Candidate Gene Product for Diabetic Nephropathy Protein 4.1O Locates to Ribosomes and Silences C-Myc Transactivation

Background: Protein synthesis and its control are an important contributor to the pathogenesis of diabetic nephropathy. Previously FRMD3 coding for Protein 4.1O was identified as a potential target gene causing diabetic nephropathy in type 1 diabetes in a GWAS of the north American population. We were interested in the cellular functions of the Protein 4.1O. Methods: Protein 4.1O isoforms and mutants were expressed HEK 293T cells and cos7 cells. Subcelluar fractionation was performed to track the appearance of Protein 4.1O and its mutants using a ultracentrifuge with 186.000xg. The samples of the different cellular compartments were subjected to SDS-PAGE and western blot analysis. Cos7 cells expressing Protein 4.1O were stained for Protein 4.1O and cellular organell markers by appropriate antibodies. Secondary antibodies with immunofluorecent dyes were used for visulization with an immunofluorecent microscope. Luciferase reporter assays were used to assess the c-myc transactivation in the presence of increasing doses of Protein 4.1O. Results: Protein 4.1O its isoforms and mutants were detected in the ribosomal compartment by western blot which was generated by cellular farctionation. The rps3 was used as a positive control for adequate ribosomal preparation. By immunofluorecence Protein 4.1O was detected in the cytosol, ER and in submebranous regions. Protein 4.1O mutants were enriched in the nucleus sparing the nuceoli. Luciferase reporter assays show a dose dependent reduction for c-myc transactivation by Protein 4.1O. Conclusion: Protein 4.1O can be deteced in the ribosomal fraction by subcellular fractionation with cosedimentation with established ribosomal markers. Immunofluorecent imaging confirms Protein 4.1O apperance in the ER. Increasing doses of Protein 4.1O silence c-myc transactivation in a dose dependet fashion. These finding shed new light on the potential cellular signficance of Protein 4.1O in the pathomechanism of diabetic nephropathy. Funding: Private Foundation Support