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removed for further analysis. Statistical Analysis Unless otherwise stated calculations of statistical significance in this work was performed according to Student’s t test. Results Bag-1 Interacts with GRP78/BiP GST-pull-down Experiments Expression of GST fusion proteins for GST-pull-down experiments were performed essentially as described previously. Proapoptotic Action of a GRP78/BiP Peptidic Ligand cence experiment where we could show colocalization of Bag-1 with an ER tracker. To determine the domain of GRP78/BiP involved in its binding to Bag-1, we used GST-fusion constructs of the two main regions of GRP78/BiP in pull-down experiments with HEK-293 cells overexpressing Bag-1. Western blot analysis showed that Bag-1 interacted not only with the full length GRP78/BiP but also with its ATPase and SBD. As Bag-1 is reported to bind to the ATPase binding domain of the molecular chaperone Hsp70/ Hsp70 and GRP78/BiP belongs to this family of chaperones, our finding that the SBD of GRP78/BiP is also bound by Bag-1 is rather intriguing and identifies a novel interaction site of Bag-1 in the molecular chaperone family. We purchase RAF265 therefore used the SBD of GRP78/BiP in further characterization of the interaction of GRP78/BiP with Bag-1. GST-pull down analyses were carried out with the SBD of GRP78/BiP and lysate of HEK293 cell expressing the wild type Bag-1, Bag-1DC47, a C-terminal deletion mutant or Bag1D68mer, an internal deletion mutant. These studies identified an internal PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22211439 sequence of 68 amino acids as a target of interaction of Bag-1 with GRP78/BiP. Deletion of this sequence abolished the interaction of Bag-1 with GRP78/BiP while expression of the 68 amino acid sequence alone showed that it is indeed required for binding the SBD of GRP78/BiP. This finding was further confirmed in an in vivo co-immunoprecipitation experiment where an HA-tagged Bag-1 peptide expressed in HEK 293 cells interacted with endogenous GRP78/BiP protein. Several studies have shown that GRP78/BiP cooperates with PDI in refolding denatured proteins in vitro. To determine whether GRP78/BiP also possesses the ability to fold denatured proteins in vivo, we adopted a refolding assay used previously to determine the chaperone activity of Hsp70 in vivo In this assay, HEK-293 cells were transfected with a plasmid encoding a luciferase gene and an expression plasmid for GRP78/BiP. The cells were briefly heat shocked and thereafter the luciferase activity of the transfected cells expressing GRP78/BiP was compared with that of the non-GRP78/BiP expressing cells. This study showed that overexpression of GRP78/BiP significantly enhanced luciferase activity after the heat shock demonstrating the ability of GRP78/BiP to refold denatured luciferase in vivo. If the cells were additionally transfected with Bag-1 or the 68 amino acid Bag-1 peptide that binds GRP78/BiP, the refolding activity of GRP78/BiP was reduced to the control level. This was not the case when Bag-1D68mer that does not bind GRP78/BiP was cotransfected. These studies demonstrate that the Bag-1 peptide interferes with the refolding activity of GRP78/BiP. However the Bag-1 peptide did not have any effect on the ATPase activity of GRP78/BiP although Bag-1 increased this enzymatic activity. This indicates that the Bag-1 peptide functions differently from the full length Bag-1. We therefore embarked on the characterization of this peptide as a ligand for GRP78/BiP for future therapeutic use. Utilization of a Bag-1 Peptide
