Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Citrullination alters immunomodulatory function of LL-37 essential for prevention of endotoxin-induced sepsis

doi: 10.4049/jimmunol.1303062

Figure Lengend Snippet: Human MDMs (A) and mouse RAW 264.7 (B) macrophages were stimulated with 10 ng/mL LPS in the presence of native or citrullinated LL-37 at the indicated concentrations (0.1–10 μg/mL). Citrullinated LL-37 was obtained by treatment of the native peptide with human PAD2 or PAD4 at 23.3 U/mg peptide). The level of TNF-α (A) and NO (B) in the culture supernatants was determined using ELISA or the Griess assay at 6 or 20 h post-stimulation, respectively. Since neither the LL-37 nor the PAD enzymes alone induced the release of NO or TNF-α, for the sake of clarity these controls are not shown in the figure. Data represent the mean ± SD of three independent experiments. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Recombinant human PAD2 and PAD4 were obtained from Modiquest (The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Griess Assay

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: PAD activity in NAWM of MS patients. (A) Quantification of PAD2 protein in white matter from normal and MS brain by immunoslot blot ( n =5, P <0.0001). (B) Citrullinated protein in white matter from normal and MS brains by immunoslot blot as pixel density ( n =4, P <0.01). (C) PAD enzyme activity in normal and MS tissue, with or without pre-incubation with 2CA ( n =5, P <0.05). Each dot represents one patient analysed three times. The means (horizontal bar) for all the MS patients were compared with the normal.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Activity Assay, Incubation

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: Interaction of PAD with 2CA. (A) PAD2 and PAD4 inhibition curves in the presence of increasing 2CA concentrations. Insert: PAD1-4 enzymes contain a common C-terminal active-site cysteine residue (Cys656) bound by 2CA, confirmed by ESI mass spectrometry of tryptic digests of PAD2-acetamidine adducts. (B) Schematic of the nucleophilic reaction between 2CA and the Cys656 residue in the active site of PAD2 . (C) Tabular summary of peptide fragment atomic masses for 2CA-modified and native PAD2.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Inhibition, Residue, Mass Spectrometry, Modification

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: mAb4E12 (anti-2CA adduct) immunogold labeled optic nerve cryosections from control and 2CA-treated PAD2 transgenic mice. Minimal labeling in untreated mice (black arrows). Numerous gold particles (white arrows) in nuclei (N) and cytoplasm of oligodendrocytes, myelin and axonoplasm (Ax) of 2CA-treated mice. Scale bars: 500 nm.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Labeling, Control, Transgenic Assay

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: 2CA attenuates demyelinating disease in ND4 mice. (A) ND4 mice treated with PBS, 2CA (5 mg/kg), or 2CA+B12 (5 mg/kg and 10 mg/kg) starting at 2 months before disease onset ( n =5, P <0.0001). (B) ND4 mice treated at disease onset ( n =4, P <0.0001). (C) Stopping 2CA, but continuing B12 at 3.5 months in ND4 mice ( n =5, P <0.0001) demonstrates that B12 alone does not attenuate disease. (D) PAD activity in brains from animals shown in ( n =5, P <0.05). The first four bars represent results from animals at 6 months of age, whereas the post-treatment animals were 8 months of age. (E) PAD2 RT PCR in white matter extracts from normal, PBS-, 2CA- and 2CA+B12-treated ND4 mice ( n =9, P <0.05). (F) LFB and hematoxylin stain of cerebella from normal, PBS-, 2CA- and 2CA+B12-treated ND4 mice (40×). WM, white matter; GM, grey matter.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: 2CA attenuates PAD2 overexpressor. (A) Demyelinating disease in PAD2 transgenic mice treated with PBS, 2CA or 2CA+B12 starting at 6 months of age ( n =5, P <0.0001). (B) PAD activity in brain extracts of PAD2 transgenic mice treated with PBS, 2CA or 2CA+B12, and non-transgenic littermates ( n =4, P <0.05). PAD activity is reduced to normal levels by treatment.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Transgenic Assay, Activity Assay

Journal: bioRxiv

Article Title: Development of Motif-Specific Monoclonal Antibodies for Global Protein Citrullination Detection with Minimal Cross-Reactivity to Homocitrullination

doi: 10.1101/2025.03.27.645732

Figure Lengend Snippet: (A) Reactivity of motif-specific monoclonal antibodies 12A2 and 21F10 against in vitro citrullinated or homocitrullinated HeLa proteomes. (-), untreated HeLa lysate. (B) Reactivity of three commercially available anti-pan citrullination antibodies (07-377, ab100932, and MABN328 (F95)) against citrullinated and homocitrullinated HeLa lysates. (-), untreated HeLa lysate. (C) Reactivity of motif-specific monoclonal antibodies 12A2 and 21F10 toward HeLa proteomes treated with different PAD isozymes (PAD1, PAD2, PAD3, PAD4). (-), untreated HeLa lysate.

Article Snippet: For comparison of the citrullination patterns of different PAD isozymes, 200 μg of HeLa lysate (5 μg/μL) was diluted with 160 μL PAD buffer and incubated with recombinant PAD1 (Cay10784, Biomol), PAD3 (Cay10786, Biomol), PAD2, or PAD4 (both kindly provided by Prof. Hui-Chih Hung) enzyme at 1:100 enzyme:protein ratio overnight at 37 °C with shaking at 600 rpm.

Techniques: Bioprocessing, In Vitro

Journal: Cell death & disease

Article Title: Peptidylarginine deiminase 2 plays a key role in osteogenesis by enhancing RUNX2 stability through citrullination.

doi: 10.1038/s41419-023-06101-7

Figure Lengend Snippet: Fig. 4 PADI2 protects RUNX2 from ubiquitin-proteasomal degradation pathway. A–C MC3T3-E1 cells were transfected with Strep-Runx2 with or without Flag-PADI2, and then cultivated in osteogenic medium for 3 days. On day 3, 4 μg/mL Actinomycin D was treated and incubated for 0, 3, and 6 h. The half-life of Runx2 mRNA and RUNX2 protein was determined by RT-qPCR (A) and western blot analysis (B), respectively. The intensities of Strep-RUNX2 protein levels were normalized against each GAPDH by ImageJ. The normalized values at 0 h were set as 1, and relative levels are shown (C). D, E MC3T3-E1 cells were transfected with Strep-Runx2 together with or without Flag-PADI2, and then cultivated in osteogenic medium for 3 days. 20 μg/mL cycloheximide was treated on the last day and incubated for 0, 3, and 6 h. The half-life of RUNX2 protein was determined by western blot analysis (D). The intensities of Strep-RUNX2 protein levels were normalized against each GAPDH by ImageJ and relative levels are shown (E). F Padi2 control and KO pOB cells were cultured in osteogenic media for 2 days, and 20 μM MG132 or DMSO as vehicle was treated for 6 h before harvesting cells, and western blot analysis followed. β-Actin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with β-Actin. The red arrow indicates PADI2. G MC3T3-E1 cells were transfected with siCont or siPadi2 #2 and then cultivated in osteogenic media for an additional 2 days. 20 μM MG132 or DMSO as the vehicle was treated for 6 h before harvesting cells followed by western blot analysis. α-Tubulin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with α-Tubulin. The red arrow indicates PADI2. H hMSCs were transfected with siCont or siPADI2 and then cultivated in osteogenic media for an additional 2 days. 20 μM MG132 or DMSO as the vehicle was treated for 6 h before harvesting cells followed by western blot analysis. β-Actin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with β-Actin. The red arrow indicates PADI2. Western blot data were collected from at least two or three independent experiments; the representative results are shown here. I Strep-Runx2, Flag-PADI2, and HA-ubiquitin were transfected into 293 T cells. 3 days after transfection, cells were treated with 20 μM MG132 for 6 h, lysed, immunoprecipitated with Strep-Tag II magnetic beads, and immunoblotted with anti-HA or anti-RUNX2 antibody. Ubiquination assay was performed in three independent experiments; the representative results are shown here.

Article Snippet: For in vitro citrullination assay, 0.5 μg recombinant human RUNX2 protein (rhRUNX2) (Origene) and one unit recombinant human PAD2 (#10785; Cayman Chemical, Ann Arbor, MI, USA) were incubated with reaction buffer (50mM HEPES (pH7.5), 5mM DTT, 10mM CaCl2, 50mM NaCl) at 37 °C for 2 h. After in vitro citrullination, the samples were labeled with Biotin-PG (Cayman) according to previously reported methods [25, 26].

Techniques: Ubiquitin Proteomics, Transfection, Incubation, Quantitative RT-PCR, Western Blot, Control, Cell Culture, Software, Immunoprecipitation, Strep-tag, Magnetic Beads

Journal: Cell death & disease

Article Title: Peptidylarginine deiminase 2 plays a key role in osteogenesis by enhancing RUNX2 stability through citrullination.

doi: 10.1038/s41419-023-06101-7

Figure Lengend Snippet: Fig. 5 PADI2 citrullinates RUNX2 via physical interaction. A 293 T cells were transfected with 3xHA-Runx2 together with or without Flag- PADI2 and cultured for 3 days after the transfection. Cells were lysed, immunoprecipitated with anti-HA antibody and protein G-conjugated magnetic beads, and immunoblotted with indicated antibodies. The red arrow indicates predicted citrullinated RUNX2. Co-IP experiment was performed in three independent experiments; the representative results are shown here. B Workflow showing the detection of citrullinated RUNX2 by Biotin-PG labeling (left). Recombinant human RUNX2 (rhRUNX2) isoform c (NP_004339) was in vitro citrullinated by rhPADI2. The samples were labeled with Biotin-PG and then separated by SDS-PAGE followed by transfer to PVDF membrane. The membrane was incubated with Streptavidin conjugated with horseradish peroxidase (Streptavidin-HRP) or was immunoblotted with anti-RUNX2 antibody (right). A red asterisk indicates citrullinated RUNX2. This experiment was performed in three independent experiments; the representative results are shown here. C In vitro citrullinated rhRUNX2 isoform c was analyzed by LC-MS/MS. The ten arginine (R) sites of rhRUNX2 citrullinated by PADI2 were identified (left column). The mouse RUNX2 isoform 1 was used for site-directed mutagenesis of the 10 R sites and, for this, the R site in mouse RUNX2 isoform 1 matching the corresponding each R site in hRUNX2 isoform c is shown in the right column.

Article Snippet: For in vitro citrullination assay, 0.5 μg recombinant human RUNX2 protein (rhRUNX2) (Origene) and one unit recombinant human PAD2 (#10785; Cayman Chemical, Ann Arbor, MI, USA) were incubated with reaction buffer (50mM HEPES (pH7.5), 5mM DTT, 10mM CaCl2, 50mM NaCl) at 37 °C for 2 h. After in vitro citrullination, the samples were labeled with Biotin-PG (Cayman) according to previously reported methods [25, 26].

Techniques: Transfection, Cell Culture, Immunoprecipitation, Magnetic Beads, Co-Immunoprecipitation Assay, Labeling, Recombinant, In Vitro, SDS Page, Membrane, Incubation, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

Journal: Cell death & disease

Article Title: Peptidylarginine deiminase 2 plays a key role in osteogenesis by enhancing RUNX2 stability through citrullination.

doi: 10.1038/s41419-023-06101-7

Figure Lengend Snippet: Fig. 6 PADI2-catalyzed citrullination of RUNX2 is required for RUNX2 stabilization. A MC3T3-E1 cells were transfected with empty vector (EV), Strep-Runx2 wild type (Wt), or R-to-K mutants and cultured in osteogenic media for 3 days after transfection. Cells were lysed and western blot analysis was performed. β-Actin was used as a loading control. Strep-RUNX2 level was quantified using ImageJ software and normalized with β-Actin. The arrow indicates non-specific bands (n.s). Western blot data were collected from three independent experiments; the representative results are shown here. B, C pOB cells and hMSCs were transfected with EV, Strep-Runx2 WT, or R381K mutant plasmids and cultured in osteogenic media for 2 days. Cells were lysed and western blot analysis was performed. β-Actin was used as a loading control. Strep-RUNX2 level was quantified and normalized with β-Actin using ImageJ software. Western blot data were collected from two independent experiments; the representative results are shown here. D 293 T cells were transfected with Strep-Runx2 Wt or R-to-K mutants with or without Myc-Cbfβ plasmids and then incubated for 3 days after transfection. Cells were lysed, immunoprecipitated with Strep-Tag II magnetic beads, and immunoblotted with indicated antibodies. Co-IP experiment was performed in three independent experiments; the representative results are shown here. E MC3T3-E1 cells were transfected with Strep-Runx2 Wt or R-to-K mutants and cultured for 2 days after transfection. Cells were fixed with 4% PFA, permeabilized, and then immunofluorescent staining was performed using anti-Strep-Tag II antibody. DAPI was used for the nucleus. Three independent experiments were performed and the representative results are shown here. Scale bar, 20 μm.

Article Snippet: For in vitro citrullination assay, 0.5 μg recombinant human RUNX2 protein (rhRUNX2) (Origene) and one unit recombinant human PAD2 (#10785; Cayman Chemical, Ann Arbor, MI, USA) were incubated with reaction buffer (50mM HEPES (pH7.5), 5mM DTT, 10mM CaCl2, 50mM NaCl) at 37 °C for 2 h. After in vitro citrullination, the samples were labeled with Biotin-PG (Cayman) according to previously reported methods [25, 26].

Techniques: Transfection, Plasmid Preparation, Cell Culture, Western Blot, Control, Software, Mutagenesis, Incubation, Immunoprecipitation, Strep-tag, Magnetic Beads, Co-Immunoprecipitation Assay, Staining