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BMC Biochemistry (2)
Vorum, Henrik (2)
Andersen, Olav M (1)
Jacobsen, Christian (1)
Ludvigsen, Maja (1)
Thøgersen, Hans C (1)
Østergaard, Morten (1)
Year of Publication
Identification and characterization of endonuclein binding proteins: evidence of modulatory effects on signal transduction and chaperone activity
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.
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), Aα 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 β-1 glycoproteins (PSGs). Surface plasmon resonance analysis and confocal fluorescence microscopy were used to further characterize the interactions.
Our results demonstrate that endonuclein interacts with several proteins indicating a broad function including signal transduction and chaperone activity.
Ca2+ binding to complement-type repeat domains 5 and 6 from the low-density lipoprotein receptor-related protein
Andersen, Olav M
Thøgersen, Hans C
The binding of ligands to clusters of complement-type repeat (CR)-domains in proteins of the low-density lipoprotein receptor (LDLR) family is dependent on Ca2+ ions. One reason for this cation requirement was identified from the crystal structure data for a CR-domain from the prototypic LDLR, which showed the burial of a Ca2+ ion as a necessity for correct folding and stabilization of this protein module. Additional Ca2+ binding data to other CR-domains from both LDLR and the LDLR-related protein (LRP) have suggested the presence of a conserved Ca2+ cage within CR-domains from this family of receptors that function in endocytosis and signalling.
We have previously described the binding of several ligands to a fragment comprising the fifth and the sixth CR-domain (CR56) from LRP, as well as qualitatively described the binding of Ca2+ ions to this CR-domain pair. In the present study we have applied the rate dialysis method to measure the affinity for Ca2+, and show that CR56 binds 2 Ca2+ ions with an average affinity of KD = 10.6 microM, and there is no indication of additional Ca2+ binding sites within this receptor fragment.
Both CR-domains of CR56 bind a single Ca2+ ion with an affinity of 10.6 microM within the range of affinities demonstrated for several other CR-domains.
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