Department of Pediatric Surgery, Erasmus MC-Sophia, Rotterdam, The Netherlands.
Department of Cell Biology, Erasmus MC, Faculty Building, Room Ee-1034B, Wytemaweg 80, 3015 CN, Rotterdam, The Netherlands.
J Biomed Sci. 2024 Nov 4;31(1):100. doi: 10.1186/s12929-024-01088-5.
Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a fatal congenital lung disorder strongly associated with genomic alterations in the Forkhead box F1 (FOXF1) gene and its regulatory region. However, little is known about how FOXF1 genomic alterations cause ACD/MPV and what molecular mechanisms are affected by these mutations. Therefore, the effect of ACD/MPV patient-specific mutations in the FOXF1 gene on the molecular function of FOXF1 was studied.
Epitope-tagged FOXF1 constructs containing one of the ACD/MPV-associated mutations were expressed in mammalian cell lines to study the effect of FOXF1 mutations on protein function. EMSA binding assays and luciferase assays were performed to study the effect on target gene binding and activation. Immunoprecipitation followed by SDS‒PAGE and western blotting were used to study protein‒protein interactions. Protein phosphorylation was studied using phos-tag western blotting.
An overview of the localization of ACD/MPV-associated FOXF1 mutations revealed that the G91-S101 region was frequently mutated. A three-dimensional model of the forkhead DNA-binding domain of FOXF1 showed that the G91-S101 region consists of an α-helix and is predicted to be important for DNA binding. We showed that FOXF1 missense mutations in this region differentially affect the DNA binding of the FOXF1 protein and influence the transcriptional regulation of target genes depending on the location of the mutation. Furthermore, we showed that some of these mutations can affect the FOXF1 protein at the posttranscriptional level, as shown by altered phosphorylation by MST1 and MST2 kinases.
Missense mutations in the coding region of the FOXF1 gene alter the molecular function of the FOXF1 protein at multiple levels, such as phosphorylation, DNA binding and target gene activation. These results indicate that FOXF1 molecular pathways may be differentially affected in ACD/MPV patients carrying missense mutations in the DNA-binding domain and may explain the phenotypic heterogeneity of ACD/MPV.
肺静脉错位的肺泡毛细血管发育不良（ACD/MPV）是一种致命的先天性肺部疾病，与叉头框 F1（FOXF1）基因及其调控区的基因组改变密切相关。然而，对于 FOXF1 基因组改变如何导致 ACD/MPV 以及这些突变影响哪些分子机制，人们知之甚少。因此，研究了 FOXF1 基因中与 ACD/MPV 相关的突变对 FOXF1 分子功能的影响。
在哺乳动物细胞系中表达含有 ACD/MPV 相关突变之一的 FOXF1 表位标记构建体，以研究 FOXF1 突变对蛋白质功能的影响。进行 EMSA 结合测定和荧光素酶测定，以研究对靶基因结合和激活的影响。免疫沉淀 followed by SDS-PAGE 和 Western blotting 用于研究蛋白-蛋白相互作用。使用 phos-tag Western blot 研究蛋白质磷酸化。
对 ACD/MPV 相关 FOXF1 突变的定位进行概述，发现 G91-S101 区域经常发生突变。FOXF1 叉头 DNA 结合域的三维模型表明，G91-S101 区域由一个α-螺旋组成，预测对 DNA 结合很重要。我们表明，该区域的 FOXF1 错义突变会导致 FOXF1 蛋白的 DNA 结合能力不同，并根据突变的位置影响靶基因的转录调控。此外，我们表明，这些突变中的一些可以在转录后水平影响 FOXF1 蛋白，如 MST1 和 MST2 激酶改变的磷酸化。
FOXF1 基因编码区的错义突变会在多个水平上改变 FOXF1 蛋白的分子功能，如磷酸化、DNA 结合和靶基因激活。这些结果表明，携带 DNA 结合域错义突变的 ACD/MPV 患者中 FOXF1 分子途径可能受到不同程度的影响，这可能解释了 ACD/MPV 的表型异质性。
