Identifying G-quadruplex-interacting proteins in cancer-related gene promoters
Simona Marzano, Gabriella Pinto, Anna Di Porzio, Jussara Amato, Antonio Randazzo, Angela Amoresano, Bruno Pagano
Abstract
G-quadruplexes (G4s) are noncanonical DNA or RNA secondary structures involved in numerous biological processes. Their recognition by G4-related proteins (G4RPs) is essential for modulating biological pathways, particularly those associated with transcription and cancer progression. Identifying G4RPs is crucial for understanding their role in diseases like cancer, as these proteins may represent promising therapeutic targets. In this study, a proteomic-based fishing-for-partners approach was employed to identify putative interactors of G4-forming DNA sequences from the promoter regions of cancer-related genes DAP, HIF-1α, JAZF-1, and PDGF-A. A total of eighty-six G4RPs were identified, including nineteen known RNA and/or DNA G4 interactors. Notably, fourteen proteins were identified as potential interactors of all four investigated G4-forming DNA, seven of which were novel G4RPs. Direct interactions with G4s were validated for five of these proteins (AHNAK, GAPDH, HNRNP M, LMNA, and PPIA) using surface plasmon resonance experiments, which showed nanomolar binding affinities. This study not only validated known G4RPs but also led to the discovery of new G4/protein interactions, providing the basis for further investigation into their biological significance and potential implications in disease-associated pathways. G-quadruplexes (G4s) are noncanonical four-stranded secondary structures of DNA or RNA that play key roles in numerous biological processes, however, G4-related proteins (G4RPs) remain understudied. Here, the authors use a proteomics-based fishing-for-partners approach to profile the interactors of G4-forming DNA sequences from the promoter regions of four cancer-related genes, identifying 86 G4RPs and validating five of them through an in vitro binding assay.