Journal: Redox biochemistry and chemistry

Article Title: Deacetylation of SOD3 by sirtuins restores furin cleavage

doi: 10.1016/j.rbc.2025.100062

Figure Lengend Snippet: SOD3 contains an N-terminal signal peptide that marks it for excretion into the extracellular matrix. Prior to excretion SOD3 forms homotetramers, mainly through interactions between a loop near the N-terminus (residues 51–60). N-linked glycosylation on N89 improves SOD3 extracellular secretion and solubility. There exist two intra-subunit disulfide bonds in the active structure, between C45 and C190, and C107 and C189. Disulfide exchange with C195 forms the inactive form. The Cu–Zn catalytic domain ranges from residues 96–193. SOD3 has a heparin binding domain at its C-terminal end consisting of a region of polybasic amino acid residues (R210-R215) that can act as a recognition site for furin, which will cleave at the C-terminal side of R215. Prior to proteolytic processing C219 residues can bond together forming a homodimer.

Article Snippet: SOD3 activity was determined using an assay (S311-10) from Dojindo (Rockville, MD) per the manufacturer’s instructions.

Techniques: Glycoproteomics, Solubility, Binding Assay

Journal: Redox biochemistry and chemistry

Article Title: Deacetylation of SOD3 by sirtuins restores furin cleavage

doi: 10.1016/j.rbc.2025.100062

Figure Lengend Snippet: (A) Treatment with 0.2–5 mM sNHSAc for 30 min at RT dose-dependently acetylated recombinant SOD3 as evaluated by (I) anti-acetylated-lysine immunoblotting. (II) TCE labeling was used to monitor protein loading and transfer. (B) No change was observed in SOD3 activity when treated with 1 mM sNHSAc. Activity was measured by the decrease in the rate of absorbance of WST-1 formazan at 450 nm with the addition of SOD3. Consumption of superoxide by SOD3 inhibits the reduction of WST-1, preventing WST-1 formazan formation. Error bars depict the standard deviation (n = 3).

Article Snippet: SOD3 activity was determined using an assay (S311-10) from Dojindo (Rockville, MD) per the manufacturer’s instructions.

Techniques: Recombinant, Western Blot, Labeling, Activity Assay, Standard Deviation

Journal: Redox biochemistry and chemistry

Article Title: Deacetylation of SOD3 by sirtuins restores furin cleavage

doi: 10.1016/j.rbc.2025.100062

Figure Lengend Snippet: Treatment of SOD3 and AcSOD3 with 0.1 mM H 2 O 2 or 1 mM βME followed by incubation with furin overnight at 37 °C was performed to evaluate the impacts of reduction and oxidation on furin cleavage. Proteolysis was monitored by electrophoretic mobility in a reducing Western blot visualized by TCE labeling (I), the uncropped blot with molecular weight markers is included in . Neither H 2 O 2 nor βME affected furin cleavage or the prevention of cleavage by acetylation. Anti-acetyl-lysine immunoblotting confirmed acetylation of SOD3 (II).

Article Snippet: SOD3 activity was determined using an assay (S311-10) from Dojindo (Rockville, MD) per the manufacturer’s instructions.

Techniques: Incubation, Western Blot, Labeling, Molecular Weight

Journal: Redox biochemistry and chemistry

Article Title: Deacetylation of SOD3 by sirtuins restores furin cleavage

doi: 10.1016/j.rbc.2025.100062

Figure Lengend Snippet: SOD3 acetylated with 1 mM sNHSAc and then deacetylated by SIRT1 or SIRT3 was incubated with furin overnight at 37 °C. All samples shown in (A) are incubated with furin, acetylation blocks furin cleavage as seen by a change in electrophoretic mobility in SDS-PAGE, and SIRT3 restores this shift (AI), the uncropped blot is included in . SIRT3 has an observed MW of 33.5 kDa and can be seen as a band above cleaved SOD3. Anti-acetylated-lysine immunoblotting confirms acetylation and deacetylation of these samples (AII). The shift in SOD3 mass was also evaluated by intact mass spectrometry, integration of the deconvoluted spectra from 25700 to 27900 amu was performed and divided by the integration of 28075–28925 amu. This cleavage ratio for the AcSOD3 no-furin sample was subtracted from the cleavage ratio for each treatment and normalized to the SOD3 +furin sample. Percent cleavage is therefore relative to defining the SOD3 + furin cleavage ratio as 100 % or complete cleavage. SIRT1 restored 5.9 % of furin cleavage and SIRT3 restored 85.0 % (B).

Article Snippet: SOD3 activity was determined using an assay (S311-10) from Dojindo (Rockville, MD) per the manufacturer’s instructions.

Techniques: Incubation, SDS Page, Western Blot, Mass Spectrometry

Journal: Redox biochemistry and chemistry

Article Title: Deacetylation of SOD3 by sirtuins restores furin cleavage

doi: 10.1016/j.rbc.2025.100062

Figure Lengend Snippet: Acetylated SOD3 was incubated with SIRT1 or SIRT3 and 2 mM NAD + for 30 min at 37 °C. Global deacetylation of SOD3 was evaluated by anti-acetylated-lysine immunoblotting (AI) and quantified by densitometry with normalization to total protein visualized with TCE labeling (AII). Data are expressed as mean with SD; a one-way ANOVA was performed on triplicates *p < 0.05, **p < 0.01. Site specific deacetylation was quantified by proteomics of trypsin digests in terms of fold change compared to acetylated SOD3 (B). ND indicates that acetylation at this site was not found in Ctrl SOD3.

Article Snippet: SOD3 activity was determined using an assay (S311-10) from Dojindo (Rockville, MD) per the manufacturer’s instructions.

Techniques: Incubation, Western Blot, Labeling