Journal: iScience

Article Title: LRP1 activated by AT2 cell-secreted MDK inhibits fibrotic ferroptosis in idiopathic pulmonary fibrosis

doi: 10.1016/j.isci.2026.116212

Figure Lengend Snippet: OTUB1 targets SLC7A11 and stabilizes its expression level (A) Molecular docking of OTUB1 and SCL7A11. (B and C) IP and co-IP assays of OTUB1 and SCL7A11 in human primary lung fibroblasts and 293T cells. ( n = 3 biological replicates for each group). (D) Laser confocal imaging of SLC7A11 and PTUB1 subcellular localization. ( n = 3 biological replicates for each group; Scale bars = 50 μm). (E) Protein structures of OTUB1 and SLC7A11. (F) Flag-tagged OTUB1-FL (1–272), OTUB1-M1 (1–85), OTUB1-M2 (86–195), or OTUB1-M3 (196–272) were transfected into HEK293T cells with Myc-SLCA11 and were then subjected to IP with anti-Flag (Left). Myc-tagged FL-SLC7A11 (1–501), SLC7A11-M1 (1–159), SLC7A11-M2 (160–291), or SLC7A11-M3 (292–501) were transfected into HEK293T cells with Flag-tagged FL-OTUB1 and were then subjected to IP with anti-Myc (Right). (G) Fibroblasts transfected with two independent OTUB1 shRNAs were treated with or without the proteasome inhibitor MG132 (20 μM, 8 h); subsequently, OTUB1 and SLC7A11 were assessed by western blotting. ( n = 3 biological replicates for each group). (H) Increasing concentrations of OTUB1 (WT) or OTUB1 (CA) were transfected into HEK293T cells, and SLC7A11 expression was assessed by western blotting. ( n = 3 biological replicates for each group). (I) Fibroblasts transfected with the shCtrl or shOTUB1-2 were treated with CHX (10 μg/mL) and collected at the indicated time points for western blotting. ( n = 3 biological replicates for each group). (J) Fibroblasts transfected with the vector control, OTUB1 (WT) or OTUB1 (CA) were treated with CHX (10 μg/mL) and collected at the indicated time points for western blotting. ( n = 3 biological replicates for each group).

Article Snippet: The structures of SLC7A11 (Protein DataBank code 7CCS) and OTBU1 (Protein DataBank code 4DDL) were used for docking simulations.

Techniques: Expressing, Co-Immunoprecipitation Assay, Imaging, Transfection, Western Blot, Plasmid Preparation, Control

Journal: iScience

Article Title: LRP1 activated by AT2 cell-secreted MDK inhibits fibrotic ferroptosis in idiopathic pulmonary fibrosis

doi: 10.1016/j.isci.2026.116212

Figure Lengend Snippet: OTUB1 deubiquitinates SLC7A11 and directly cleaves the Lys 48-polyubiquitin chain (A) Fibroblasts treated with 20 μM MG132 for 8 h were co-transfected with HA-Ub and shCtrl, shOTUB1-1, or shOTUB1-2, and cell lysates were subjected to IP with SLC7A11 antibody followed by IB with the indicated antibodies. ( n = 3 biological replicates for each group). (B) Fibroblasts treated with 20 μM MG132 for 8 h were co-transfected with Myc-SLC7A11, HA-Ub, and Flag-OTUB1 (WT) or Flag-OTUB1 (CA), and cell lysates were subjected to IP with Myc antibody followed by IB with the indicated antibodies. ( n = 3 biological replicates for each group). (C) Unubiquitylated or ubiquitylated His-SLC7A11 was incubated with GST-OTUB1 (WT) or GST-OTUB1 (CA) and HA-Ub coupled to glutathione Sepharose beads. His-SLC7A11 was subjected to IP with His beads followed by IB with antibodies against HA and His. ( n = 3 biological replicates for each group). (D) HEK293T cells were co-transfected with Myc-SLC7A11, Flag-OTUB1 (CA), and HA-Ub (Lys0), HA-Ub (Lys48), or HA-Ub (Lys63) plasmids, and the SLC7A11 poly-ubiquitylation linkage was analyzed. ( n = 3 biological replicates for each group). (E) Fibroblasts were co-transfected with Myc-SLC7A11, Flag-OTUB1, and HA-Ub (WT) or HA-Ub (Lys48R) plasmids, and the SLC7A11 poly-ubiquitylation linkage was analyzed. ( n = 3 biological replicates for each group). (F) Unubiquitylated or ubiquitylated His-SLC7A11 was incubated with GST-OTUB1 (WT) or GST-OTUB1 (CA) and HA-Ub coupled to glutathione Sepharose beads. His-SLC7A11 was subjected to IP with His beads followed by IB with antibodies against HA and His. ( n = 3 biological replicates for each group). (G) Fibroblasts transfected with WT or Lys48-Res were cultured for 72 h in the presence of control or sh-OTUB1-2. Cell lysates were analyzed by IB using antibodies against SLC7A11 and OTUB1. shOTUB1: small hairpin of OTUB1, shCtrl: small hairpin control. ( n = 3 biological replicates for each group).

Article Snippet: The structures of SLC7A11 (Protein DataBank code 7CCS) and OTBU1 (Protein DataBank code 4DDL) were used for docking simulations.

Techniques: Transfection, Incubation, Cell Culture, Control

Journal: iScience

Article Title: LRP1 activated by AT2 cell-secreted MDK inhibits fibrotic ferroptosis in idiopathic pulmonary fibrosis

doi: 10.1016/j.isci.2026.116212

Figure Lengend Snippet: SLC7A11 knockdown in fibroblasts partially reverses pulmonary fibrosis in vivo (A–C) Representative images of H&E staining (A), Masson staining (B), and IHC of hydroxyproline (C) for pulmonary tissues from BLM mice and BLM mice with OTUB1 overexpression and/or SLC7A11 knockdown ( n = 6 biological replicates for each group; mean ± SD; one way ANOVA; Scale bars = 200 μm). (D) Quantification of the concentrations of hydroxyproline from C and the Ashcroft score evaluating pulmonary fibrosis of mouse lungs ( n = 6 biological replicates for each group; mean ± SD; one way ANOVA; ∗ p < 0.05). (E–H) The protein levels of hub ferroptosis regulators (E), the level of lipid oxidation (F and G), the GSH/GSSG ratio and MDA level (H) in fibroblasts extracted from the mice. ( n = 6 biological replicates for each group; mean ± SD; one way ANOVA; Scale bars = 50 μm ∗ p < 0.05).

Article Snippet: The structures of SLC7A11 (Protein DataBank code 7CCS) and OTBU1 (Protein DataBank code 4DDL) were used for docking simulations.

Techniques: Knockdown, In Vivo, Staining, Over Expression

Journal: Protein engineering, design & selection : PEDS

Article Title: Structure-activity correlations in pentachlorobenzene oxidation by engineered cytochrome P450cam.

doi: 10.1093/protein/gzm028

Figure Lengend Snippet: Fig. 2. (a) Stereo view of the active site structure of the PeCB complex of the F87W/Y96F/L244A/V247L mutant in molecule A showing the electron density (2Fo–Fc contoured at 1s) and fit for the PeCB substrate. One PeCB chlorine is coordinated to the heme iron while another chlorine is located in the cavity generated by the L244A mutation. The W87 indole NH points upward and form a hydrogen bond with the carbonyl of M184. The figure was generated with the program PyMOL. (b) Overlay of the active site structure of the PeCB complex of the mutant (carbons in yellow) with the camphor complex of wild type P450cam (carbons in grey) showing movements of A244, G248 and T252. Camphor has been omitted for clarity. (c) Overlay of the structure of the PeCB complex of the F87W/Y96F/ L244A/V247L mutant (carbons in yellow) with the TCB complex of the F87W/Y96F/V247L mutant (carbons in grey) showing the rotation of PeCB relative to TCB about the carbon closest to the heme iron and the altered conformations of A244, G248 and T252 in the PeCB complex. Figure (b) and (c) were generated with the program ViewerPro.

Article Snippet: The structure was solved by molecular replacement, based on the crystal structure of wild type P450cam (protein databank code: 2CPP) but with the camphor removed.

Techniques: Mutagenesis, Generated