-
Leukemia May 2024TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in...
TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.
Topics: Humans; Leukemia, Myeloid, Acute; Animals; Mice; Disease Progression; TNF Receptor-Associated Factor 6; N-Acetylglucosaminyltransferases; Glycolysis; Cell Line, Tumor; Metabolic Reprogramming; Intracellular Signaling Peptides and Proteins
PubMed: 38609495
DOI: 10.1038/s41375-024-02245-3 -
Targeting Fatty Acid Desaturase I Inhibits Renal Cancer Growth Via ATF3-mediated ER Stress Response.BioRxiv : the Preprint Server For... Mar 2024Monounsaturated fatty acids (MUFAs) play a pivotal role in maintaining endoplasmic reticulum (ER) homeostasis, an emerging hallmark of cancer. However, the role of...
Monounsaturated fatty acids (MUFAs) play a pivotal role in maintaining endoplasmic reticulum (ER) homeostasis, an emerging hallmark of cancer. However, the role of polyunsaturated fatty acid (PUFAs) desaturation in persistent ER stress driven by oncogenic abnormalities remains elusive. Fatty Acid Desaturase 1 (FADS1) is a rate-limiting enzyme controlling the bioproduction of long-chain PUFAs. Our previous research has demonstrated the significant role of FADS1 in cancer survival, especially in kidney cancers. We explored the underlying mechanism in this study. We found that pharmacological inhibition or knockdown of the expression of FADS1 effectively inhibits renal cancer cell proliferation and induces cell cycle arrest. The stable knockdown of FADS1 also significantly inhibits tumor formation . Mechanistically, we show that while FADS1 inhibition induces ER stress, its expression is also augmented by ER-stress inducers. Notably, FADS1-inhibition sensitized cellular response to ER stress inducers, providing evidence of FADS1's role in modulating the ER stress response in cancer cells. We show that, while FADS1 inhibition-induced ER stress leads to activation of ATF3, ATF3-knockdown rescues the FADS1 inhibition-induced ER stress and cell growth suppression. In addition, FADS1 inhibition results in the impaired biosynthesis of nucleotides and decreases the level of UPD-N-Acetylglucosamine, a critical mediator of the unfolded protein response. Our findings suggest that PUFA desaturation is crucial for rescuing cancer cells from persistent ER stress, supporting FADS1 as a new therapeutic target.
PubMed: 38586033
DOI: 10.1101/2024.03.23.586426 -
Advanced Science (Weinheim,... Jun 2024Glycans are complex biomolecules that encode rich information and regulate various biological processes, such as fertilization, host-pathogen binding, and immune...
Glycans are complex biomolecules that encode rich information and regulate various biological processes, such as fertilization, host-pathogen binding, and immune recognition, through interactions with glycan-binding proteins. A key driving force for glycan-protein recognition is the interaction between the π electron density of aromatic amino acid side chains and polarized C─H groups of the pyranose (termed the CH-π interaction). However, the relatively weak binding affinity between glycans and proteins has hindered the application of glycan detection and imaging. Here, computational modeling and molecular dynamics simulations are employed to design a chemical strategy that enhances the CH-π interaction between glycans and proteins by genetically incorporating electron-rich tryptophan derivatives into a lectin PhoSL, which specifically recognizes core fucosylated N-linked glycans. This significantly enhances the binding affinity of PhoSL with the core fucose ligand and enables sensitive detection and imaging of core fucosylated glycans in vitro and in xenograft tumors in mice. Further, the study showed that this strategy is applicable to improve the binding affinity of GafD lectin for N-acetylglucosamine-containing glycans. The approach thus provides a general and effective way to manipulate glycan-protein recognition for glycoscience applications.
Topics: Polysaccharides; Animals; Mice; Molecular Dynamics Simulation; Lectins; Protein Binding; Humans; Disease Models, Animal
PubMed: 38582526
DOI: 10.1002/advs.202308522 -
Cell Death & Disease Apr 2024The immunosuppressive microenvironment caused by several intrinsic and extrinsic mechanism has brought great challenges to the immunotherapy of pancreatic cancer. We...
The immunosuppressive microenvironment caused by several intrinsic and extrinsic mechanism has brought great challenges to the immunotherapy of pancreatic cancer. We identified GFPT2, the key enzyme in hexosamine biosynthesis pathway (HBP), as an immune-related prognostic gene in pancreatic cancer using transcriptome sequencing and further confirmed that GFPT2 promoted macrophage M2 polarization and malignant phenotype of pancreatic cancer. HBP is a glucose metabolism pathway leading to the generation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which is further utilized for protein O-GlcNAcylation. We confirmed GFPT2-mediated O-GlcNAcylation played an important role in regulating immune microenvironment. Through cellular proteomics, we identified IL-18 as a key downstream of GFPT2 in regulating the immune microenvironment. Through CO-IP and protein mass spectrum, we confirmed that YBX1 was O-GlcNAcylated and nuclear translocated by GFPT2-mediated O-GlcNAcylation. Then, YBX1 functioned as a transcription factor to promote IL-18 transcription. Our study elucidated the relationship between the metabolic pathway of HBP in cancer cells and the immune microenvironment, which might provide some insights into the combination therapy of HBP vulnerability and immunotherapy in pancreatic cancer.
Topics: Humans; Glycosylation; Interleukin-18; Pancreatic Neoplasms; Proteins; Biosynthetic Pathways; Hexosamines; Tumor Microenvironment; Y-Box-Binding Protein 1; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
PubMed: 38575607
DOI: 10.1038/s41419-024-06589-7 -
Disease Models & Mechanisms Apr 2024The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to proteins (referred to as O-GlcNAcylation) is a modification that is crucial for vertebrate development....
The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to proteins (referred to as O-GlcNAcylation) is a modification that is crucial for vertebrate development. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). Missense variants of OGT have recently been shown to segregate with an X-linked syndromic form of intellectual disability, OGT-linked congenital disorder of glycosylation (OGT-CDG). Although the existence of OGT-CDG suggests that O-GlcNAcylation is crucial for neurodevelopment and/or cognitive function, the underlying pathophysiologic mechanisms remain unknown. Here we report a mouse line that carries a catalytically impaired OGT-CDG variant. These mice show altered O-GlcNAc homeostasis with decreased global O-GlcNAcylation and reduced levels of OGT and OGA in the brain. Phenotypic characterization of the mice revealed lower body weight associated with reduced body fat mass, short stature and microcephaly. This mouse model will serve as an important tool to study genotype-phenotype correlations in OGT-CDG in vivo and for the development of possible treatment avenues for this disorder.
Topics: Animals; N-Acetylglucosaminyltransferases; Intellectual Disability; Disease Models, Animal; Brain; Phenotype; Mice; Neurodevelopmental Disorders; beta-N-Acetylhexosaminidases; Glycosylation; Body Weight
PubMed: 38566589
DOI: 10.1242/dmm.050671 -
Journal of the American Chemical Society Apr 2024Protein O-linked β--acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of the homeostasis...
Protein O-linked β--acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of the homeostasis of O-GlcNAcylation has been linked to various human diseases, including cancer, diabetes, and neurodegeneration. However, there are limited chemical tools for protein- and site-specific O-GlcNAc modification, rendering the precise study of the O-GlcNAcylation challenging. To address this, we have developed heterobifunctional small molecules, named O-GlcNAcylation TArgeting Chimeras (OGTACs), which enable protein-specific O-GlcNAcylation in living cells. OGTACs promote O-GlcNAcylation of proteins such as BRD4, CK2α, and EZH2 by recruiting FKBP12-fused O-GlcNAc transferase (OGT), with temporal, magnitude, and reversible control. Overall, the OGTACs represent a promising approach for inducing protein-specific O-GlcNAcylation, thus enabling functional dissection and offering new directions for O-GlcNAc-targeting therapeutic development.
Topics: Humans; Nuclear Proteins; Transcription Factors; Protein Processing, Post-Translational; Neoplasms; N-Acetylglucosaminyltransferases; Acetylglucosamine; Bromodomain Containing Proteins; Cell Cycle Proteins
PubMed: 38561350
DOI: 10.1021/jacs.3c14380 -
Glycoconjugate Journal Apr 2024Molluscs are intermediate hosts for several parasites. The recognition processes, required to evade the host's immune response, depend on carbohydrates. Therefore, the...
Determination, expression and characterization of an UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) from the Pacific oyster, Crassostrea gigas.
Molluscs are intermediate hosts for several parasites. The recognition processes, required to evade the host's immune response, depend on carbohydrates. Therefore, the investigation of mollusc glycosylation capacities is of high relevance to understand the interaction of parasites with their host. UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) is the key enzyme for the biosynthesis of hybrid and complex type N-glycans catalysing the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the α-1,3 Man antenna of ManGlcNAc. Thereby, the enzyme produces a suitable substrate for further enzymes, such as α-mannosidase II, GlcNAc-transferase II, galactosyltransferases or fucosyltransferases. The sequence of GnT- I from the Pacific oyster, Crassostrea gigas, was obtained by homology search using the corresponding human enzyme as the template. The obtained gene codes for a 445 amino acids long type II transmembrane glycoprotein and shared typical structural elements with enzymes from other species. The enzyme was expressed in insect cells and purified by immunoprecipitation using protein A/G-plus agarose beads linked to monoclonal His-tag antibodies. GnT-I activity was determined towards the substrates Man5-PA, MM-PA and GnM-PA. The enzyme displayed highest activity at pH 7.0 and 30 °C, using Man5-PA as the substrate. Divalent cations were indispensable for the enzyme, with highest activity at 40 mM Mn, while the addition of EDTA or Cu abolished the activity completely. The activity was also reduced by the addition of UDP, UTP or galactose. In this study we present the identification, expression and biochemical characterization of the first molluscan UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I, GnT-I, from the Pacific oyster Crassostrea gigas.
Topics: Animals; N-Acetylglucosaminyltransferases; Crassostrea; Amino Acid Sequence; Molecular Sequence Data; Cloning, Molecular; Substrate Specificity; Phylogeny; Spodoptera
PubMed: 38557922
DOI: 10.1007/s10719-024-10148-9 -
BMC Research Notes Mar 2024O-GlcNAcylation is a nutrient-sensing post-translational modification process. This cycling process involves two primary proteins: the O-linked N-acetylglucosamine...
O-GlcNAcylation is a nutrient-sensing post-translational modification process. This cycling process involves two primary proteins: the O-linked N-acetylglucosamine transferase (OGT) catalysing the addition, and the glycoside hydrolase OGA (O-GlcNAcase) catalysing the removal of the O-GlCNAc moiety on nucleocytoplasmic proteins. This process is necessary for various critical cellular functions. The O-linked N-acetylglucosamine transferase (OGT) gene produces the OGT protein. Several studies have shown the overexpression of this protein to have biological implications in metabolic diseases like cancer and diabetes mellitus (DM). This study retrieved 159 SNPs with clinical significance from the SNPs database. We probed the functional effects, stability profile, and evolutionary conservation of these to determine their fit for this research. We then identified 7 SNPs (G103R, N196K, Y228H, R250C, G341V, L367F, and C845S) with predicted deleterious effects across the four tools used (PhD-SNPs, SNPs&Go, PROVEAN, and PolyPhen2). Proceeding with this, we used ROBETTA, a homology modelling tool, to model the proteins with these point mutations and carried out a structural bioinformatics method- molecular docking- using the Glide model of the Schrodinger Maestro suite. We used a previously reported inhibitor of OGT, OSMI-1, as the ligand for these mutated protein models. As a result, very good binding affinities and interactions were observed between this ligand and the active site residues within 4Å of OGT. We conclude that these mutation points may be used for further downstream analysis as drug targets for treating diabetes mellitus.
Topics: Humans; Molecular Docking Simulation; Ligands; Mutation; Point Mutation; Diabetes Mellitus; Protein Processing, Post-Translational
PubMed: 38539217
DOI: 10.1186/s13104-024-06744-5 -
Neuroscience May 2024GABAergic interneurons and perineuronal nets (PNNs) are important regulators of plasticity throughout life and their dysfunction has been implicated in the pathogenesis...
Alterations of Perineuronal Net Expression and Abnormal Social Behavior and Whisker-dependent Texture Discrimination in Mice Lacking the Autism Candidate Gene Engrailed 2.
GABAergic interneurons and perineuronal nets (PNNs) are important regulators of plasticity throughout life and their dysfunction has been implicated in the pathogenesis of several neuropsychiatric conditions, including autism spectrum disorders (ASD). PNNs are condensed portions of the extracellular matrix (ECM) that are crucial for neural development and proper formation of synaptic connections. We previously showed a reduced expression of GABAergic interneuron markers in the hippocampus and somatosensory cortex of adult mice lacking the Engrailed2 gene (En2-/- mice), a mouse model of ASD. Since alterations in PNNs have been proposed as a possible pathogenic mechanism in ASD, we hypothesized that the PNN dysfunction may contribute to the neural and behavioral abnormalities of En2-/- mice. Here, we show an increase in the PNN fluorescence intensity, evaluated by Wisteria floribunda agglutinin, in brain regions involved in social behavior and somatosensory processing. In addition, we found that En2-/- mice exhibit altered texture discrimination through whiskers and display a marked decrease in the preference for social novelty. Our results raise the possibility that altered expression of PNNs, together with defects of GABAergic interneurons, might contribute to the pathogenesis of social and sensory behavioral abnormalities.
Topics: Animals; Homeodomain Proteins; Nerve Tissue Proteins; Mice, Knockout; Social Behavior; Vibrissae; Male; Mice, Inbred C57BL; Extracellular Matrix; Interneurons; Disease Models, Animal; Mice; Somatosensory Cortex; Discrimination, Psychological; Receptors, N-Acetylglucosamine; GABAergic Neurons; Brain; Plant Lectins
PubMed: 38537894
DOI: 10.1016/j.neuroscience.2024.03.023 -
Plant Direct Mar 2024Plant genomes contain numerous genes encoding chitinase-like (CTL) proteins, which have a similar protein structure to chitinase belonging to the glycoside hydrolase...
Plant genomes contain numerous genes encoding chitinase-like (CTL) proteins, which have a similar protein structure to chitinase belonging to the glycoside hydrolase (GH) family but lack the chitinolytic activity to cleave the -1,4-glycosidic bond in chitins, polymers of -acetylglucosamine. mutations found in rice and have caused pleiotropic developmental defects, including altered cell wall composition and decreased abiotic stress tolerance, likely due to reduced cellulose content. In this study, we identified () as a genetic suppressor of the mutation in . The mutation in restored almost all examined defects to nearly wild-type levels or at least partially. encodes a Golgi-located type II membrane protein with glycosyltransferase (GT) activity, and its mutations lead to a reduction in cellulose content and hypersensitivity to cellulose biosynthesis inhibitors, although to a lesser extent than mutation. The promoter fused with the GUS reporter gene exhibited GUS activity in interfascicular fibers and xylem in stems; meanwhile, the mutation significantly increased this activity. Our findings provide genetic and molecular evidence that the antagonistic activities of CTL1 and SUH1 play an essential role in assembling the cell wall in .
PubMed: 38525472
DOI: 10.1002/pld3.580