Differential proteomic analysis of nuclear extracts from thyroid cell lines.
Nuclear proteins play a major role in controlling cell functions. Differential proteomic analysis of nuclear proteins by combined 2D gel electrophoresis (2D-E) and mass spectrometry procedures can provide useful information to understand the control of cell proliferation and differentiation. To identify proteins involved in dedifferentiation, we used a differential proteomics approach by comparing nuclear extracts from the differentiated rat thyroid cell line FRTL-5 and the derived undifferentiated Ki-mol cell line, obtained by transformation with the Ki-ras oncogene. Thirteen proteins were identified as differently expressed in the nuclear compartment between the two cell lines. RT-PCR analysis performed on seven differently expressed genes showed that only in two cases the difference may be ascribable to a transcriptional mechanism. Since one of the identified proteins, namely apurinic apyrimidinic endonuclease/redox effector factor-1 (APE1/Ref-1), is suspected to play a role in thyroid tumorigenesis, we used a glutathione S-transferase (GST)-pulldown assay coupled to a 2D electrophoretic/matrix assisted laser desorption ionization-time of flight (MALDI-TOF)-mass spectrometry (MS) analysis to detect and identify its interacting partners. We show here that beta-actin directly interacted with APE1/Ref-1, as confirmed by co-immunoprecipitation assays and that this interaction was enhanced by oxidative stress on FRTL-5 cells.
Salzano
Anna Maria
AM
Proteomics and Mass Spectrometry Laboratory, I.A.B.B.A.M., National Research Council, via Argine 1085, 80147 Naples, Italy.
Paron
Igor
I
Pines
Alex
A
Bachi
Angela
A
Talamo
Fabio
F
Bivi
Nicoletta
N
Vascotto
Carlo
C
Damante
Giuseppe
G
Quadrifoglio
Franco
F
Scaloni
Andrea
A
Tell
Gianluca
G
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D017931:DNA Primers; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D009687:Nuclear Proteins / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013961:Thyroid Gland / Q000737:chemistry* / Q000166:cytology
D017931:DNA Primers; D009687:Nuclear Proteins; D011994:Recombinant Proteins