Screening of S100A7 Interacting Proteins Using BioID Technology

doi:10.13523/j.cb.2203048

China Biotechnology

2022, Vol. 42

Issue (8): 52-62 DOI: 10.13523/j.cb.2203048

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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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

Received: 21 March 2022 Published: 07 September 2022

ZTFLH:

Q819

Corresponding Authors: Ling-yun HUANG,Xue-yuan XIAO E-mail: lyhuang@bnu.edu.cn;xyxiao@bnu.edu.cn

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	Jun-hao WANG
	Fei KONG
	Yun-guang LI
	Jin LIU
	En-ze HU
	Rui WANG
	Ling-yun HUANG
	Xue-yuan XIAO

Cite this article:

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.

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

Table 1 Gene primers for PCR

Fig.1 HEK293WT cells were transfected with S100A7-BioID recombinant plasmid to verify the expression of mRNA and protein (a)S100A7 specific primers were used to detect the mRNA expression level of S100A7 in the cells. Agarose gel electrophoresis showed that the experimental group was significantly increased (b)The protein level expression of S100A7-BioID recombinant plasmid was detected by Western blot. The arrows indicate the modified biotin ligase (BriA^*) and S100A7-BriA^* fusion proteins respectively, indicating that the two fusion proteins and their corresponding empty biotin ligase can be normally expressed in cells

Fig.2 Subcellular localization of S100A7 and S100A7-BioID fusion protein HEK293WT cells were transiently transfected with S100A7 BioID recombinant plasmid (pcDNA3.1-Myc -BriA^* - S100A7) and normal S100A7 overexpression plasmid (pcDNA3.1 - S100A7). S100A7 specific antibody showed S100A7, red fluorescence; DAPI specific staining of DNA showed nuclei with blue fluorescence. S100A7 and its fusion protein showed cytoplasmic diffuse distribution

Fig.3 Verification of BioID system by Western blot HEK293WT cells were transiently transfected with S100A7 BioID recombinant plasmid (pcDNA3.1-Myc-BriA^* - S100A7, pCI2- S100A7-BriA^* - HA) and corresponding empty plasmid, Biotin and its control group were established. Strepavidin HRP can covalently bind to biotin and display Western blot bands

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

Table 2 Results of mass spectrometry for Myc-BriA^*-S100A7 fusion protein

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

Table 3 Results of mass spectrometry for pCI2-S100A7-BriA^*-HA fusion protein

Fig.4 Co-IP verification of the interaction between S100A7 and AnxA2 in HEK293WT cells (a)The overexpression plasmids of two isoforms of AnxA2 and the corresponding empty plasmids were transfected into HEK293WT cells respectively. The overexpression effect was verified by Western blot (b)HEK293WT cells were transfected with S100A7 and AnxA2 overexpression plasmids and their corresponding empty plasmids, FLAG labeled antibody immunoprecipitation target protein, and Co-IP was used to verify their interaction (c)HEK293WT cells were transfected with S100A7 and AnxA2 overexpression plasmids and their corresponding empty plasmids, S100A7 antibody immunoprecipitation target protein, and Co-IP was used to verify their interaction

Fig.5 Co-localization verification of S100A7 and AnxA2 S100A7 overexpression plasmid (pcDNA3.1 - S100A7) and AnxA2 overexpression plasmid (pCMV14 AnxA2 flag). S100A7 specific antibody showed S100A7 with red fluorescence; FLAG tag antibody showed AnxA2 with green fluorescence; DAPI specific staining of DNA showed nuclei with blue fluorescence. S100A7 and AnxA2 were co-located in the cytoplasm and cell membrane


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