Grant amount：\3100000 （ Direct Cost: \3100000 ）

In recent years, podocytes have been shown to play a central role in maintaining the filtration barrier of the glomerulus. Several podocyte-related proteins, such as nephrin and podocin, have been identified and analyzed for their function. To identify novel proteins that work specifically in podocytes, we searched for proteins that interact with podocin. We screened a mouse kidney cDNA library using the yeast two-hybrid system and identified a novel protein. The deduced amino acid sequence of the protein consisted of 306 residues with a molecular weight of 34 kDa. Sequence analysis revealed that the protein does not have a distinct functional motif. Reverse transcription-PCR findings showed that the protein was expressed strongly in the kidney, weakly in the brain and heart. The human ortholog of the protein was identified. Amino acid sequences of the protein is highly conserved between mouse and human (81% identity, 92% similarity). We established stable transformants of NIH3T3 cells that expressed podocin and the novel protein added FLAG epitope. Podocin was co-immunoprecipitated with the novel protein by anti-FLAG antibody. Furthermore, the direct association between podocin and the protein was confirmed by GST pull down assay. Using the yeast two-hybrid system to search for proteins that bind to the novel protein, we obtained the Aha1 (activator of HSP90 ATPase). Aha1 is known to bind to HSP90 (heat shock protein 90) and increase the ATPase activity of HSP90. Recently, it was shown that HSP90 regulates the structure of actin filaments. Actin cytoskeleton plays an important role in maintaining the structure of podocytes. The novel protein identified in this study might recruit HSP90 near podocin through the binding to Aha1 and play some roles for actin cytoskeletal formation.

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