NGFN-PLUS
Proteomics of NB master regulators
| Coordinator: | PD Dr. Alexander Schramm | |
| Institution: | Universitätsklinikum Essen, Onkologisches Labor, Kinderklinik | |
| Homepage: | www.kinderklinik3.wtz-essen.de |
In this subproject, we addressed two important areas of neuroblastoma research using proteomics methods. First, we identified interaction partners of the key transcription factors SOX10 und MYCN and secondly, we analyse the role of NB-relevant miRNAs by monitoring the proteomic changes upon overexpression of miRNAs identified in TP6. Interaction partners of the transcription factors SOX10 and MYCN were analysed in human and murine cell culture models of neuroblastoma.
For this purpose we deployed affinity-based purification of protein complexes and subsequent mass spectrometry. Suitable constructs for overexpressing N- or C-terminal Step/Flag-Tag-fusion proteins of SOX 10 and MYCN as well as a control
(GFP) were cloned and the expression of fusion proteins in the human SY5Y neuroblastoma cells was confirmed by Western Blotting. Moreover, we established the identification of interaction partners by affinity purification and subsequent mass spectrometry, so that this method can be routinely deployed. As a further step to confirm specificity of interaction partners, isotope labelling of proteins (SILAC) was established in SY5Y cells. The contribution of putative SOX10 interaction partners, which include also known DNA-binding proteins, to maintain the undifferentiated state of neuroblastoma cells in vitro were analysed.
In order to identify MYCN interacting proteins, we performed co-immunoprecipitation using three different antibodies as well as controls followed by high resolution mass spectrometric identification and quantification of proteins. Using this approach several putative MYCN interaction partners were identified, and those with identification cores comparable to the known-MYCN interaction partner MAX are currently further validated. Additionally, proteome studies were conducted to elucidate the role of NB-relevant miRNAs on the protein level. In cooperation with WP6 we studied the consequences of miR-137 (Althoff et al., Int J Cancer, 2013) as well as miR 542-3p overexpression. Using label-free techniques and subsequent mass spectrometry-based quantification, 15941 features were identified by MS/MS, corresponding to 1731 quantified proteins. In total, 38 proteins were found to be regulated upon overexpression of miR137 and 29 proteins, which were regulated upon overexpression of miR542-3p.
Althoff et al: http://www.ncbi.nlm.nih.gov/pubmed/23400681
Further Coordinators:
For this purpose we deployed affinity-based purification of protein complexes and subsequent mass spectrometry. Suitable constructs for overexpressing N- or C-terminal Step/Flag-Tag-fusion proteins of SOX 10 and MYCN as well as a control
(GFP) were cloned and the expression of fusion proteins in the human SY5Y neuroblastoma cells was confirmed by Western Blotting. Moreover, we established the identification of interaction partners by affinity purification and subsequent mass spectrometry, so that this method can be routinely deployed. As a further step to confirm specificity of interaction partners, isotope labelling of proteins (SILAC) was established in SY5Y cells. The contribution of putative SOX10 interaction partners, which include also known DNA-binding proteins, to maintain the undifferentiated state of neuroblastoma cells in vitro were analysed.
In order to identify MYCN interacting proteins, we performed co-immunoprecipitation using three different antibodies as well as controls followed by high resolution mass spectrometric identification and quantification of proteins. Using this approach several putative MYCN interaction partners were identified, and those with identification cores comparable to the known-MYCN interaction partner MAX are currently further validated. Additionally, proteome studies were conducted to elucidate the role of NB-relevant miRNAs on the protein level. In cooperation with WP6 we studied the consequences of miR-137 (Althoff et al., Int J Cancer, 2013) as well as miR 542-3p overexpression. Using label-free techniques and subsequent mass spectrometry-based quantification, 15941 features were identified by MS/MS, corresponding to 1731 quantified proteins. In total, 38 proteins were found to be regulated upon overexpression of miR137 and 29 proteins, which were regulated upon overexpression of miR542-3p.
Althoff et al: http://www.ncbi.nlm.nih.gov/pubmed/23400681
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