Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Instituto Politécnico Nacional 2508, San Pedro Zacatenco, Gustavo A. Madero, 07360, Mexico City, Mexico.
Programa Institucional en Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico Nacional, Guillermo Massieu Helguera 239, La Escalera, Gustavo A. Madero, 07320, Mexico City, Mexico.
Microb Pathog. 2022 Jan;162:105349. doi: 10.1016/j.micpath.2021.105349. Epub 2021 Dec 2.
The heat shock response is a conserved mechanism that allows cells to respond and survive stress damage and is transcriptionally regulated by the heat shock factors and heat shock elements. The P-glycoprotein confer the multidrug resistance phenotype; Entamoeba histolytica has the largest multidrug resistance gene family described so far; one of these genes, the EhPgp5 gene, has an emetine-inducible expression. A functional heat shock element was localized in the EhPgp5 gene promoter, indicating transcriptional regulation by heat shock factors. In this work, we determined the oligomer state of EhHSTF7 and the recognition of the heat shock element of the EhPgp5 gene. The EhHSTF7 recombinant protein was obtained as monomer and oligomer. In silico molecular docking predicts protein-DNA binding between EhHSTF7 and 5'-GAA-3' complementary bases. The rEhHSTF7 protein specifically binds to the heat shock element of the EhPgp5 gene in gel shift assays. The competition assays with heat shock element mutants indicate that 5'-GAA-3' complementary bases are necessary for the rEhHSTF7 binding. Finally, the siRNA-mediated knockdown of Ehhstf7 expression causes downregulation of EhPgp5 expression, suggesting that EhHSTF7 is likely to play a key role in the E. histolytica multidrug resistance. This is the first report of a transcription factor that recognizes a heat shock element from a gene involved in drug resistance in parasites. However, further analysis needs to demonstrate the biological relevance of the EhHSTF7 and the rest of the heat shock factors of E. histolytica, to understand the underlying regulation of transcriptional control in response to stress.
热休克反应是一种保守的机制，使细胞能够对应激损伤做出反应并存活下来，其转录调控由热休克因子和热休克元件进行。P-糖蛋白赋予多药耐药表型；溶组织内阿米巴拥有迄今为止描述的最大的多药耐药基因家族；这些基因之一，EhPgp5 基因，具有依米丁诱导的表达。一个功能性的热休克元件被定位在 EhPgp5 基因启动子中，表明热休克因子的转录调控。在这项工作中，我们确定了 EhHSTF7 的寡聚状态和 EhPgp5 基因热休克元件的识别。EhHSTF7 重组蛋白以单体和寡聚体形式获得。计算机分子对接预测 EhHSTF7 与 5'-GAA-3'互补碱基之间的蛋白质-DNA 结合。rEhHSTF7 蛋白在凝胶迁移分析中特异性结合 EhPgp5 基因的热休克元件。与热休克元件突变体的竞争分析表明，5'-GAA-3'互补碱基是 rEhHSTF7 结合所必需的。最后，siRNA 介导的 Ehhstf7 表达敲低导致 EhPgp5 表达下调，表明 EhHSTF7 可能在溶组织内阿米巴多药耐药中发挥关键作用。这是第一个报道识别寄生虫中参与耐药基因的热休克元件的转录因子的报告。然而，进一步的分析需要证明 EhHSTF7 以及其余热休克因子在溶组织内阿米巴中的生物学相关性，以了解应激反应中转录控制的潜在调节。
