Journal: Acta Pharmaceutica Sinica. B

Article Title: Augmentation of PRDX1–DOK3 interaction alleviates rheumatoid arthritis progression by suppressing plasma cell differentiation

doi: 10.1016/j.apsb.2025.06.006

Figure Lengend Snippet: DOK3 serves as a PRDX1-interacting protein in B cells. (A) Immunoblots were performed in NALM-6 cells upon stable expression of PRDX1, with GAPDH serving as the loading control. (B) A volcano plot depicts differentially expressed proteins identified in co-immunoprecipitation assays of flag-PRDX1 in NALM-6 cells treated with anti-human CD40 antibody, human IL-21, and IL-4 for 48 h. (C) Co-immunoprecipitation of flag-PRDX1 in NALM-6 cells. (D) Representative images of Duolink in situ PLA using rabbit anti-PRDX1, mouse anti-DOK3 antibody, and PLA probes in WT and Prdx1 -OE mouse B cells from the spleen. Scale bar, 50 μm. (E) Quantification of relative intensity of fluorescence in mouse WT and Prdx1 -OE B cells using Duolink in situ PLA ( n = 3). (F) Pull-down assay was performed using HIS-PRDX1 and GST-DOK3 in the purified form. (G) The interaction between His-PRDX1 and AVI-DOK3 was evaluated by BLI. K D represents the dissociation constant ( n = 3). (H) The Line graph shows deuterium uptake plots for peptide 218–237 aa of DOK3 in the presence and absence of PRDX1. (I) The 3D structure of DOK3(1–330 aa) was predicted using Alphafold to illustrate the perturbation of hydrogen–deuterium exchange upon interaction with recombinant PRDX1 protein. Peptide differences with % D between –5% and 5% were considered not significant and are shown in grey, while green–blue represents decreased hydrogen–deuterium exchange rate. Data are plotted as percentage deuterium uptake versus time on a logarithmic scale. (J) The line graph shows the deuterium uptake profile of peptide 43–49 aa of PRDX1 in the presence and absence of DOK3. (K) Differential HDX consolidation view was mapped to the PRDX1 crystal structure (PDB ID: 7WET) to demonstrate hydrogen–deuterium exchange perturbations upon interaction of the recombinant PRDX1 protein and the recombinant DOK3(1–330 aa) protein. (L) Pull-down assay was performed using His-PRDX1mutants (L46A, D47A, F48A, T49A) and GST-DOK3 in the purified form. (M) Relative protein levels of His-PRDX1(Wild type and mutants) in the (L) pull-down assay ( n = 3). Data are shown as mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, and ns indicates no significance.

Article Snippet: After in vitro stimulation of B cells, the Duolink assay was performed according to the manufacturer's instructions using Duolink Detection reagents Far Red (Sigma–Aldrich, St. Louis, MO, USA) and anti-PRDX1 rabbit (Proteintech, 15816-1-AP, Wuhan, China) and anti-DOK3 mouse primary antibodies (Santa Cruz Biotechnology, SC-390007, Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing, Control, Immunoprecipitation, In Situ, Fluorescence, Pull Down Assay, Purification, Recombinant

Journal: Acta Pharmaceutica Sinica. B

Article Title: Augmentation of PRDX1–DOK3 interaction alleviates rheumatoid arthritis progression by suppressing plasma cell differentiation

doi: 10.1016/j.apsb.2025.06.006

Figure Lengend Snippet: PRDX1 suppresses plasma cell differentiation by reducing the degradation of DOK3 in the autophagy–lysosome pathway. (A–D) WT B cells were treated with vehicle or siDOK3 and induced with anti-mouse CD40 antibody, murine IL-21, and IL-4. (A) The proportion of plasma cells was analyzed by flow cytometry. (B) A quantitative analysis of plasma cell counts in (A) ( n = 3). (C) Immunoblot analysis was performed to assess the efficiency of DOK3 knockdown and JNK phosphorylation. (D) Relative protein levels of DOK3 and P-JNK in the (C) ( n = 3). (E–H) Representative Western blot showing DOK3, phosphor-JNK, total JNK, and PRDX1 expression in WT, Prdx1- OE (E), and Prdx1 −/− B cells (G) treated with anti-mouse CD40 antibody, murine IL-21, and IL-4 for 5 days, with GAPDH as a loading control. (F, H) Relative protein levels of DOK3, phosphor-JNK in WT, Prdx1 -OE and Prdx1 −/− B cells ( n = 3). (I) Representative images of Duolink in situ PLA using rabbit anti-PRDX1, mouse anti-DOK3 antibody, and PLA probes in B cells from the spleen of the WT and PRDX1 -OE mice, followed by anti-mouse CD40 antibody, murine IL-21, and IL-4. Scale bar, 50 μm. (J) Relative intensity of fluorescence of Duolink in situ PLA in B cells from the spleen of the WT and Prdx1 -OE B cells ( n = 3). (K) WT B cells were stimulated with anti-mouse CD40 antibody, murine IL-2, and IL-4 for the indicated times, and immunoblot analysis was performed to assess DOK3 and JNK phosphorylation. (L) Relative protein levels of DOK3 and P-JNK in WT B cells stimulated with anti-mouse CD40 antibody, murine IL-21, and IL-4 for the indicated times ( n = 3). (M) WT B cells were treated with the lysosome inhibitor CQ following stimulation with anti-mouse CD40 antibody, murine IL-21, and IL-4 for 2 h, and immunoblot analysis was performed to assess DOK3. (N) Relative protein levels of DOK3 in WT B cells treated with the lysosome inhibitor CQ ( n = 3). Data are shown as mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, and ns indicates no significance.

Article Snippet: After in vitro stimulation of B cells, the Duolink assay was performed according to the manufacturer's instructions using Duolink Detection reagents Far Red (Sigma–Aldrich, St. Louis, MO, USA) and anti-PRDX1 rabbit (Proteintech, 15816-1-AP, Wuhan, China) and anti-DOK3 mouse primary antibodies (Santa Cruz Biotechnology, SC-390007, Santa Cruz, CA, USA).

Techniques: Clinical Proteomics, Cell Differentiation, Flow Cytometry, Western Blot, Knockdown, Phospho-proteomics, Expressing, Control, In Situ, Fluorescence

Journal: Acta Pharmaceutica Sinica. B

Article Title: Augmentation of PRDX1–DOK3 interaction alleviates rheumatoid arthritis progression by suppressing plasma cell differentiation

doi: 10.1016/j.apsb.2025.06.006

Figure Lengend Snippet: Salvianolic acid B functions as a molecular glue-like compound enhancing the interaction between PRDX1 and DOK3. (A) The chemical structure of salvianolic acid B is depicted. (B) Pull-down assay of GST-DOK3(1–330 aa) and His-PRDX1 protein incubated with 100, 10, and 1 μmol/L salvianolic acid B, respectively. (C) The binding affinity of AVI-PRDX1 with SAB was measured using a BLI binding kinetic assay. K D stands for the dissociation constant ( n = 3). (D) The binding affinity of AVI-DOK3(1–330 aa) with SAB was measured using a BLI binding kinetic assay. K D stands for the dissociation constant ( n = 3). (E) Electrostatic potential of the SAB–PRDX1 complex crystal structure and the binding site of SAB. The interior of SAB's binding site is electronegative (colored in blue), while its exterior is electropositive (colored in red). SAB was shown in the wheat stick. Unbiased F o – F c density map contoured at 2.5 Å of a crystal structure for PRDX1 in complex with SAB. (F) The hydrogen bond network formed between SAB and residues of PRDX1 is shown. The hydrogen bond network consists of ten hydrogen bonds, displayed as magenta dashed lines, while water molecules are represented as red spheres. (G) HDX analysis of PRDX1 comparing deuterium exchange rates before and after incubation with DOK3(1–330 aa) and after incubation of PRDX1 and DOK3(1–330 aa) with and without the addition of SAB. (H) HDX analysis of DOK3(1–330 aa) was performed to compare the rate of deuterium exchange before and after incubation with PRDX1, as well as the DOK3(1–330 aa) and PRDX1 complexes with SAB versus without SAB. (I) The affinity of the ternary complex of PRDX1, DOK3(1–330 aa), and SAB was measured by BLI binding kinetic assay. K D stands for the dissociation constant ( n = 3). (J) Co-immunoprecipitation of flag-PRDX1 in NALM-6 cells treated with 60 μmol/L SAB is shown. (K) The relative Protein levels of DOK3 in (J) ( n = 3). (L) Pull-down assay was performed using GST-DOK3(1–330 aa) and His-PRDX1 mutant protein (D47A, F48A, T49A) incubated with 50 μmol/L salvianolic acid B. (M) Relative protein levels of His-PRDX1 (Wild type and mutants) in the (L) pull-down assay ( n = 3). (N) The co-immunoprecipitation of flag-PRDX1 and its mutants wih myc-DOK3 in HEK-293T cells treated with 60 μmol/L SAB is shown. (O) The relative Protein levels of DOK3 in (N) ( n = 3). Data are shown as mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, and ns indicates no significance.

Article Snippet: After in vitro stimulation of B cells, the Duolink assay was performed according to the manufacturer's instructions using Duolink Detection reagents Far Red (Sigma–Aldrich, St. Louis, MO, USA) and anti-PRDX1 rabbit (Proteintech, 15816-1-AP, Wuhan, China) and anti-DOK3 mouse primary antibodies (Santa Cruz Biotechnology, SC-390007, Santa Cruz, CA, USA).

Techniques: Pull Down Assay, Incubation, Binding Assay, Kinetic Assay, Immunoprecipitation, Mutagenesis

Journal: Acta Pharmaceutica Sinica. B

Article Title: Augmentation of PRDX1–DOK3 interaction alleviates rheumatoid arthritis progression by suppressing plasma cell differentiation

doi: 10.1016/j.apsb.2025.06.006

Figure Lengend Snippet: Salvianolic acid B prevents the progression of collagen-induced arthritis by enhancing the interaction between PRDX1 and DOK3. (A) Clinical scores of arthritis in mice treated with vehicle, 1.5 mg/kg TF, and 15 mg/kg or 30 mg/kg SAB, and immunized with collagen in complete Freund's adjuvant ( n = 6). (B) Representative micro-CT images of the hind paws from mice treated with vehicle, TF, and SAB. (C) Joint destruction was graded based on the CT score in B ( n = 3). (D, E) histological score of the inflammation area in mice treated with vehicle, TF, and SAB ( n = 6) (D) and representative images of hematoxylin and eosin (H&E)-stained paw sections (E). Scale bar, 100 μm. (F) Serum titer of IgG2A analyzed from vehicle, TF, and SAB by ELISA ( n = 6). (G, H) The frequency of plasma cells (G) and GC B cells (H) in the spleen from mice treated with vehicle, TF, and SAB was analyzed by flow cytometry ( n = 6). (I) GSEA enrichment profiles of the B cell receptor pathway and CD40 pathway in the SAB-H treatment and model groups. (J) Heatmap depicting the expression levels of the B cell receptor signaling pathway and RA defined by the differentially downregulated genes in the SAB-H treatment group compared to the WT model group. (K) The biological process analysis of B cells for downregulated genes in the SAB-H treatment group compared to the model group. (L) Representative immunofluorescence staining images of PRDX1 (green) and DOK3 (red) in the spleen from the model and SAB treatment groups. Scale bar, 50 μm. (M) Representative immunofluorescence staining images of CD19 (green), P-JNK (red) in the spleen from the model and SAB treatment groups. Scale bar, 50 μm. (N) Relative fluorescence intensity of stained PRDX1 and DOK3 in the spleen from the model and SAB treatment groups ( n = 3). (O) Relative fluorescence intensity of stained CD19 and P-JNK in the spleen from the model and SAB treatment groups ( n = 3). Data are presented as mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, and ns indicates no significance.

Article Snippet: After in vitro stimulation of B cells, the Duolink assay was performed according to the manufacturer's instructions using Duolink Detection reagents Far Red (Sigma–Aldrich, St. Louis, MO, USA) and anti-PRDX1 rabbit (Proteintech, 15816-1-AP, Wuhan, China) and anti-DOK3 mouse primary antibodies (Santa Cruz Biotechnology, SC-390007, Santa Cruz, CA, USA).

Techniques: Adjuvant, Micro-CT, Staining, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Flow Cytometry, Expressing, Immunofluorescence, Fluorescence