Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
In Vitro Capture and Identification of Proteins that Bind to a GGA-Rich Sequence from the ERBB2 Gene Promoter Region
Human epidermal growth factor receptor 2 (ErbB2) is a key breast cancer marker which is over-expressed in about 30% of human breast cancer. The nuclease hypersensitive site of the promoter region of ERBB2 contains a 28 bp GGA-TCC repeat sequence (from -69 to -42bp), referred to hereinafter as “Pu28-mer”, that has been reported to play a role in down-regulation of the ERBB2 gene.
In the present work, we used circular dichroism (CD) spectroscopy to investigate conformational similarities between the Pu28-mer and the G4 structure of c-MYB, followed by in vitro protein capture studies at Pu28-mer modified surfaces. Nuclear extract (NE) from cultured MCF-7 cancer cells was incubated on indium tin oxide (ITO)-coated glass slides that were modified with Pu28-mer and captured proteins were analyzed directly at the modified surfaces using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We then performed affinity capture using magnetic beads and the captured proteins were separated on polyacrylamide gels. Proteins that were selectively captured by Pu28-mer as indicated by the gel bands were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a first step toward protein identification. Western blotting experiments were performed to confirm the identities of a few of the captured proteins.
The results of this work indicate that Pu28-mer forms a G4 structure in vitro that selectively captures transcription factors including Ku70, Ku80 and PURA from MCF-7 cell nuclear extracts. These selectively captured proteins may exert their regulatory role on the transcription of the ERBB2 gene through their interaction with non-duplex structures, possibly an intramolecular G4, formed by the Pu28-mer.
This research has been supported by NSF under CHE-0911108
Department of Chemistry & Chemical Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy NY, 12180