The analysis of the nuclear localization signal of hepatocyte nuclear factor 1-α transcription factor

Maturity-onset diabetes of the young (MODY) is a monogenic subtype of diabetes mellitus. MODY3 associated with HNF1A gene mutations is the most common form of MODY. HNF1A protein is a transcription factor which is expressed mainly in pancreas, liver and kidney. Functional characterization of HNF1A gene mutations revealed that they affect DNA binding, transactivation function and nuclear localization of HNF1A. In previous studies R271W and S345Y mutant HNF1A were found to be defective in nuclear localization, but molecular mechanisms of this transport defect was not clarified yet. Importin-α receptor family are responsible for translocating proteins such as transcription factors (HNF1A) into the nucleus. Importin proteins recognize and bind to nuclear localization signal (NLS) motifs which are found on cargo proteins. Although classic NLS motifs are known to consist of basic amino acids, non-classical NLS motifs were also reported. Importin α proteins are categorized into three groups based on the identity of amino acid sequence and function. To date, no single import signal in the HNF1A protein has been identified, but there are three regions that fit the description of an NLS: region A (amino acids 158-171), region B (amino acids 197-205) and region C (amino acids 271-282). However, importin α subtype which recognizes and binds to HNF1A has not been reported yet. One of the aims of this study is to identify the importin α subgroup which is responsible for the transport of HNF1A protein into the nucleus. For this purpose the interactions of HNF1A with three candidate importin α proteins were be evaluated by co-immunoprecipitation technique. In this study, homolog mouse karyopherin α proteins (Kpna2, 4 and 6) were used. The results of co-IP revealed that HNF1A protein is recognized and bound by all three importin α subgroup proteins. The strongest band was observed with Kpna6 protein, therefore in the following part of the experiment the interactions with Kpna6 protein were analyzed. To confirm this interaction, Kpna6 was knockdown by siRNA in mouse pancreatic cells (MIN6) and the location of HNF1A was analyzed by immunochemical staining techniques. The results showed that there is 10% reduction in the nuclear localization of HNF1A protein when the Kpna6 protein is silenced. Second purpose of the study was to analyze the interaction between predetermined importin α subgroup and mutant HNF1A proteins (R271W and S345Y) which were identified in MODY patients. R271W mutation resides in a region similar to classical NLS motif of HNF1A protein. Therefore, the interaction between mutant HNF1A and importin α will be evaluated by co-immunoprecipitation technique. A reduced interaction was observed when compared to the interaction with the wild type and this confirmed that R271W mutation resides in the classical NLS motif in HNF1A protein. S345Y mutation is on the other hand, does not reside in a classical NLS, but was shown to have nuclear localization defect in functional studies. A reduced interaction of S345Y mutant protein with Kpna6 protein was detected and this result showed that this region is a possible non-classical NLS motif in HNF1A protein. As a conclusion, the results of this study clarified the molecular pathway of nuclear import of HNF1A protein and molecular pathogenesis of HNF1A mutations in MODY3 patients. Also the identification of non-classical NLS motifs in HNF1A protein will contribute to the literature and also will provide a preliminary result for further projects.