Structural analysis of full-length lysine acetyltransferase 7 by cryo-electron microscopy

doi:10.3969/j.issn.1674-8115.2023.09.004

Abstract

Abstract:

Objective ·To analyze the full-length protein structure of human-derived lysine acetyltransferase 7 (KAT7) using cryo-electron microscopy (Cryo-EM) and to obtain the profile information of human-derived KAT7. Methods ·The recombinant protein expression plasmid pGEX-4T1-GST-KAT7 was constructed by using the pGEX-4T1 vector and the full-length gene of human-derived KAT7, and the KAT7 protein was expressed in the prokaryotic protein expression system BL21 (DE3). The GST-KAT7 recombinant protein was obtained by using GST affinity chromatography. After removing the GST protein tag with TEV protease, KAT7 was further isolated and purified by HiLoad 16/600 Superdex 75 pg volume exclusion chromatography. The obtained protein samples were identified by Western blotting, and the samples were screened. The protein morphology was observed under negative-stain electron microscopy, and data were collected by using Cryo-EM. The protein particles were selected and the spatial structure of the full-length KAT7 was analyzed with the Cryo-EM analysis software CryoSparc. The MYST structural domain model (5GK9) in the Protein Data Bank (PDB) and AlphaFold prediction model of KAT7 were matched with the generated structural model by UCSF Chimera software. Results ·The full-length protein of human-derived KAT7 was successfully purified by affinity chromatography, and high purity KAT7 was obtained by volume exclusion chromatography. After identifying KAT7 by Western blotting, the spatial structure of KAT7 full-length protein was initially resolved by Cryo-EM and single-particle reconstruction techniques, and a preliminary three-dimensional structure model with a resolution of about 10 ? was obtained by three-dimensional optimization. The spatial structure of KAT7 full-length protein was irregular and semi-loop-shaped, and the existing MYST domain model (PDB: 5GK9) can be matched into the C-terminal part of the KAT7 full-length model. The adjusted AlphaFold prediction model can also match the KAT7 full-length structure model. Conclusion ·A preliminary analysis of the spatial structure model of full-length protein of human-derived KAT7 is performed by using Cryo-EM.

Key words: lysine acetyltransferase, post-translational protein modification, acetylation, cryo-electron microscopy

CLC Number:

Q518.2

ZHENG Guopei, CAO Qin, SHEN Jianfeng. Structural analysis of full-length lysine acetyltransferase 7 by cryo-electron microscopy[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(9): 1099-1106.

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References 26

1	SUGANUMA T, WORKMAN J L. Signals and combinatorial functions of histone modifications[J]. Annu Rev Biochem, 2011, 80: 473-499.
2	KIM M S, CHO H I, PARK S H, et al. The histone acetyltransferase Myst2 regulates Nanog expression, and is involved in maintaining pluripotency and self-renewal of embryonic stem cells[J]. FEBS Lett, 2015, 589(8): 941-950.
3	BOBDE R C, KUMAR A, VASUDEVAN D. Plant-specific HDT family histone deacetylases are nucleoplasmins[J]. Plant Cell, 2022, 34(12): 4760-4777.
4	WU Q S, HUANG Q Y, GUAN H L, et al. Comprehensive genome-wide analysis of histone acetylation genes in roses and expression analyses in response to heat stress[J]. Genes, 2022, 13(6): 980.
5	MARMORSTEIN R, ZHOU M M. Writers and readers of histone acetylation: structure, mechanism, and inhibition[J]. Cold Spring Harb Perspect Biol, 2014, 6(7): a018762.
6	XING G F, JIN M S, QU R F, et al. Genome-wide investigation of histone acetyltransferase gene family and its responses to biotic and abiotic stress in foxtail millet (Setaria italica[L.]P. Beauv)[J]. BMC Plant Biol, 2022, 22(1): 292.
7	WANG W, ZHENG Y X, SUN S H, et al. A genome-wide CRISPR-based screen identifies KAT7 as a driver of cellular senescence[J]. Sci Transl Med, 2021, 13(575): eabd2655.
8	HEINLEIN M, GANDOLFO L C, ZHAO K L, et al. The acetyltransferase KAT7 is required for thymic epithelial cell expansion, expression of AIRE target genes, and thymic tolerance[J]. Sci Immunol, 2022, 7(67): eabb6032.
9	HAN Z, WU H, KIM S, et al. Revealing the protein propionylation activity of the histone acetyltransferase MOF (males absent on the first)[J]. J Biol Chem, 2018, 293(9): 3410-3420.
10	NIIDA H, MATSUNUMA R, HORIGUCHI R, et al. Phosphorylated HBO1 at UV irradiated sites is essential for nucleotide excision repair[J]. Nat Commun, 2017, 8: 16102.
11	YAN M S, TURGEON P J, MAN H S J, et al. Histone acetyltransferase 7 (KAT7)-dependent intragenic histone acetylation regulates endothelial cell gene regulation[J]. J Biol Chem, 2018, 293(12): 4381-4402.
12	NEWMAN D M, VOSS A K, THOMAS T, et al. Essential role for the histone acetyltransferase KAT7 in T cell development, fitness, and survival[J]. J Leukoc Biol, 2017, 101(4): 887-892.
13	STERNER D E, BERGER S L. Acetylation of histones and transcription-related factors[J]. Microbiol Mol Biol Rev, 2000, 64(2): 435-459.
14	SAKSOUK N, AVVAKUMOV N, CHAMPAGNE K S, et al. HBO1 HAT complexes target chromatin throughout gene coding regions via multiple PHD finger interactions with histone H3 tail[J]. Mol Cell, 2009, 33(2): 257-265.
15	MIOTTO B, STRUHL K. JNK1 phosphorylation of Cdt1 inhibits recruitment of HBO1 histone acetylase and blocks replication licensing in response to stress[J]. Mol Cell, 2011, 44(1): 62-71.
16	TAO Y, ZHONG C, ZHU J J, et al. Structural and mechanistic insights into regulation of HBO1 histone acetyltransferase activity by BRPF2[J]. Nucleic Acids Res, 2017, 45(10): 5707-5719.
17	AU Y Z, GU M X, DE BRAEKELEER E, et al. KAT7 is a genetic vulnerability of acute myeloid leukemias driven by MLL rearrangements[J]. Leukemia, 2021, 35(4): 1012-1022.
18	MCCULLOUGH C E, MARMORSTEIN R. Molecular basis for histone acetyltransferase regulation by binding partners, associated domains, and autoacetylation[J]. ACS Chem Biol, 2016, 11(3): 632-642.
19	FENG Y P, VLASSIS A, ROQUES C, et al. BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation[J]. EMBO J, 2016, 35(2): 176-192.
20	YAN K Z, YOU L Y, DEGERNY C, et al. The chromatin regulator BRPF3 preferentially activates the HBO1 acetyltransferase but is dispensable for mouse development and survival[J]. J Biol Chem, 2016, 291(6): 2647-2663.
21	KUEH A J, BERGAMASCO M I, QUAGLIERI A, et al. Stem cell plasticity, acetylation of H3K14, and de novo gene activation rely on KAT7[J]. Cell Rep, 2023, 42(1): 111980.
22	MACPHERSON L, ANOKYE J, YEUNG M M, et al. HBO1 is required for the maintenance of leukaemia stem cells[J]. Nature, 2020, 577(7789): 266-270.
23	XIAO Y H, LI W J, YANG H, et al. HBO1 is a versatile histone acyltransferase critical for promoter histone acylations[J]. Nucleic Acids Res, 2021, 49(14): 8037-8059.
24	IIZUKA M, SARMENTO O F, SEKIYA T, et al. Hbo1 links p53-dependent stress signaling to DNA replication licensing[J]. Mol Cell Biol, 2008, 28(1): 140-153.
25	XIE C, SHEN H, ZHANG H, et al. Quantitative proteomics analysis reveals alterations of lysine acetylation in mouse testis in response to heat shock and X-ray exposure[J]. Biochim Biophys Acta Proteins Proteom, 2018, 1866(3): 464-472.
26	LAN R F, WANG Q Q. Deciphering structure, function and mechanism of lysine acetyltransferase HBO1 in protein acetylation, transcription regulation, DNA replication and its oncogenic properties in cancer[J]. Cell Mol Life Sci, 2020, 77(4): 637-649.

[1]	DAI Qin, WANG Weiming. Inflammatory expression in IgA nephropathy cell model and anti-inflammatory effect of sodium valproate [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(6): 751-757.
[2]	DUAN Yujuan, HUANG Jing. Negative-stain electron microscopic study of the nucleosome remodeling and deacetylase complex [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(4): 455-463.
[3]	Ya-nan LAI, Yu-feng YAO, Xiao-kui GUO, Jin-jing NI. Acetylation involved in bacterial susceptibility to ribosome-targeting antibiotics [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(3): 308-313.
[4]	DAI Qin1, WANG Wei-ming2. Role of histone acetylation in the pathogenesis of IgA nephropathy [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2020, 40(8): 1063-1068.
[5]	YANG Shuang-shuang1, GAO Tian-xing2, HE Xuan1, ZHANG Rui1, ZHANG Yong-fang1. Regulation on brain-derived neurotrophic factor and relevant mechanism of anemarrhena saponin in H2O2-induced SH-SY5Y cells [J]. , 2019, 39(6): 578-.
[6]	YANG Yi-qi, TANG Ting-ting. SIRT1 signaling pathway in bone metabolism [J]. , 2019, 39(11): 1335-.
[7]	LIU Li-jie1, HONG Xi1, HUANG Xian-yu3, LUO Jing3, YU Jian-jun1, 2. Global levels of lysine crotonylation in prostate cancer and its correlation with clinical stages and pathologic grades [J]. , 2018, 38(7): 788-.
[8]	YANG Xiao, DU Yun-lan, GUAN Yang-tai. Research advances in acetylation modification of α-synuclein in Parkinsons disease [J]. , 2018, 38(11): 1381-.
[9]	FENG Hai-zhong. Histone modification and glioma tumorigenesis [J]. , 2018, 38(1): 1-.
[10]	ZHANG Lin, HE Ming. Roles of Sirtuins in occurrence and development of chronic kidney disease [J]. , 2015, 35(4): 594-.
[11]	REN Jie, SANG Yu, YAO Yu-feng, et al. Effects of acetylation on acid tolerance response of Salmonella typhimurium [J]. , 2014, 34(5): 584-.
[12]	OU Mei-xian, SHI Yi-ming, LIU Hui-zhong, et al. Synthesis of tropane compounds and their antagonistic activity to M3 receptors [J]. , 2011, 31(7): 909-.