Defining Protein Interactions in Ewing’s Sarcoma: EWS/FLI1 and its Protein Partners NKX2.2, TCF4, and KLF15
Date
2020-05
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Publisher
University of Delaware
Abstract
Ewing’s Sarcoma (ES) is the second most prevalent pediatric soft tissue sarcoma. ES is caused by a chromosomal translocation which fuses the EWS gene of chromosome 22 to the FLI1 gene of chromosome 11 resulting in the formation of an internally disordered EWS/FLI1 (EF) fusion protein. EF can bind with numerous protein partners to gain the stability to function as an oncoprotein. EF cannot be directly targeted, so characterization of its protein partners is critical for further understanding the biology of ES. Three transcription factors have been previously identified as possible protein partners for EF: NKX2.2, TCF4 and KLF15. The goal of this research is to confirm the interaction between EF and these transcription factors and understand their mechanism of action. Due to a highly unstable structure, EF is difficult to purify for biochemical studies. Proximity Ligation Assay was utilized to allow in vivo visualization of protein-protein interactions without requiring purification. For this method, Ewing’s Sarcoma patient derived cell lines were grown on glass coverslips, fixed, permeabilized, blocked and incubated with a set of primary antibodies specific for each protein. DNA oligos attached to secondary antibodies when in close proximity, get ligated to form circular DNA. This DNA is amplified through rolling circle amplification (RCA). With the addition of fluorescent probes specific to the product of RCA, the interacting proteins can be visualized using a fluorescence microscope. The results show that each of the suspected protein interact directly with EF and that this reaction is dependent on DNA. For all of the proteins tested, the majority of the PLA spots were seen in the nucleus of the cell. As transcription factors this was expected and strengthens our findings. Likewise, this reactions reliance on DNA was investigated to further understand the interactions. The
results show that each NKX2.2, TCF4, and KLF15 rely on DNA to interact with EF. Further research will be done to confirm these findings and enhance our understanding of this protein interaction and its function in tumor progression in Ewing’s Sarcoma.
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Keywords
medical science, Ewing's sarcoma, NKX2.2, TCF4, KLF15