O-GlcNAcylation of Sox2 at threonine 258 regulates the self-renewal and early cell fate of embryonic stem cells

Kim, Dong-Keon; Lee, Jang-Seok; Lee, Eun Young; Jang, Hansol; Han, Shuang; Kim, Hee Yeon; Hwang, Inyoung; Choi, Ji-Woong; Shin, Hyun Mu; You, Hye Jin; Youn, Hwa Young; Jang, Hyonchol

doi:10.1038/s12276-021-00707-7

Cited by 18 publications

(19 citation statements)

References 54 publications

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“…The obtained peptide fragments were analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) and several proteins were identified. Among these proteins, we focused on the transcription factor Sox2 because this protein was reported to be involved in the self-renewal of ESCs, and its O-GlcNAcylation was also discussed [ 17 , 18 ]. We then tried to identify O-GlcNAcylated sites on their tryptic fragments and successfully identified an O-GlcNAcylated site on Sox2.…”

Section: Resultsmentioning

confidence: 99%

Disaccharide-tag for highly sensitive identification of O-GlcNAc-modified proteins in mammalian cells

et al. 2022

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O-GlcNAcylation is the only sugar modification for proteins present in the cytoplasm and nucleus and is thought to be involved in the regulation of protein function and localization. Currently, several methods are known for detecting O-GlcNAcylated proteins using monoclonal antibodies or wheat germ agglutinin, but these methods have some limitations in their sensitivity and quantitative comparison. We developed a new disaccharide-tag method to overcome these problems. This is a method in which a soluble GalNAc transferase is expressed intracellularly, extended to a disaccharide of GalNAc-GlcNAc, and detected using a Wisteria japonica agglutinin specific to this disaccharide. We verified the method using human c-Rel protein and also highly sensitively compared the difference in O-GlcNAc modification of intracellular proteins associated with differentiation from embryonic stem cell (ESC) to epiblast-like cells (EpiLC). As one example of such a modification, a novel O-GlcNAc modification was found in the transcription factor Sox2 at residue Ser263, and the modification site could be identified by nano liquid chromatography-mass spectrometry.

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Section: Resultsmentioning

confidence: 99%

Disaccharide-tag for highly sensitive identification of O-GlcNAc-modified proteins in mammalian cells

et al. 2022

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“…A heatmap of DEGs was created using a MultiExperiment Viewer (The Institute of Genomic Research, Rockville, MD, USA). DEGs were subjected to core analysis using Ingenuity Pathway Analysis (IPA) software (Qiagen, Hilden, Germany), as described previously [ 23 , 24 ].…”

Section: Methodsmentioning

confidence: 99%

“…Statistical analyses were performed as reported previously [ 24 ]. Numerical values are expressed as the mean ± standard deviation, and p -values were calculated using the Student’s t -test calculator ( ) (accessed on 24 July 2022).…”

Section: Methodsmentioning

confidence: 99%

Cholesterol Synthesis Is Important for Breast Cancer Cell Tumor Sphere Formation and Invasion

et al. 2022

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Breast cancer has a high risk of recurrence and distant metastasis after remission. Controlling distant metastasis is important for reducing breast cancer mortality, but accomplishing this goal remains elusive. In this study, we investigated the molecular pathways underlying metastasis using cells that mimic the breast cancer distant metastasis process. HCC1143 breast cancer cells were cultured under two-dimensional (2D)-adherent, tumor sphere (TS), and reattached (ReA) culture conditions to mimic primary tumors, circulating tumor cells, and metastasized tumors, respectively. ReA cells demonstrated increased TS formation and enhanced invasion capacity compared to the original 2D-cultured parental cells. In addition, ReA cells had a higher frequency of ESA+CD44+CD24− population, which represents a stem-cell-like cell population. RNA sequencing identified the cholesterol synthesis pathway as one of the most significantly increased pathways in TS and ReA cells compared to parental cells, which was verified by measuring intracellular cholesterol levels. Furthermore, the pharmacological inhibition of the cholesterol synthesis pathway decreased the ability of cancer cells to form TSs and invade. Our results suggest that the cholesterol synthesis pathway plays an important role in the distant metastasis of breast cancer cells by augmenting TS formation and invasion capacity.

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“…Cells were harvested and washed in PBS and then lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM EDTA, a protease inhibitor cocktail (genDEPOT, USA), and a phosphatase inhibitor cocktail (genDEPOT, USA). Western blotting was performed according to a modified version of a previously described method 25 . After SDS–PAGE and protein transfer, the desired protein was detected using an iBind™ Automated Western system (Thermo Fisher Scientific, USA).…”

Section: Methodsmentioning

confidence: 99%

BMS794833 inhibits macrophage efferocytosis by directly binding to MERTK and inhibiting its activity

et al. 2022

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Myeloid epithelial reproductive proto-oncogene tyrosine kinase (MERTK) plays an essential role in modulating cancer immune tolerance by regulating macrophage efferocytosis. Studies are underway to develop small-molecule chemicals that inhibit MERTK as cancer immunotherapeutic agents, but these efforts are in their early stages. This study identified BMS794833, whose primary targets are MET and VEGFR2, as a potent MERTK inhibitor and developed a real-time efferocytosis monitoring system. The X-ray cocrystal structure revealed that BMS794833 was in contact with the ATP-binding pocket and the allosteric back pocket, rendering MERTK inactive. Homogeneous time-resolved fluorescence kinetic and Western blotting analyses showed that BMS794833 competitively inhibited MERTK activity in vitro and inhibited the autophosphorylation of MERTK in macrophages. We developed a system to monitor MERTK-dependent efferocytosis in real time, and using this system, we confirmed that BMS794833 significantly inhibited the efferocytosis of differentiated macrophages. Finally, BMS794833 significantly inhibited efferocytosis in vivo in a mouse model. These data show that BMS794833 is a type II MERTK inhibitor that regulates macrophage efferocytosis. In addition, the real-time efferocytosis monitoring technology developed in this study has great potential for future applications.

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O-GlcNAcylation of Sox2 at threonine 258 regulates the self-renewal and early cell fate of embryonic stem cells

Cited by 18 publications

References 54 publications

Disaccharide-tag for highly sensitive identification of O-GlcNAc-modified proteins in mammalian cells

Disaccharide-tag for highly sensitive identification of O-GlcNAc-modified proteins in mammalian cells

Cholesterol Synthesis Is Important for Breast Cancer Cell Tumor Sphere Formation and Invasion

BMS794833 inhibits macrophage efferocytosis by directly binding to MERTK and inhibiting its activity

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