Identification and characterization of endonuclein binding proteins: evidence of modulatory effects on signal transduction and chaperone activity.

BACKGROUND:We have previously identified endonuclein as a cell cycle regulated WD-repeat protein that is up-regulated in adenocarcinoma of the pancreas. Now, we aim to investigate its biomedical functions. RESULTS:Using the cDNA encoding human endonuclein, we have expressed and purified the recombinant protein from Escherichia coli using metal affinity chromatography. The recombinant protein was immobilized to a column and by affinity chromatography several interacting proteins were purified from several litres of placenta tissue extract. After chromatography the eluted proteins were further separated by two-dimensional gel electrophoresis and identified by tandem mass spectrometry. The interacting proteins were identified as; Tax interaction protein 1 (TIP-1), Aalpha fibrinogen transcription factor (P16/SSBP1), immunoglobulin heavy chain binding protein (BiP), human ER-associated DNAJ (HEDJ/DNAJB11), endonuclein interaction protein 8 (EIP-8), and pregnancy specific beta-1 glycoproteins (PSGs). Surface plasmon resonance analysis and confocal fluorescence microscopy were used to further characterize the interactions. CONCLUSIONS:Our results demonstrate that endonuclein interacts with several proteins indicating a broad function including signal transduction and chaperone activity.

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