Abstract 1918 Functional Analysis of the Lysine Specific Demethylase 1 (LSD1) Enzyme Complex With Ews-FLI1 Protein Constructs

Document Type

Article

Publication Date

5-2025

Publisher

American Society for Biochemistry and Molecular Biology

Source Publication

Journal of Biological Chemistry

Source ISSN

0021-9258

Original Item ID

DOI: 10.1016/j.jbc.2025.109269

Abstract

Lysine specific demethylase 1 (LSD1) is an essential epigenetic regulator involved in chromatin remodeling that requires the co-repressor element-1 silencing transcription factor (CoREST) to catalyze the removal of mono- and dimethyl groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with >100 proteins and plays a critical role in embryogenesis. Importantly, its misregulation is associated with cancer progression, including Ewing sarcoma. To study the biochemical properties and potential therapeutic relevance of LSD1, we overexpressed a form of LSD1 that lacks the N-terminus region (ΔN LSD1) and is in complex with CoREST. After transformation, cells were cultured and harvested to extract the ΔN LSD1-CoREST complex. Protein purification was carried out using Nickel-Nitrilotriacetic Acid (Ni-NTA) and glutathione (GSH) affinity chromatography, followed by size-exclusion chromatography to achieve high purity. The functional integrity of the purified ΔN LSD1-CoREST complex was verified through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic activity assays. We also performed protein crystallization of the ΔN LSD1 CoREST complex to facilitate structural studies through X-ray crystallography. Lastly, we investigated the effect of the EWS-FlI1 oncofusion protein on LSD1 activity through Western blot analysis, highlighting the association of LSD1 upregulation in Ewing sarcoma. This work provides valuable insight into LSD1's novel protein interaction pathways with potential applications to develop targeted therapies for Ewing Sarcoma (EWS). Mr. Zach Zablocki will present this poster. Zach acknowledges support from the Chemistry Department's Summer Undergraduate Research Fellowship at Marquette. Research in the Reiter Lab is supported by NIH R01 Grant (GM120572 - NJR). We also acknowledge sup

Comments

Journal of Biological Chemistry, Vol. 301, No. 5, Sup (May 2025). DOI.

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