Screening and Identification of Proteins Interacting with the N-terminal 301~650 Peptide Fragment of BLM Helicase
ZHAO Jia-Fu1,2,*, YANG Yuan-Qing3, WEN Xiao-Yan3, ZHANG Xi-Lu1,2, CHEN Yi1,2, PAN Zhi-Ren1,2, WEI Zhong-Yi1,2
1 Key Laboratory of Genetic Breeding and Reproduction of Highland Mountain Animals of Ministry of Education/Key Laboratory of Animal Genetic Breeding and Reproduction of Guizhou Province, Guizhou University, Guiyang 550025, China; 2 School of Animal Science, Guizhou University, Guiyang 550025, China; 3 Bijie Livestock Station, Bijie 551700, China
摘要布卢姆综合征突变蛋白(Bloom syndrome mutated, BLM)作为人类重组Q (recombination Q, RecQ)解旋酶家族的重要一员,在DNA复制、修复、重组、转录、端粒维持等细胞代谢过程中发挥作用。对BLM解旋酶的结构研究发现,其除了RecQ解旋酶家族所包含的解旋酶结构域(helicase domain, HD)、RecQ解旋酶C端结构域(RecQ C-terminal domain, RecQ-Ct)和解旋酶与RNase D羧基端结构域(helicase and RNase D C-terminal domain, HRDC) 3个典型保守区域外,在羧基端(C端)还发现了核定位信号(nuclear localization signal, NLS),但在氨基端(N端)的研究较少,本课题组对BLM N端结构分析发现,在619~843 bp处有许多苏氨酸磷酸化位点,推测其在BLM解旋酶的翻译后修饰(post-translational modifications, PTMs)中发挥了重要作用,但哪些细胞蛋白参与了这一生理过程目前并不完全清楚。为筛选与BLM解旋酶N端301~650肽段(BLM301~650)具有相互作用的细胞蛋白,并鉴定其与BLM解旋酶N端的相互作用,本研究采用免疫沉淀-高效液相色谱串联质谱(IP-HPLC-MS/MS)技术筛选前列腺癌细胞系3 (prostate cancer 3, PC3)中与BLM301~650具有相互作用的细胞蛋白,通过注释、可视化与整合发现数据库(database for annotation, visualization and integrated discovery, DAVID)和基因/蛋白质相互作用检索工具(search tool for the retrieval of interacting genes/proteins, STRING)对筛选得到的互作蛋白进行生物信息学分析,利用免疫荧光验证BLM301~650与互作蛋白的亚细胞共定位,通过Pull-down实验证实互作蛋白间的互作关系。IP-HPLC-MS/MS结果显示,筛选到172个与BLM301~650特异性结合的互作蛋白,KEGG分析显示这些互作蛋白主要富集于核糖体通路;蛋白互作网络分析发现,核糖体蛋白S27a (ribosomal protein S27a, RPS27A)被鉴定为BLM301~650的关键互作因子;免疫荧光和Pull-down实验证实BLM301~650与RPS27A在细胞核中共定位,且两者之间为直接靶向关系。综上,RPS27A与BLM解旋酶N端的直接相互作用,为核糖体功能与DNA修复通路的交叉调控提供了新的分子桥梁,对阐释基因组稳定性维持、肿瘤发生及DNA损伤应答机制具有重要的科学价值。
Abstract：As a vital member of the human recombination Q (RecQ) helicase family, Bloom syndrome mutated protein (BLM) plays essential roles in multiple cellular metabolic processes, including DNA replication, repair, recombination, transcription, and telomere maintenance. Structural studies on BLM helicase have revealed that, in addition to the 3 canonical conserved domains characteristic of the RecQ helicase family—the helicase domain (HD), RecQ C-terminal domain (RecQ-Ct) and helicase and RNase D C-terminal domain (HRDC)—a nuclear localization signal (NLS) is also identified at its carboxyl terminus (C-terminus).However, research on its amino terminus (N-terminus) remains limited. This research group's structural analysis of the BLM N-terminus has found that a large number of threonine phosphorylation sites were present in the region spanning 619~843 bp. It was hypothesized that these sites play a critical function in the post-translational modifications (PTMs) of BLM helicase, yet the cellular proteins involved in this physiological process remain incompletely elucidated. To screen for cellular proteins that interact with the N-terminal 301~650 peptide fragment of BLM helicase (BLM301~650) and further characterize their interaction with the BLM N-terminus, this present study employed immunoprecipitation-high-performance liquid chromatography tandem mass spectrometry (IP-HPLC-MS/MS) to identify BLM301~650-interacting proteins in the prostate cancer (PC3) cell line. Bioinformatic analyses of the screened interacting proteins were performed using the database for annotation, visualization and integrated discovery (DAVID) and search tool for the retrieval of interacting genes/proteins (STRING). The subcellular colocalization between BLM301~650 and its interacting proteins was validated via immunofluorescence assay, while the interaction relationship was confirmed by Pull-down assays. IP-HPLC-MS/MS results demonstrated that a total of 172 proteins specifically binding to BLM301~650 were identified. KEGG pathway enrichment analysis indicated that these interacting proteins were predominantly enriched in the ribosome pathway. Protein-protein interaction (PPI) network analysis further identified ribosomal protein S27a (RPS27A) as a key interacting factor of BLM301~650. Immunofluorescence and Pull-down assays verified that BLM301~650 and RPS27A colocalized in the nucleoplasm and exhibited a direct targeting relationship. Collectively, the direct interaction between RPS27A and the N?terminal region of BLM helicase serves as a novel molecular bridge for the crosstalk between the ribosomal pathway and the DNA repair pathway, and is of great scientific significance for elucidating the mechanisms underlying genome stability maintenance, tumorigenesis, and DNA damage response.
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