title: Identification of cyclophilin-40-interacting proteins reveals potential cellular function of cyclophilin-40.
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.affiliation: Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA 95211, USA. mpark@pacific.edu
authors.forename: Miki Susanto
authors.initials: MS
authors.lastname: Park
authors.forename: Feixia
authors.initials: F
authors.lastname: Chu
authors.forename: Jinghang
authors.initials: J
authors.lastname: Xie
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Pompeya
authors.initials: P
authors.lastname: Bhattacharya
authors.forename: William K
authors.initials: WK
authors.lastname: Chan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D051903:Nuclear Factor 45 Protein / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D000075370:Receptors for Activated C Kinase; D012156:Reticulocytes; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D029681:Arabidopsis Proteins; D000081406:Peptidyl-Prolyl Isomerase F; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C463462:KU70 protein, Arabidopsis; D051903:Nuclear Factor 45 Protein; D010455:Peptides; C501334:RPS3 protein, S cerevisiae; D000075370:Receptors for Activated C Kinase; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C070301:peptide I; D021983:Cyclophilins