Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: This figure presents the computational prediction of the interaction between matrix metalloproteinase-9 (MMP9) binding to superoxide dismutase-3 (SOD3), achieved through molecular docking using ClusPro and AutoDock software. In visualization, MMP9 is depicted in green, and SOD3 is represented in purple. The Prodigy webserver was utilized to analyze their binding affinity, revealing a strong interaction characterized by a ΔG (kcal mol−1) value of −16.2 and a Kd (M) value of 1.4e-12, indicative of high binding affinity. Further examination identified a potential cleavage site for MMP9 on SOD3 amino acid at position 173AA with a significant score of 0.906. This suggests a probable proteolytic interaction between the two proteins. The molecular interaction and the structural remodeling of these protein complexes were visualized and assessed using Pymol and ChimeraX. Collectively, the computational analysis, binding affinity evaluation, and identification of a potential cleavage site provide insights into the protein–protein interaction between MMP9 and SOD3, highlighting their intricate molecular relationship.

Article Snippet: SOD3 was detected using an antibody from Proteintech (Cat No. 14316-1-AP).

Techniques: Binding Assay, Software

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: This figure presents a compelling visualization of the regulatory effects catalytic inactive matrix metalloproteinase-9 (MMP9) exerts on superoxide dismutase-3 (SOD3) through protein–protein interactions. (A) The representative Western blot image illustrates the protein levels of SOD3, approximately 30 kD, across various experimental conditions: a control group (CT), MMP9 cells activated by phorbol myristate acetate (PMA), MMP9 cells treated with a specific inhibitor, and cells transfected with a catalytically inactive MMP9 mutant plasmid. To ensure accuracy in protein quantification, total protein served as the loading control. The accompanying bar graph provides a detailed densitometric analysis of the Western blot bands, allowing for a quantitative comparison of SOD3 expression across the different treatment groups. This analysis represents data from six separate experiments (N = 6), ensuring a robust and reliable assessment of the experimental outcomes. (B) The co-immunoprecipitation results provide evidence of the interaction between MMP9 and SOD3 proteins. We conducted immunoprecipitation with an MMP9 antibody and subsequent immunoblotting with a SOD3 antibody on cell lysates. The detection of SOD3 in complexes with mutant MMP9 underscores a direct protein-protein interaction between MMP9 and SOD3. The accompanying bar graph offers a densitometric analysis of the SOD3 bands from the Western blot, quantitatively depicting the extent of this interaction. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test, with significance levels denoted as ** for P < 0.001 and **** for P < 0.0001. ’ns’ indicates non-significant differences. This assay was repeated thrice (N = 3) to ensure data robustness. Each point represents one sample.

Article Snippet: SOD3 was detected using an antibody from Proteintech (Cat No. 14316-1-AP).

Techniques: Protein-Protein interactions, Western Blot, Control, Transfection, Mutagenesis, Plasmid Preparation, Comparison, Expressing, Immunoprecipitation

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: The proximal ligation assay (PLA) effectively demonstrated a direct protein-protein interaction between matrix metalloproteinase-9 (MMP9) and superoxide dismutase-3 (SOD3). In this assay, we fixed the experimental cells and introduced specific antibodies targeting SOD3 and MMP9. The close proximity of these proteins was evidenced by the formation of red fluorescent dots, signifying their interaction. To confirm the assay’s specificity, we included a negative control using only one of the primary antibodies. Scale bar = 10 μm.

Article Snippet: SOD3 was detected using an antibody from Proteintech (Cat No. 14316-1-AP).

Techniques: Ligation, Negative Control

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: The Western blots and accompanying bar graphs delineate the levels of superoxide dismutase-3 (SOD3) and matrix metalloproteinase-9 (MMP9) in the culture medium of HEK293 cells. The experiment encompassed three distinct groups: a control (CT), cells transfected with an MMP9 overexpressing plasmid followed by phorbol myristate acetate (PMA) treatment to activate MMP9, and cells transfected with a catalytically inactive mutant MMP9 plasmid. The Western blot data, presented in two sections, (A) highlight MMP9 levels and (B) focus on SOD3 levels. For each sample, total protein was utilized as the loading control. We conducted statistical analyses using one-way ANOVA followed by Tukey’s post-hoc test across the groups, with each data point representing an individual sample (N = 6). Significance levels are marked as ** (P < 0.01) and **** (P < 0.0001), with ’ns’ indicating non-significant results.

Article Snippet: SOD3 was detected using an antibody from Proteintech (Cat No. 14316-1-AP).

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Mutagenesis

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: The schematic illustrates the distinct regulatory roles of matrix metalloproteinase-9 (MMP9) on superoxide dismutase-3 (SOD3). It showcases how active MMP9 potentially enhances SOD3 levels, likely through transcriptional upregulation. In contrast, catalytically inactive MMP9 seems to reduce SOD3 levels through direct protein-protein interactions and possibly via a unique proteolytic degradation mechanism. This differential regulation by MMP9 leads to increased secretion of SOD3 from cells in its active form, while the catalytically inactive MMP9 impedes SOD3 secretion. Phorbol myristate acetate (PMA), identified as an MMP9 activator in the schematic, plays a crucial role in modulating these interactions.

Article Snippet: SOD3 was detected using an antibody from Proteintech (Cat No. 14316-1-AP).

Techniques: Protein-Protein interactions