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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2022, Vol. 42 Issue (8): 52-62    DOI: 10.13523/j.cb.2203048
技术与方法     
运用BioID技术筛选S100A7互作蛋白*
王俊豪,孔菲,李允广,刘进,胡恩泽,王睿,黄凌云**(),肖雪媛**()
北京师范大学生命科学学院 细胞增殖及调控生物学教育部重点实验室 北京 100875
Screening of S100A7 Interacting Proteins Using BioID Technology
WANG Jun-hao,KONG Fei,LI Yun-guang,LIU Jin,HU En-ze,WANG Rui,HUANG Ling-yun**(),XIAO Xue-yuan**()
Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education,College of Life Sciences, Beijing Normal University, Beijing 100875, China
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摘要:

目的: 通过邻近生物素鉴定(BioID)技术筛选S100A7互作蛋白并进行验证。方法: 采用邻近生物素BioID和质谱相结合的方法,筛选S100A7互作蛋白。通过免疫荧光和免疫共沉淀方法,对相互作蛋白进行验证。结果: 与对照组比对,通过BioID技术共获得94个可能与 S100A7 相互作用的候选蛋白质。选取 Annexin A2(AnxA2)蛋白在 HEK293 细胞中进行了验证,结果表明S100A7 与 AnxA2 存在直接的相互作用,且二者共定位于细胞质和细胞膜。结论: 可将BioID技术作为互作蛋白筛选的一项新技术,通过该技术发现S100A7和AnxA2存在相互作用。

关键词: S100A7BioIDAnnexin A2互作蛋白    
Abstract:

Objective: To investigate and verify the candidate interactors of S100A7. Methods: The candidate interactors of S100A7 are identified using BioID and mass spectrum and verified by Co-IP and immunofluorescence. Results: 94 candidate proteins are identified in two groups by mass spectrum. Interaction between Annexin A2(AnxA2) and S100A7 was then verified using Co-IP in HEK293WT cells. It is demonstrated that AnxA2 interacts with S100A7 directly, and the immunofluorescence results show that S100A7 and AnxA2 are collocating in the cytoplasm. Conclusion: BioID is a new technology for screening S100A7 interactors and the interaction between S100A7 and AnxA2 is discovered using BioID.

Key words: S100A7    BioID    Annexin A2    Interaction proteins
收稿日期: 2022-03-21 出版日期: 2022-09-07
ZTFLH:  Q819  
基金资助: * 国家重点基础研究计划(2011CB915504);国家自然科学基金(81172216)
通讯作者: 黄凌云,肖雪媛     E-mail: lyhuang@bnu.edu.cn;xyxiao@bnu.edu.cn
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引用本文:

王俊豪,孔菲,李允广,刘进,胡恩泽,王睿,黄凌云,肖雪媛. 运用BioID技术筛选S100A7互作蛋白*[J]. 中国生物工程杂志, 2022, 42(8): 52-62.

WANG Jun-hao,KONG Fei,LI Yun-guang,LIU Jin,HU En-ze,WANG Rui,HUANG Ling-yun,XIAO Xue-yuan. Screening of S100A7 Interacting Proteins Using BioID Technology. China Biotechnology, 2022, 42(8): 52-62.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2203048        https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I8/52

质粒 pcDNA3.1-Myc-BriA*-S100A7 pCI2-S100A7-BriA*-HA
Forward 5'-CGAATTCATGAGCAACACTCAAGCTGAGAGG (EcoRI) 5'-CGAATTCATGAGCAACACTCAAGCTGAGAGG (EcoRI)
Reverse 5'-TTGCTCGAGTCACTGGCTGCCCCCGGAACAGGG (XhoI) 5'-TTGCTCGAGGCCTCCCTGGCTGCCCCCGGAACAGGG (XhoI)
表1  用于PCR的基因引物
图1  HEK293WT 细胞转染 S100A7-BioID 重组质粒验证 mRNA 和蛋白质表达
图2  S100A7 与 S100A7-BioID 融合蛋白亚细胞定位
图3  Western blot 验证 BioID 体系
Accession # AAs MW/kDa pI Description
J3QL15 128 15.0 11.97 核糖体蛋白L19(片段)
Q02878 288 32.7 10.58 60S核糖体蛋白L6
H0YB56 224 24.5 9.76 蛋白 LYRIC (片段)
F8W1N5 71 7.8 9.63 新生多肽相关复合物亚单位α(片段)
Q5JW30 494 54.7 9.51 双链RNA结合蛋白Staufen同源物1
B2ZWH1 50 5.9 9.41 钙周期素结合蛋白(片段)
Q9NWB6-3 198 23.8 9.20 富含精氨酸和谷氨酸的蛋白质1的亚型3
Q9UHR4 511 56.8 8.68 脑特异性血管生成抑制剂1相关蛋白2
M0R080 183 20.5 8.56 DnaJ同源亚家族B成员1(片段)
H0Y8K3 188 21.0 8.34 蛋白质CDV3同源物(片段)
Q96HC4 596 63.9 8.21 PDZ和LIM结构域蛋白5
E7ETU6 137 15.8 8.19 基质膜相关蛋白1
P30419-2 416 48.1 8.12 甘氨酰肽N-十四烷酰转移酶1的亚型
A1L3A6 120 14.6 8.07 NUFIP2蛋白
P48668 564 60.0 8.00 角蛋白,II型细胞骨架6C
P07355 339 38.6 7.75 膜联蛋白A2
Q9UGI8-2 412 46.9 7.59 睾丸素亚型2
P20290-2 162 17.7 7.50 转录因子BTF3的亚型2
Q06124-3 460 52.8 7.44 酪氨酸蛋白磷酸酶非受体11型亚型3
F8WBG8 128 13.7 7.21 脑发育蛋白样蛋白
P18669 254 28.8 7.18 磷酸甘油酸变位酶1
Q15365 356 37.5 7.09 聚(rC)结合蛋白1
O00151 329 36.0 7.02 PDZ和LIM结构域蛋白1
P13639 858 95.3 6.83 翻译延长因子2
P31151 101 11.5 6.77 蛋白质S100-A7
Q9Y385 318 35.2 6.74 泛素结合酶E2 J1
P46109 303 33.8 6.74 Crk样蛋白
P98170 497 56.6 6.65 E3泛素蛋白连接酶XIAP
Q9UQ80 394 43.8 6.55 增殖相关蛋白2G4
P81605 110 11.3 6.54 皮霉素
P55196-2 1612 181.9 6.52 极性蛋白AF6亚型1
P33176 963 109.6 6.51 驱动蛋白-1重链
F8WCH0 52 5.6 6.49 肌动蛋白,γ肠平滑肌
Q9Y5K6 639 71.4 6.40 CD2相关蛋白
O75534-2 767 85.7 6.20 冷休克域蛋白E1的亚型2
P14923 745 81.7 6.14 连接蛋白
F5H8K9 500 53.9 5.92 角蛋白,II型细胞骨架4
Q08188 693 76.6 5.86 蛋白质谷氨酰胺γ谷氨酰转移酶E
Accession # AAs MW/kDa pI Description
P49959-2 680 77.6 5.82 双链断裂修复蛋白MRE11A的亚型2
Q9Y5X1 595 66.5 5.58 排序nexin-9
P31944 242 27.7 5.58 胱天蛋白酶-14
G3V226 76 8.9 5.54 钙调素
Q08554-2 840 93.8 5.53 桥粒蛋白-1的1B亚型
F5H5A9 277 31.7 5.50 SKP1 G2等位基因抑制子
V9GZ37 476 51.9 5.49 热休克70 kDa蛋白1A/1B O
P05783 430 48.0 5.45 角蛋白,I型细胞骨架18
K7EP07 169 19.2 5.34 微管蛋白折叠辅因子B(片段)
M0R3A4 183 20.8 5.29 含PIH1结构域的蛋白质1(片段)
F5GWP8 591 66.3 5.19 连接蛋白
S4R457 77 8.8 4.88 异质核核糖核蛋白K
G8JLF3 614 66.4 4.88 Rab样蛋白6
P19012 456 49.2 4.77 角蛋白,I型细胞骨架15
M0R3I1 213 23.3 4.65 表皮生长因子受体底物15样1(片段)
Q96K17-2 100 10.9 4.50 转录因子BTF3同源物4的亚型2
表2  Myc-BriA*-S100A7融合蛋白生物素亲和纯化质谱鉴定结果
Accession # AAs MW/kDa pI Description
P50990 548 59.6 5.60 T复合蛋白1亚单位θ
P48668 564 60.0 8.00 角蛋白,II型细胞骨架6C
P49959 708 80.5 5.90 双链断裂修复蛋白MRE11A
P08107 641 70.0 5.66 热休克70 kDa蛋白1A/1B
F8VZY9 391 43.7 5.35 角蛋白,I型细胞骨架18
Q04695 432 48.1 5.02 Ke角蛋白,I型细胞骨架17
Q5XKE5 535 57.8 7.20 角蛋白,II型细胞骨架79
B4DS13 572 64.8 6.13 真核翻译起始因子4B
Q9Y265-2 386 42.1 6.38 IsRuvB样1的亚型2
Q9Y266 331 38.2 5.38 核迁移蛋白nudC
C9JX92 1743 197.5 6.54 极性蛋白AF6
O75534-2 767 85.7 6.20 冷休克域蛋白E1的亚型2
P08727 400 44.1 5.14 角蛋白,I型细胞骨架19
P07355 339 38.6 7.75 膜联蛋白A2
Q14157-4 976 103.1 6.87 泛素相关蛋白2样亚型4
Q9Y5K6 639 71.4 6.40 CD2相关蛋白
Q9NWB6 198 23.8 9.20 富含精氨酸和谷氨酸的蛋白质1的亚型3
Q9UBC2-3 754 83.2 5.08 表皮生长因子受体底物15样1的亚型3
Q71U36-2 416 46.3 5.08 微管蛋白α-1A链的亚型2
Q08554-2 840 93.8 5.53 桥粒蛋白-1的1B亚型
Accession # AAs MW/kDa pI Description
H3BMT0 115 12.2 8.19 血液学和神经系统表达1-样蛋白
P31151 101 11.5 6.77 S100A7蛋白
Q9H2G2-2 1204 138.9 5.10 STE20样丝氨酸/苏氨酸蛋白激酶亚型2
Q5HY57 219 24.9 5.02 伊默菌素
K7EIJ4 155 17.0 5.00 Ran结合蛋白3(片段)
O15173 223 23.8 4.88 膜相关孕酮受体成分2
Q9Y2J2-3 612 69.5 6.24 带4.1样蛋白3的亚型3
Q7Z417 695 76.1 8.70 核脆性X智力低下相互作用蛋白2
F8W7C6 163 18.6 9.95 60S核糖体蛋白L10
Q99456 494 53.5 4.78 角蛋白,I型细胞骨架12
K7ENK9 68 7.8 8.79 囊泡相关膜蛋白2
P48634-4 1533 162.9 7.02 蛋白PRRC2A的亚型4
F8WBG8 128 13.7 7.21 脑发育蛋白样蛋白
P05109 93 10.8 7.03 S100A8蛋白
P32119 198 21.9 5.97 过氧氧化还原酶-2
C9JW34 43 4.5 10.13 复制因子C亚单位4(片段)
E9PJ90 118 13.5 8.66 HBS1样蛋白
P18206-2 1066 116.6 6.09 黏着斑蛋白亚型1
E7EMM2 984 111.2 8.10 AP-3复合亚单位δ-1
H0YMP5 130 14.0 4.54 Arpin蛋白
表3  pCI2-S100A7-BriA*-HA融合蛋白标记质谱鉴定结果
图4  HEK293WT 细胞中S100A7 与 AnxA2 相互作用关系 Co-IP 验证
图5  S100A7与 AnxA2免疫荧光共定位验证
[1] Varnaitė R, MacNeill S A. Meet the neighbors: mapping local protein interactomes by proximity-dependent labeling with BioID. Proteomics, 2016, 16(19): 2503-2518.
doi: 10.1002/pmic.201600123 pmid: 27329485
[2] Choi-Rhee E, Schulman H, Cronan J E. Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. Protein Science, 2004, 13(11): 3043-3050.
pmid: 15459338
[3] Roux K J. Marked by association: techniques for proximity-dependent labeling of proteins in eukaryotic cells. Cellular and Molecular Life Sciences, 2013, 70(19): 3657-3664.
doi: 10.1007/s00018-013-1287-3
[4] Roux K J, Kim D I, Raida M, et al. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. The Journal of Cell Biology, 2012, 196(6): 801-810.
doi: 10.1083/jcb.201112098
[5] Kim D I, Birendra K C, Zhu W H, et al. Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(24): E2453-E2461.
[6] Comartin D, Gupta G D, Fussner E, et al. CEP120 and SPICE 1 cooperate with CPAP in centriole elongation. Current Biology, 2013, 23(14): 1360-1366.
doi: 10.1016/j.cub.2013.06.002 pmid: 23810536
[7] Firat-Karalar E N, Stearns T. Probing mammalian centrosome structure using BioID proximity-dependent biotinylation. Methods in Cell Biology, 2015, 129: 153-170.
doi: S0091-679X(15)00102-8 pmid: 26175438
[8] Couzens A L, Knight J D R, Kean M J, et al. Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions. Science Signaling, 2013, 6(302): rs15.
[9] Kim D I, Jensen S C, Noble K A, et al. An improved smaller biotin ligase for BioID proximity labeling. Molecular Biology of the Cell, 2016, 27(8): 1188-1196.
doi: 10.1091/mbc.E15-12-0844
[10] Madsen P, Rasmussen H H, Leffers H, et al. Molecular cloning, occurrence, and expression of a novel partially secreted protein "psoriasin" that is highly up-regulated in psoriatic skin. The Journal of Investigative Dermatology, 1991, 97(4): 701-712.
doi: 10.1111/1523-1747.ep12484041
[11] Emberley E D, Murphy L C, Watson P H. S100A7 and the progression of breast cancer. Breast Cancer Research: BCR, 2004, 6(4): 153-159.
doi: 10.1186/bcr816
[12] Alowami S, Qing G F, Emberley E, et al. Psoriasin (S100A7) expression is altered during skin tumorigenesis. BMC Dermatology, 2003, 3: 1.
pmid: 12600274
[13] Kataoka K, Ono T, Murata H, et al. S100A 7 promotes the migration and invasion of osteosarcoma cells via the receptor for advanced glycation end products. Oncology Letters, 2012, 3(5): 1149-1153.
pmid: 22783409
[14] Fukuzawa H, Kiyoshima T, Kobayashi I, et al. Transcription promoter activity of the human S100A 7 gene in oral squamous cell carcinoma cell lines. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 2006, 1759(3-4): 171-176.
doi: 10.1016/j.bbaexp.2006.03.004
[15] Kong F, Li Y G, Hu E Z, et al. The characteristic of S100A 7 induction by the hippo-YAP pathway in cervical and glossopharyngeal squamous cell carcinoma. PLoS One, 2016, 11(12): e0167080.
doi: 10.1371/journal.pone.0167080
[16] Li Y G, Kong F, Shao Q R, et al. YAP expression and activity are suppressed by S100A7 via p65/NFκB-mediated repression of ΔNp63. Molecular Cancer Research: MCR, 2017, 15(12): 1752-1763.
doi: 10.1158/1541-7786.MCR-17-0349
[17] Brandherm I, Disse J, Zeuschner D, et al. cAMP-induced secretion of endothelial von Willebrand factor is regulated by a phosphorylation/dephosphorylation switch in annexin A2. Blood, 2013, 122(6): 1042-1051.
doi: 10.1182/blood-2012-12-475251 pmid: 23757730
[18] de Graauw M, Tijdens I, Smeets M B, et al. Annexin A2 phosphorylation mediates cell scattering and branching morphogenesis via cofilin Activation. Molecular and Cellular Biology, 2008, 28(3): 1029-1040.
doi: 10.1128/MCB.01247-07 pmid: 18070928
[19] Lokman N A, Elder A S F, Ween M P, et al. Annexin A2 is regulated by ovarian cancer-peritoneal cell interactions and promotes metastasis. Oncotarget, 2013, 4(8): 1199-1211.
doi: 10.18632/oncotarget.1122 pmid: 23945256
[20] Cui H Y, Wang S J, Miao J Y, et al. CD147 regulates cancer migration via direct interaction with Annexin A2 and DOCK3-β-catenin-WAVE2 signaling. Oncotarget, 2016, 7(5): 5613-5629.
doi: 10.18632/oncotarget.6723
[21] Jia J W, Li K L, Wu J X, et al. Clinical significance of annexin II expression in human non-small cell lung cancer. Tumor Biology, 2013, 34(3): 1767-1771.
doi: 10.1007/s13277-013-0715-1
[22] Moreau K, Ghislat G, Hochfeld W, et al. Transcriptional regulation of Annexin A 2 promotes starvation-induced autophagy. Nature Communications, 2015, 6: 8045.
doi: 10.1038/ncomms9045 pmid: 26289944
[23] Lokman N A, Pyragius C E, Ruszkiewicz A, et al. Annexin A2 and S100A10 are independent predictors of serous ovarian cancer outcome. Translational Research, 2016, 171: 83-95.e2.
doi: 10.1016/j.trsl.2016.02.002 pmid: 26925708
[24] 王英, 赵晓航. Psoriasin(S 100A7)蛋白的结构与功能. 癌症进展, 2003, 1(1): 10-13.
Wang Y, Zhao X H. The structure and functions of psoriasin (S 100A7). Oncology Progress, 2003, 1(1): 10-13.
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