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.
Ludvigsen
Maja
M
Department of Medical Biochemistry, Aarhus University, Ole Worms Allé 3, Building 1170, Aarhus, DK-8000 Aarhus C, Denmark. maja@biokemi.au.dk
Østergaard
Morten
M
Vorum
Henrik
H
Jacobsen
Christian
C
Honoré
Bent
B
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D005972:Glutaminase / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D024101:Mitochondrial Proteins; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011268:Pregnancy-Specific beta 1-Glycoproteins / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D053719:Tandem Mass Spectrometry
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C494648:DNAJB11 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D055767:Immobilized Proteins; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D009687:Nuclear Proteins; C447901:PWP1 protein, human; D011268:Pregnancy-Specific beta 1-Glycoproteins; D011994:Recombinant Proteins; C504091:NABP2 protein, human; C421249:TAX1BP3 protein, human; D005972:Glutaminase