Project description:Histone modifications are typically recognized by chromatin-binding protein modules (referred to as “readers”) to mediate fundamental processes such as transcription. Lysine β-hydroxybutyrylation (Kbhb) is a new type of histone mark that couples metabolism to gene expression. However, the readers that prefer histone Kbhb remain elusive. This knowledge gap must be filled in order to reveal the molecular mechanism of this epigenetic regulation. Herein, we developed a chemical proteomic approach, relying upon multivalent photoaffinity probes to capture binders of the mark and identified ENL as a novel target of H3K9bhb. Biochemical studies and CUT&Tag analysis further suggested that ENL favorably binds to H3K9bhb, and co-localizes with it on promoter regions to modulate gene expression. Notably, disrupting the interaction between H3K9bhb and ENL via structure-based mutation leads to the suppressed expression of the gene like MYC that drives cell proliferation.
Project description:Histone modifications are typically recognized by chromatin-binding protein modules (referred to as “readers”) to mediate fundamental processes such as transcription. Lysine β-hydroxybutyrylation (Kbhb) is a new type of histone mark that couples metabolism to gene expression. However, the readers that prefer histone Kbhb remain elusive. This knowledge gap must be filled in order to reveal the molecular mechanism of this epigenetic regulation. Herein, we developed a chemical proteomic approach, relying upon multivalent photoaffinity probes to capture binders of the mark and identified ENL as a novel target of H3K9bhb. Biochemical studies and CUT&Tag analysis further suggested that ENL favorably binds to H3K9bhb, and co-localizes with it on promoter regions to modulate gene expression. Notably, disrupting the interaction between H3K9bhb and ENL via structure-based mutation leads to the suppressed expression of the gene like MYC that drives cell proliferation.
Project description:Olaparib is a widely used PARP inhibitor for the treatment of BRCA-mutated cancers. To comprehensively understand the drug's clinical impact, measuring its interactions with intended on- and off-targets is crucial. In this study, olaparib's on- and off-targets were profiled using photoaffinity labeling, a powerful, proteome-wide method for studying the direct interactions between a drug and its protein targets. A novel photoaffinity probe was designed and used in a proteomic screening to discover novel targets of olaparib in the human proteome. The probe, incorporating a pre-installed biotin group, bypasses the limitations of using a copper(I)-catalyzed click reaction in cell lysates for reporter group conjugation and revealed a broad range of olaparib interactors, including previously unreported proteins, in a quantitative mass spectrometry-based proteomic screening using HeLa whole cell lysate. The study contributes to our current understanding of the pharmacology of olaparib and provides a valuable tool for elucidating drug interactors within cell lysates, potentially guiding the development of more targeted therapeutics with fewer off-targets.
Project description:Alzheimer's disease (AD) and most of other tauopathies are incurable neurodegenerative diseases with unpleasant symptoms and consequences. The common hallmark of all these diseases is tau pathology but its connection with disease progress has not been completely understood so far. Therefore, uncovering novel tau interacting partners and pathology affected molecular pathways can reveal the causes of diseases as well as potential targets for development of AD treatment. Despite of large amount of known tau interacting partners, limited number of studies focused on in vivo tau interactions in disease or healthy conditions are available. Here, we applied an in vivo crosslinking approach, capable of capturing weak and transient protein-protein interactions, to a unique transgenic rat model of progressive tau pathology similar to human AD. We have identified 175 potential novel and known tau interacting proteins by MALDI-TOF mass spectrometry. Several of the most promising candidates for potential drug development were selected for validation by coimmunoprecipitation and colocalization experiments in animal and cellular models.
Project description:p53 is an essential tumor suppressor, whose activity is finely tuned by the posttranslational modifications. Previous research has reported that β-hydroxybutyrate (BHB) induces β-hydroxybutyrylation (Kbhb), which is a novel histone posttranslational modification. Here we report that p53 is modified by kbhb and that this modification occurs at lysines 120, 319, and 370 of p53. We demonstrate that the level of p53 kbhb is dramatically increased in cultured cells treated with BHB and in thymus tissues of fasted mice, and that CBP catalyze p53 kbhb. We show that p53 kbhb results in lower levels of p53 acetylation and reduced expression of the p53 downstream genes p21 and PUMA, as well as reduced cell growth arrest and apoptosis in cultured cells under p53-activating conditions. Similar results were observed in mouse thymus tissue under starvation conditions, which result in increased concentrations of serum BHB, and in response to genotoxic stress caused by γ-irradiation to activate p53. Our findings thus show that BHB-mediated p53 kbhb is a novel mechanism of p53 activity regulation, which may explain the link between ketone bodies and tumor, and which may provide promising therapeutic target for cancer treatment.
Project description:The purpose of this study was to identify genes in keratinocytes and fibroblasts in human skin equivalents that changed expression in response to the burrowing of live scabies mites. Four biological replicates for the uninfested control condition and five biological replicates for the treatment conditions (live mites, mite extract) were processed for gene expression analysis using Affymetrix Human Gene 1.0 ST arrays.
Project description:Drosophila preblastoderm embryo extract assisted chromatin assembly with or without 16mer DNA mimic. Pull down of chromatin fiber to reveal chromatin binders in the system.