Identification of cyclophilin-40-interacting proteins reveals potential cellular function of cyclophilin-40.

Academic Article

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.
  • Authors

  • Park, Miki Susanto
  • Chu, Feixia
  • Xie, Jinghang
  • Wang, Yu
  • Bhattacharya, Pompeya
  • Chan, William K
  • Status

    Publication Date

  • March 15, 2011
  • Published In

    Keywords

  • Amino Acid Sequence
  • Animals
  • Arabidopsis Proteins
  • Cell Line
  • Cyclophilin D
  • Cyclophilins
  • DNA-Binding Proteins
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Mass Spectrometry
  • Molecular Sequence Data
  • Nuclear Factor 45 Protein
  • Peptides
  • Protein Binding
  • Rabbits
  • Receptors for Activated C Kinase
  • Reticulocytes
  • Ribosomal Proteins
  • Saccharomyces cerevisiae Proteins
  • Tandem Mass Spectrometry
  • Digital Object Identifier (doi)

    Start Page

  • 257
  • End Page

  • 265
  • Volume

  • 410
  • Issue

  • 2