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

Anal Biochem. 2011 Mar 15;410(2):257-65. doi: 10.1016/j.ab.2010.12.007. Epub 2010 Dec 10.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arabidopsis Proteins / chemistry*
  • Arabidopsis Proteins / metabolism
  • Cell Line
  • Cyclophilin D
  • Cyclophilins / chemistry*
  • Cyclophilins / genetics
  • Cyclophilins / isolation & purification
  • Cyclophilins / metabolism*
  • DNA-Binding Proteins / chemistry*
  • 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 / chemistry*
  • Nuclear Factor 45 Protein / metabolism
  • Peptides / chemistry*
  • Peptides / metabolism
  • Protein Binding
  • Rabbits
  • Receptors for Activated C Kinase
  • Reticulocytes
  • Ribosomal Proteins / chemistry*
  • Ribosomal Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Tandem Mass Spectrometry / methods

Substances

  • Arabidopsis Proteins
  • Cyclophilin D
  • DNA-Binding Proteins
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • KU70 protein, Arabidopsis
  • Nuclear Factor 45 Protein
  • Peptides
  • RPS3 protein, S cerevisiae
  • Receptors for Activated C Kinase
  • Ribosomal Proteins
  • Saccharomyces cerevisiae Proteins
  • peptide I
  • Cyclophilins