| NDRG gene family is a new gene family which has been identified in recent years. This family includes four related members, NDRG 1 to 4. NDRG2 has two subtypes which are composed of 357 and 371 amino acids. The two subtypes can be distinguished by fourteen amino acids of N-terminal. NDRG2(357), which was first identified and cloned from normal human whole brain cDNA library in our lab in 1999.Database search and phylogenetic analysis revealed that NDRG gene family is highly conserved in mammals.Moreover, mammalian NDRG family members show high homology to each other, except for their C-and N-terminal regions, pointing out the important functions of this gene family. Bioinformation analysis does not indicate any known motifs or domains in NDRG2 and other members of NDRG family. The precise molecular and cellular functions of NDRG2 are still unknow so far. Some preliminary researches suggest that NDRG2 may play a role in growth arrest and cell differentiation, and it may be regulated by cellular stress. So identification of NDRG2 interaction proteins may provide important information for its function research.For this purpose, we performed yeast two-hybrid screening of fetal brain cDNA library using NDRG2(371) as a bait protein. 121 clones were obtained after 1.05 x106 clones were screened by reporter genes. 12 clones were got after interactions were retested in yeast, which represents 9 different gene fragments. We retested the interactions between NDRG2(371) and 12 positive clones in AH109, and using mammalian two-hybrid system we confirmed these interactions in HEK293 cells.Of these 9 final isolates, three fragments encoded Na+/K+ ATPaseβ1, so it was chosen for the further investigations. The Na+/K+ ATPaseβ1 subunit, a 50-60 kDa glycosylated protein with a single membrane-spanning domain, may act as a molecular chaperone or otherwise regulate the membrane targeting and function of theαsubunit of Na+/K+ ATPase. To delineate the interaction region(s) of NDRG2(371) and Na+/K+ ATPaseβ1, various deletions of two proteins were engineered and subjected to analyze in yeast two hybrid assay. We found that the 141-272aa of NDRG2(371) is responsible for the interaction with Na+/K+ ATPaseβ1, while 244-304aa of Na+/K+ ATPaseβ1 are responsible for its interaction with NDRG2(371). Co-immunoprecipitation further showed that NDRG2(371) and Na+/K+ ATPaseβ1 possessed physical interactions in vivo.The two-hybrid analysis is of exceptional value for the detection of pairwise and transient associations. However, yeast two-hybrid approaches do not seem to be particularly suited for characterization of protein complexes.This supports the view that complex formation is more than the sum of binary interactions. Success of the TAP/MS approach for the characterization of protein complexes relies on its maintaining protein concentration, localization and post-translational modification in a manner that closely approximates normal physiology. Yeast two-hybrid analysis and TAP/MS method are ideally complementary. In this research, we successfully constructed adenovirus vectors which could express the NDRG-TAP fusion proteins. This work built up the basis of identifying interaction protein complexes of NDRG family with TAP method.In summary, we performed yeast two-hybrid screening using NDRG2(371) as a bait protein and obtained 12 positive clones. Among these positive clones, the Na+/K+ ATPaseβ1 was chosen for the further investigation. Physical interaction between them was proved in vivo. Next, to complement the result of the yeast two hybrid screening, we developed an sepcific mammalian TAP expression system for NDRG family. Our results provide important clues for the function research of NDRG2(371).
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