ABSTRACT
Cyclophilin-40 (CyP40) is part of the immunophilin family and is found in Hsp90-containing protein complexes. We were interested in identifying proteins that interact with CyP40. CyP40-interacting proteins in HeLa cells were identified using the tandem affinity purification approach. Adenovirus expressing human CyP40 protein (Ad-CyP40), fused with streptavidin and calmodulin binding peptides at the N terminus, was generated. Proteins were separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel after tandem affinity purification. Here 10 silver-stained protein bands that were enriched in the Ad-CyP40-infected lysate and the corresponding regions in the control lysate were excised, digested by trypsin, and identified by tandem mass spectrometric analysis. Of 11 interacting proteins that were identified, 4 (RACK1, Ku70, RPS3, and NF45) were expressed in rabbit reticulocyte lysate, bacteria, and MCF-7 cells. We confirmed that these proteins interact with CyP40. We observed that RACK1 suppressed the cobalt chloride-induced, hypoxia response element-dependent luciferase activity in MCF-7 cells but not in MCF-7 stable cells expressing approximately 10% of the cellular CyP40 content. In addition, RACK1 reduced the HIF-1α protein accumulation after cobalt chloride treatment, which was not observed when the CyP40 content was down-regulated. Collectively, we conclude that reduction of the HIF-1 α protein by RACK1 is CyP40-mediated.
Subject(s)
Arabidopsis Proteins/chemistry , Cyclophilins/chemistry , Cyclophilins/metabolism , DNA-Binding Proteins/chemistry , Nuclear Factor 45 Protein/chemistry , Peptides/chemistry , Ribosomal Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Amino Acid Sequence , Animals , Arabidopsis Proteins/metabolism , Cell Line , Peptidyl-Prolyl Isomerase F , Cyclophilins/genetics , Cyclophilins/isolation & purification , DNA-Binding Proteins/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/chemistry , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mass Spectrometry/methods , Molecular Sequence Data , Nuclear Factor 45 Protein/metabolism , Peptides/metabolism , Protein Binding , Rabbits , Receptors for Activated C Kinase , Reticulocytes , Ribosomal Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Tandem Mass Spectrometry/methodsABSTRACT
Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex that contains the aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE) using baculovirus expressed proteins. Here we reported that CyP40 plays a role in the AhR signaling. When the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylchloranthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidyl-prolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer.