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.
Differential proteomic analysis of nuclear extracts from thyroid cell lines
Salzano AM;Scaloni A;
2006
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
