Dueñas Mena Dulce Liliana, Gutiérrez-Pabello José A, Quintero Chávez Kaina, Brito-Perea Mirna Del Carmen, Díaz Padilla Dania Melissa, Cortez Hernández Omar, Chávez Mendez José Román, Alcalá Zacarias Jocelyn Marcela, Vela Sancho Giselle Berenice, Landeros Sánchez Bertha
Facultad de Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja California, Tijuana 22390, Mexico.
Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, México City 04510, Mexico.
Int J Mol Sci. 2024 Nov 26;25(23):12687. doi: 10.3390/ijms252312687.
subsp. (MAP) is known to cause paratuberculosis. One notable protein, MAP3773c, plays a critical role in iron metabolism as a transcription factor. This study aims to investigate the binding affinity of MAP3773c to the chromatin of the Ferroportin1 (FPN1) gene in murine macrophage J774 A.1. We conducted a sequence alignment to identify potential interaction sites for MAP3773c. Following this, we used in silico analysis to predict binding interactions, complemented by electrophoretic mobility shift assay (EMSA) to confirm in vitro binding of MAP3773c. The map3773c gene was cloned into the pcDNA3.1 vector, with subsequent expression analysis carried out via Western blotting and real-time PCR. Chromatin immunoprecipitation (CHiP) assays were performed on transfected macrophages to confirm binding in the native chromatin context. Our in silico and in vitro analysis indicated that MAP3773c interacts with two binding motifs within the FPN1 coding region. The ChiP results provided additional validation, demonstrating the binding of MAP3773c to the FPN1 chromatin through successful amplification of the associated chromatin fragment via PCR. Our study demonstrated that MAP3773c binds to FPN1 and provides insight into the role of MAP3773c and its effect on host iron transport.
亚种（MAP）已知会导致副结核病。一种值得注意的蛋白质，MAP3773c，作为转录因子在铁代谢中起关键作用。本研究旨在调查MAP3773c与小鼠巨噬细胞J774 A.1中Ferroportin1（FPN1）基因染色质的结合亲和力。我们进行了序列比对以确定MAP3773c的潜在相互作用位点。在此之后，我们使用计算机分析来预测结合相互作用，并辅以电泳迁移率变动分析（EMSA）以确认MAP3773c的体外结合。将map3773c基因克隆到pcDNA3.1载体中，随后通过蛋白质免疫印迹和实时PCR进行表达分析。对转染的巨噬细胞进行染色质免疫沉淀（CHiP）测定，以确认在天然染色质环境中的结合。我们的计算机分析和体外分析表明，MAP3773c与FPN1编码区内的两个结合基序相互作用。CHiP结果提供了额外的验证，通过PCR成功扩增相关染色质片段证明了MAP3773c与FPN1染色质的结合。我们的研究表明MAP3773c与FPN1结合，并深入了解了MAP3773c的作用及其对宿主铁转运的影响。
