020.11.009
[34]	HYDE J L, GILLESPIE L K, MACKENZIE J M. Mouse norovirus 1 utilizes the cytoskeleton network to establish localization of the replication complex proximal to the microtubule organizing center. Journal of Virology, 2012,86(8):4110-4122. doi: 10.1128/JVI.05784-11
[35]	DIGIUSEPPE S, LUSZCZEK W, KEIFFER T R, BIENKOWSKA- HABA M, GUION L G, SAPP M J. Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis. Proceedings of the National Academy of Sciences of the United States of America, 2016,113(22):6289-6294.
[36]	LOFTUS M S, VERVILLE N, KEDES D H. A conserved leucine zipper motif in gammaherpesvirus ORF52 is critical for distinct microtubule rearrangements. Journal of Virology, 2017,91(17):e00304-17.
[37]	CHEN S, LIU M, HUANG H, LI B, ZHAO H, FENG X Q, ZHAO H P. Heat stress-induced multiple multipolar divisions of human cancer cells. Cells, 2019,8(8):888. doi: 10.3390/cells8080888
[38]	LI S, WANG Y, HOU D, GUAN Z, SHEN S, PENG K, DENG F, CHEN X, HU Z, WANG H, WANG M. Host factor heat-shock protein 90 contributes to baculovirus budded virus morphogenesis via facilitating nuclear actin polymerization. Virology, 2019,535:200-209. doi: 10.1016/j.virol.2019.07.006

引物名称Primer name	引物序列Primer sequence
BmHSP90^HA-Sac I-F	5′-CGAGCTCATGTACCCATACGATGTTCCAGATTACGCTATGCCGGAAGAAATGGAGACA-3′
BmHSP90^HA-Sac II-R	5′-TCCCCGCGGGGATTAAGCGTAATCTGGAACATCGTATGGGTAATCAACCTCCTCCATGCGA-3′
BmGolga5^Flag-Xho I-F	5′-CCGCTCGAGCATGGATTACAAGGATGACGACGATAAGATGGCTTGGTTTGCGGACTT-3′
BmGolga5^Flag-Sac II-R	5′-TCCCCGCGGCTTATCGTCGTCATCCTTGTAATCTGACTTCATCGCTCTCGTGATAT-3′
BmTbce^Flag-BamH I-F	5′-CGGGATCCATGGATTACAAGGATGACGACGATAAGATGAGTAAAGTTTTTGTGCAAG-3′
BmTbce^Flag-Not I-R	5′-GAATGCGGCCGCTCTTATCGTCGTCATCCTTGTAATCTCTGAATTTAACTAGAATAACATCG-3′

蛋白ID Protein ID	描述 Description	分子量 Molecular weight (kD)	功能 Function
XP_012553114	Serine/threonine-protein phosphatase 5 (PP5)	53.90	信号转导机制、能量代谢 Signal transduction mechanisms, energy metabolism
NP_001268820	Juvenile hormone esterase-like precursor	58.41	控制细胞周期、细胞分裂 Cell cycle control, cell division
XP_004923154	Tubulin-specific chaperone E	58.52	控制细胞周期、细胞分裂、染色体分配 Cell cycle control, cell division, chromosome partitioning
XP_004923957	60 kD heat shock protein (HSP60)	62.91	翻译后修饰、蛋白质折叠、伴侣 Posttranslational modification, protein turnover, chaperones
XP_012550337	Golgin subfamily A member 5	67.87	碳水化合物的运输和代谢 Carbohydrate transport and metabolism
XP_012545391	SAM and SH3 domain-containing protein 1-like	70.73	信号转导机制Signal transduction mechanisms
NP_001036892	Heat shock cogn protein	71.39	翻译后修饰、蛋白质折叠、伴侣 Posttranslational modification, protein turnover, chaperones