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Self‐renewal and differentiation of embryonic stem cells (ESCs) are influenced by protein O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) modification, but the underlying mechanism remains incompletely understood. Herein, we report the identification of 979 O‐GlcNAcylated proteins and 1340 modification sites in mouse ESCs (mESCs) by using a chemoproteomics method. In addition to OCT4 and SOX2, the third core pluripotency transcription factor (PTF) NANOG was found to be modified and functionally regulated by O‐GlcNAc. Upon differentiation along the neuronal lineage, the O‐GlcNAc stoichiometry at 123 sites of 83 proteins—several of which were PTFs—was found to decline. Transcriptomic profiling reveals 2456 differentially expressed genes responsive to OGT inhibition during differentiation, of which 901 are target genes of core PTFs. By acting on the core PTF network, suppression of O‐GlcNAcylation upregulates neuron‐related genes, thus contributing to mESC fate determination. [ABSTRACT FROM AUTHOR]

As an essential micronutrient element in organisms, copper controls a host of fundamental cellular functions. Recently, copper-dependent cell growth and proliferation have been defined as "cuproplasia". Conversely, "cuproptosis" represents copper-dependent cell death, in a nonapoptotic manner. So far, a series of copper ionophores have been developed to kill cancer cells. However, the biological response mechanism of copper uptake has not been systematically analyzed. Based on quantitative proteomics, we revealed the crosstalk between copper stress and cuproptosis in cancer cells, and also explored the feasibility of curcumin as anticancer copper ionophore. Copper stress not only couples with cuproptosis, but also leads to reactive oxygen species (ROS) stress, oxidative damage and cell cycle arrest. In cancer cells, a feedback cytoprotection mechanism involving cuproptosis mediators was discovered. During copper treatment, the activation of glutamine transporters and the loss of Fe–S cluster proteins are the facilitators and results of cuproptosis, respectively. Through copper depletion, glutathione (GSH) blocks the cuproptosis process, rescues the activation of glutamine transporters, and prevents the loss of Fe–S cluster proteins, except for protecting cancer cells from apoptosis, protein degradation and oxidative damage. In addition, the copper ionophore curcumin can control the metabolisms of lipids, RNA, NADH and NADPH in colorectal cancer cells, and also up-regulates positive cuproptosis mediators. This work not only established the crosstalk between copper stress and cuproptosis, but also discolored the suppression and acceleration of cuproptosis by GSH and curcumin, respectively. Our results are significant for understanding cuproptosis process and developing novel anticancer reagents based on cuproptosis. This work not only established the crosstalk between copper stress and cuproptosis, but also discolored the suppression and acceleration of cuproptosis by glutathione and curcumin, respectively. [Display omitted] • The crosstalk between copper stress and cuproptosis was disclosed. • Copper stress also leads to ROS stress, oxidative damage and cell cycle arrest. • GSH blocks the cuproptosis process, along with multiply cytoprotective mechanisms. • Curcumin controls the metabolisms of lipids, RNA, NADH and NADPH. • Curcumin up-regulates positive cuproptosis mediators in colorectal cancer cells. [ABSTRACT FROM AUTHOR]