Department of Molecular Biology and Genetics, İstanbul Arel University, 34537, Istanbul, Turkey.
Department of Chemistry, Yıldız Technical University, 34220, Istanbul, Turkey.
Protein J. 2021 Aug;40(4):512-521. doi: 10.1007/s10930-020-09959-0. Epub 2021 Jan 18.
Mutations in hepatocyte nuclear factor (HNF)1A gene cause the most common form of Maturity-onset diabetes of the young (MODY), a monogenic subtype of diabetes mellitus. Functional characterization of mutant proteins reveals that mutations may disrupt DNA binding capacity, transactivation ability and nuclear localization of HNF1A depending on the position of the mutation. Previously identified Arg271Trp and Ser345Tyr mutations in HNF1A were found to be defective in nuclear localization. Arg271 residue resides in a region similar to classical nuclear localization signal (NLS) motif, while Ser345 does not. Importin α family members recognize NLS motifs on cargo proteins and subsequently translocate them into nucleus. Here, we first investigated the nuclear localization mechanism of wild type HNF1A protein. For this purpose, we analyzed the interaction of HNF1A with three mouse homolog importin α proteins (KPNA2, KPNA4 and KPNA6) by co-immunoprecipitation assay and molecular docking simulation. Hereby, KPNA6 was identified as the main import receptor, which is responsible for the transport of HNF1A into the nucleus. Immunolocalization studies in mouse pancreatic cells (Min6) also confirmed the co-localization of HNF1A and KPNA6 in the cytoplasm. Secondly, the interaction between KPNA6 and mutant HNF1A proteins (Arg271Trp and Ser345Tyr) was assessed. Co-immunoprecipitation studies revealed a reduced interaction compared to wild type HNF1A. Our study demonstrated for the first time that HNF1A transcription factor is recognized and transported by importin/karyopherin import family, and mutations in NLS motifs may disrupt the interaction leading to nuclear localization abnormalities and MODY phenotype.
HNF1A 基因突变导致最常见的青少年发病型成年糖尿病（MODY），这是一种单基因糖尿病亚型。突变蛋白的功能特征表明，突变可能会破坏 HNF1A 的 DNA 结合能力、转录激活能力和核定位，具体取决于突变的位置。先前在 HNF1A 中发现的 Arg271Trp 和 Ser345Tyr 突变被发现核定位缺陷。Arg271 残基位于类似于经典核定位信号（NLS）基序的区域，而 Ser345 则不然。Importin α 家族成员识别货物蛋白上的 NLS 基序，随后将它们转运到细胞核内。在这里，我们首先研究了野生型 HNF1A 蛋白的核定位机制。为此，我们通过共免疫沉淀分析和分子对接模拟分析了 HNF1A 与三种小鼠同源 Importin α 蛋白（KPNA2、KPNA4 和 KPNA6）的相互作用。结果鉴定出 KPNA6 为主要的 Import 受体，负责将 HNF1A 转运到细胞核内。在小鼠胰腺细胞（Min6）中的免疫定位研究也证实了 HNF1A 和 KPNA6 在细胞质中的共定位。其次，评估了 KPNA6 与突变型 HNF1A 蛋白（Arg271Trp 和 Ser345Tyr）之间的相互作用。与野生型 HNF1A 相比，共免疫沉淀研究显示相互作用减少。我们的研究首次证明 HNF1A 转录因子被 Importin/karyopherin 家族识别和转运，NLS 基序中的突变可能会破坏相互作用，导致核定位异常和 MODY 表型。
