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Interactions between proteins are essential to any cellular process and constitute the basis for molecular networks that determine the functional state of a cell. With the technical advances in recent years, an astonishingly high number of protein–protein interactions has been revealed. However, the interactome of O-linked N-acetylglucosamine transferase (OGT), the sole enzyme adding the O-linked β-N-acetylglucosamine (O-GlcNAc) onto its target proteins, has been largely undefined. To that end, we collated OGT interaction proteins experimentally identified in the past several decades. Rigorous curation of datasets from public repositories and O-GlcNAc-focused publications led to the identification of up to 929 high-stringency OGT interactors from multiple species studied (including Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Arabidopsis thaliana, and others). Among them, 784 human proteins were found to be interactors of human OGT. Moreover, these proteins spanned a very diverse range of functional classes (e.g., DNA repair, RNA metabolism, translational regulation, and cell cycle), with significant enrichment in regulating transcription and (co)translation. Our dataset demonstrates that OGT is likely a hub protein in cells. A webserver OGT-Protein Interaction Network (OGT-PIN) has also been created, which is freely accessible.

Experience-driven synaptic plasticity is believed to underlie adaptive behavior by rearranging the way neuronal circuits process information. We have previously discovered that O-GlcNAc transferase (OGT), an enzyme that modifies protein function by attaching β–N-acetylglucosamine (GlcNAc) to serine and threonine residues of intracellular proteins (O-GlcNAc), regulates food intake by modulating excitatory synaptic function in neurons in the hypothalamus. However, how OGT regulates excitatory synapse function is largely unknown. Here we demonstrate that OGT is enriched in the postsynaptic density of excitatory synapses. In the postsynaptic density, O-GlcNAcylation on multiple proteins increased upon neuronal stimulation. Knockout of the OGT gene decreased the synaptic expression of the AMPA receptor GluA2 and GluA3 subunits, but not the GluA1 subunit. The number of opposed excitatory presynaptic terminals was sharply reduced upon postsynaptic knockout of OGT. There were also fewer and less mature dendritic spines on OGT knockout neurons. These data identify OGT as a molecular mechanism that regulates synapse maturity.

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGT’s myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGT’s enzymatic activities for cell viability. Using genetic complementation, we found that OGT’s glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGT’s catalytic and noncatalytic activities affect protein abundance, we found that OGT’s noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology.

Hepatocellular carcinoma (HCC) is a lethal and aggressive human malignancy. The present study examins the anti‐tumor effects of deubiquitylating enzymes (DUB) inhibitors in HCC. It is found that the inhibitor of ubiquitin specific peptidase 8 (USP8) and DUB‐IN‐3 shows the most effective anti‐cancer responses. Targeting USP8 inhibits the proliferation of HCC and induces cell ferroptosis. In vivo xenograft and metastasis experiments indicate that inhibition of USP8 suppresses tumor growth and lung metastasis. DUB‐IN‐3 treatment or USP8 depletion decrease intracellular cystine levels and glutathione biosynthesis while increasing the accumulation of reactive oxygen species (ROS). Mechanistical studies reveal that USP8 stabilizes O‐GlcNAc transferase (OGT) via inhibiting K48‐specific poly‐ubiquitination process on OGT protein at K117 site, and STE20‐like kinase (SLK)‐mediated S716 phosphorylation of USP8 is required for the interaction with OGT. Most importantly, OGT O‐GlcNAcylates solute carrier family 7, member 11 (SLC7A11) at Ser26 in HCC cells, which is essential for SLC7A11 to import the cystine from the extracellular environment. Collectively, this study demonstrates that pharmacological inhibition or knockout of USP8 can inhibit the progression of HCC and induce ferroptosis via decreasing the stability of OGT, which imposes a great challenge that targeting of USP8 is a potential approach for HCC treatment. Targeting USP8 suppresses HCC progression and induces ferroptosis. USP8 stabilizes OGT to O‐GlcNAcylate SLC7A11, which is essential for SLC7A11 to import the cystine from the extracellular environment. Targeting USP8 may be a promising approach for the treatment of HCC.

O-GlcNAcylation is a posttranslational modification that occurs at serine and threonine residues of protein substrates by the addition of O-linked β-d-N-acetylglucosamine (GlcNAc) moiety. Two enzymes are involved in this modification: O-GlcNac transferase (OGT), which attaches the GlcNAc residue to the protein substrate, and O-GlcNAcase (OGA), which removes it. This biological balance is important for many biological processes, such as protein expression, cell apoptosis, and regulation of enzyme activity. The extent of this modification has sparked interest in the medical community to explore OGA and OGT as therapeutic targets, particularly in degenerative diseases. While some OGA inhibitors are already in phase 1 clinical trials for the treatment of Alzheimer’s disease, OGT inhibitors still have a long way to go. Due to complex expression and instability, the discovery of potent OGT inhibitors is challenging. Over the years, the field has grappled with this problem, and scientists have developed a number of techniques and assays. In this review, we aim to highlight assays and techniques for OGT inhibitor discovery, evaluate their strength for the field, and give us direction for future bioassay methods.

O -linked-N-acetylglucosaminylation ( O -GlcNAcylation) is a type of glycosylation that occurs when a monosaccharide, O -GlcNAc, is added onto serine or threonine residues of nuclear or cytoplasmic proteins by O -GlcNAc transferase (OGT) and which can be reversibly removed by O -GlcNAcase (OGA). O -GlcNAcylation couples the processes of nutrient sensing, metabolism, signal transduction and transcription, and plays important roles in development, normal physiology and physiopathology. Cumulative studies have indicated that O -GlcNAcylation affects the functions of protein substrates in a number of ways, including protein cellular localization, protein stability and protein/protein interaction. Particularly, O -GlcNAcylation has been shown to have intricate crosstalk with phosphorylation as they both modify serine or threonine residues. Aberrant O -GlcNAcylation on various protein substrates has been implicated in many diseases, including neurodegenerative diseases, diabetes and cancers. However, the role of protein O -GlcNAcylation in immune cell lineages has been less explored. This review summarizes the current understanding of the fundamental biochemistry of O -GlcNAcylation, and discusses the molecular mechanisms by which O -GlcNAcylation regulates the development, maturation and functions of immune cells. In brief, O -GlcNAcylation promotes the development, proliferation, and activation of T and B cells. O -GlcNAcylation regulates inflammatory and antiviral responses of macrophages. O -GlcNAcylation promotes the function of activated neutrophils, but inhibits the activity of nature killer cells.

The circulatory system is a closed conduit system throughout the body and consists of two parts as follows: the cardiovascular system and the lymphatic system. Hematological malignancies usually grow and multiply in the circulatory system, directly or indirectly affecting its function. These malignancies include multiple myeloma, leukemia, and lymphoma. O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) regulates the function and stability of substrate proteins through O-GlcNAc modification. Abnormally expressed OGT is strongly associated with tumorigenesis, including hematological malignancies, colorectal cancer, liver cancer, breast cancer, and prostate cancer. In cells, OGT can assemble with a variety of proteins to form complexes to exercise related biological functions, such as OGT/HCF-1, OGT/TET, NSL, and then regulate glucose metabolism, gene transcription, cell proliferation, and other biological processes, thus affecting the development of hematological malignancies. This review summarizes the complexes involved in the assembly of OGT in cells and the role of related OGT complexes in hematological malignancies. Unraveling the complex network regulated by the OGT complex will facilitate a better understanding of hematologic malignancy development and progression.

The accumulation of dysfunctional mitochondria is an early feature of Alzheimer's disease (AD). The impaired turnover of damaged mitochondria increases reactive oxygen species production and lowers ATP generation, leading to cellular toxicity and neurodegeneration. Interestingly, AD exhibits a disruption in the global post-translational modification β-N-acetylglucosamine (O-GlcNAc). O-GlcNAc is a ubiquitous single sugar modification found in the nuclear, cytoplasmic, and mitochondrial proteins. Cells maintain a homeostatic level of O-GlcNAc by cycling the addition and removal of the sugar by O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA), respectively. We used patient-derived induced pluripotent stem cells, a transgenic mouse model of AD, SH-SY5Y neuroblastoma cell lines to examine the effect of sustained O-GlcNAcase inhibition by Thiamet-G (TMG) or OGT deficiency on mitophagy using biochemical analyses. Here, we established an essential role for O-GlcNAc in regulating mitophagy (mitochondria-selective autophagy). Stimulating mitophagy using urolithin A (UA) decreases cellular O-GlcNAc and elevates mitochondrial O-GlcNAc. Sustained elevation in O-GlcNAcylation via pharmacologically inhibiting OGA using Thiamet-G (TMG) increases the mitochondrial level of mitophagy protein PTEN-induced kinase 1 (PINK1) and autophagy-related protein light chain 3 (LC3). Moreover, we detected O-GlcNAc on PINK1 and TMG increases its O-GlcNAcylation level. Conversely, decreasing cellular O-GlcNAcylation by knocking down OGT decreases both PINK1 protein expression and LC3 protein expression. Mitochondria isolated from CAMKII-OGT-KO mice also had decreased PINK1 and LC3. Moreover, human brain organoids treated with TMG showed significant elevation in LC3 compared to control. However, TMG-treated AD organoids showed no changes in LC3 expression. Collectively, these data demonstrate that O-GlcNAc plays a crucial role in the activation and progression of mitophagy, and this activation is disrupted in AD.

O -linked β- d - N -acetylglucosamine ( O -GlcNAc) is an important post-translational modification of serine or threonine residues on thousands of proteins in the nucleus and cytoplasm of all animals and plants. In eukaryotes, only two conserved enzymes are involved in this process. O -GlcNAc transferase is responsible for adding O -GlcNAc to proteins, while O -GlcNAcase is responsible for removing it. Aberrant O -GlcNAcylation is associated with a variety of human diseases, such as diabetes, cancer, neurodegenerative diseases, and cardiovascular diseases. Numerous studies have confirmed that O -GlcNAcylation is involved in the occurrence and progression of cancers in multiple systems throughout the body. It is also involved in regulating multiple cancer hallmarks, such as metabolic reprogramming, proliferation, invasion, metastasis, and angiogenesis. In this review, we first describe the process of O -GlcNAcylation and the structure and function of O -GlcNAc cycling enzymes. In addition, we detail the occurrence of O -GlcNAc in various cancers and the role it plays. Finally, we discuss the potential of O -GlcNAc as a promising biomarker and novel therapeutic target for cancer diagnosis, treatment, and prognosis.

Recent data have linked hypoxia, a classic feature of the tumor microenvironment, to the function of specific microRNAs (miRNAs); however, whether hypoxia affects other types of noncoding transcripts is currently unknown. Starting from a genome-wide expression profiling, we demonstrate for the first time a functional link between oxygen deprivation and the modulation of long noncoding transcripts from ultraconserved regions, termed transcribed-ultraconserved regions (T-UCRs). Interestingly, several hypoxia-upregulated T-UCRs, henceforth named ‘hypoxia-induced noncoding ultraconserved transcripts’ (HINCUTs), are also overexpressed in clinical samples from colon cancer patients. We show that these T-UCRs are predominantly nuclear and that the hypoxia-inducible factor (HIF) is at least partly responsible for the induction of several members of this group. One specific HINCUT, uc.475 (or HINCUT-1) is part of a retained intron of the host protein-coding gene, O-linked N-acetylglucosamine transferase, which is overexpressed in epithelial cancer types. Consistent with the hypothesis that T-UCRs have important function in tumor formation, HINCUT-1 supports cell proliferation specifically under hypoxic conditions and may be critical for optimal O-GlcNAcylation of proteins when oxygen tension is limiting. Our data gives a first glimpse of a novel functional hypoxic network comprising protein-coding transcripts and noncoding RNAs (ncRNAs) from the T-UCRs category.