Journal: PLOS Pathogens

Article Title: O-GlcNAcylation licenses RNF166 to degrade the M protein of porcine coronaviruses

doi: 10.1371/journal.ppat.1014301

Figure Lengend Snippet: A, Schematic diagram of the hexosamine biosynthetic pathway (HBP) from glycolysis to UDP-GlcNAc. B–C , IPI-2I cells were infected with PDCoV (MOI = 2) and harvested at the indicated time points for HBP flux analysis. (B) Protein levels of GFAT, AGX, OGT, PDCoV N, and cellular O-GlcNAcylation, assessed by western blot. Ratios below the images represent the protein levels of GFAT and AGX relative to β-actin, as quantified by the ImageJ software. (C) Intracellular UDP-GlcNAc content, measured by ELISA. D-I, IPI‑2I cells were subjected to the indicated treatments, infected with PDCoV (MOI = 2), and harvested at 12 h post-infection (hpi) for viral replication analysis. (D) Cells supplemented with or without UDP‑GlcNAc (32 mM). Ratios below the images represent the protein levels of PDCoV relative to β-actin, as quantified by the ImageJ software. (E) Cells treated with the O‑GlcNAcylation agonist MK‑8719 (10 μM) or inhibitor OSMI‑1 (50 μM). (F) Wild-type versus OGT knockout (KO) IPI-2I cells. Ratios below the images represent the protein levels of PDCoV relative to β-actin, as quantified by the ImageJ software. (G–H) Wild‑type and OGT (KO) IPI-2I cells supplemented with or without UDP‑GlcNAc. (I) OGT (KO) IPI-2I cells overexpressing OGT or catalytically inactive OGT‑DP, with UDP‑GlcNAc supplementation. β-actin and GAPDH served as internal controls for western blot and RT-qPCR, respectively. Data in panels C, D, E, F, G, H, and I represent mean ± s.d. (n = 3). Statistical significance was determined by two-tailed Student’s t-test; * P < 0.05; ** P < 0.01; ns, not significant.

Article Snippet: UDP-GlcNAc (HY-112174) was purchased from MedChemExpress, China.

Techniques: Infection, Western Blot, Software, Enzyme-linked Immunosorbent Assay, Knock-Out, Quantitative RT-PCR, Two Tailed Test

Journal: PLOS Pathogens

Article Title: O-GlcNAcylation licenses RNF166 to degrade the M protein of porcine coronaviruses

doi: 10.1371/journal.ppat.1014301

Figure Lengend Snippet: A-B, HBP flux in IPI-2I cells infected with PEDV, TGEV, or SADS-CoV at indicated time points: (A) Protein levels of GFAT, AGX, OGT, viral N protein, and global O-GlcNAcylation, assessed by western blot. Ratios below the images represent the protein levels of GFAT and AGX relative to β-actin, as quantified by the ImageJ software. (B) Intracellular UDP-GlcNAc content, measured by ELISA. C, Viral replication of PEDV, TGEV, or SADS-CoV in IPI-2I cells supplemented with or without UDP-GlcNAc. D , Viral replication of PEDV, TGEV, or SADS-CoV in IPI-2I cells treated with or without the O-GlcNAcylation agonist MK-8719. E-F , Viral replication of PEDV, TGEV, or SADS-CoV in wild-type ( E ) and RNF166 (KO) (F) IPI-2I cells overexpressing RNF166 or RNF166-T157A. G-I , Western blot analysis of the degradation of PEDV M protein (G) , TGEV M protein (H) , SADS-CoV M protein ( I ) and their corresponding mutants (PEDV M-K202R, TGEV M-K239R, SADS-CoV M-K246R) in HEK293T cells transfected with FLAG-RNF166 or FLAG-RNF166-T157A. Ratios below the images represent the protein levels of HA relative to β-actin, as quantified by the ImageJ software. Data in panels B–F represent mean ± s.d. (n = 3). Statistical significance was determined by two-tailed Student’s t-test; * P < 0.05; ** P < 0.01; ns, not significant.

Article Snippet: UDP-GlcNAc (HY-112174) was purchased from MedChemExpress, China.

Techniques: Infection, Western Blot, Software, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test

Journal: PLOS Pathogens

Article Title: O-GlcNAcylation licenses RNF166 to degrade the M protein of porcine coronaviruses

doi: 10.1371/journal.ppat.1014301

Figure Lengend Snippet: Using PDCoV as a representative example, in response to porcine coronavirus infection, host cells enhance the HBP flux, leading to UDP-GlcNAc accumulation. Concurrently, OGT translocates to the cytoplasm and catalyzes the O-GlcNAcylation of RNF166 at T157. This modification licenses RNF166 to mediate the degradation of the PDCoV M protein, while it does not alter the ability of RNF166 to induce interferon production. This mechanism is highly conserved across porcine enteric coronaviruses.

Article Snippet: UDP-GlcNAc (HY-112174) was purchased from MedChemExpress, China.

Techniques: Infection, Modification

Journal: Acta Crystallographica Section D: Structural Biology

Article Title: A structural basis for the strain-dependent UDP-sugar specificity of glycosyltransferase C from the Limosilactobacillus reuteri accessory secretion system

doi: 10.1107/S2059798325008782

Figure Lengend Snippet: Crystal structures and conformational changes accompanying ligand-bound Lr GtfC 100-23 . ( a , b ) Details of the binding of UDP and the UDP-GlcNAc sugar donor (magenta C atoms), respectively, to Lr GtfC 100-23 . The ligands and neighboring residues (labeled) are shown in stick format with a cartoon representation of the enzyme. Polar interactions are indicated by black dashed lines. A ligand omit map calculated contoured at 3σ is shown as a blue mesh in both panels. Note that here and in subsequent panels, residues 1–67 have been removed for clarity and so the hydrogen-bond interaction of the α-phosphate of ligands with the side chain of Thr16 is not seen. ( c , d ) Conformational changes observed during ligand binding. ( c ) A superposition of the N-terminal acceptor-binding domains of the apo (yellow cartoon, yellow C atoms) and sugar donor-bound (magenta cartoon, magenta C atoms) structures. Bound UDP-GlcNAc is shown in stick format with a molecular-surface rendering. Residues 103 and 112 (red text) delimit a flexible region of polypeptide adjacent to the binding site in the apo structure. ( d ) A superposition of the sugar donor-bound (orange cartoon, orange C atoms) and UDP-bound (gray cartoon, gray C atoms) enzyme structures. Bound UDP is shown in stick format. For each structure selected residues are shown in stick format and with a selection of hydrogen-bond interactions indicated by black dashed lines.

Article Snippet: Finally, for the UDP-GlcNAc-bound enzyme, purified untagged Lr GtfC 100-23 in 20 m M HEPES pH 7.1, 100 m M NaCl buffer was mixed with a solution of UDP-GlcNAc disodium salt (Biosynth) in the same buffer to obtain a protein concentration of 11 mg ml −1 and a 100:1 ligand:enzyme molar ratio for crystallization screening, including the use of JCSG- plus (Newman et al. , 2005 ).

Techniques: Binding Assay, Labeling, Ligand Binding Assay, Selection

Journal: Acta Crystallographica Section D: Structural Biology

Article Title: A structural basis for the strain-dependent UDP-sugar specificity of glycosyltransferase C from the Limosilactobacillus reuteri accessory secretion system

doi: 10.1107/S2059798325008782

Figure Lengend Snippet: Locations of residue differences with Lr GtfC 53608 mapped onto the crystal structure of sugar donor-bound Lr GtfC 100-23 . The enzyme is shown in a cartoon format with the N-terminal sugar acceptor-binding domain colored green and the C-terminal sugar donor-binding domain in yellow. Bound UDP-GlcNAc is shown in stick format with a molecular-surface rendering in white. Locations of residues of Lr GtfC 100-23 which differ from those in Lr GtfC 53608 are shown in sphere representation with C atoms colored yellow and the substitution labeled. Residue Asn331 in the N-terminal domain of Lr GtfC 100-23 , which is a lysine in Lr GtfC 53608 , is found at the back of the molecule in the orientation shown and is obscured in the image. The presumed catalytic aspartate, Asp101, is shown with C atoms colored magenta.

Article Snippet: Finally, for the UDP-GlcNAc-bound enzyme, purified untagged Lr GtfC 100-23 in 20 m M HEPES pH 7.1, 100 m M NaCl buffer was mixed with a solution of UDP-GlcNAc disodium salt (Biosynth) in the same buffer to obtain a protein concentration of 11 mg ml −1 and a 100:1 ligand:enzyme molar ratio for crystallization screening, including the use of JCSG- plus (Newman et al. , 2005 ).

Techniques: Residue, Binding Assay, Labeling