Journal: Antioxidants & Redox Signaling

Article Title: The Axonal Motor Neuropathy-Related HINT1 Protein Is a Zinc- and Calmodulin-Regulated Cysteine SUMO Protease

doi: 10.1089/ars.2019.7724

Figure Lengend Snippet: HINT1 isopeptidase activity, time-course and effect of zinc ions. (A) Time-course for HINT1 activated by Ca 2+ -CaM to remove SUMO1 from the RGSZ2 protein. The data were analyzed by nonlinear regression (Sigmaplot/Sigmastat v 14.0; Systat Software, Inc.), (estimate ± SE): r = 0.97 ± 0.04, T50 = 12.35 ± 0.27 min, t = 37.21, DF total = 6, MS = 0.26, p < 0.05. Identical assay, but HINT1 was activated by the NO-donor SNAP (estimate ± SE): r = 0.99 ± 0.07, T50 = 6.31 ± 0.23 min, t = 57.79, DF total = 6, MS = 0.215, p < 0.05. “C” denotes control group without HINT1 isopeptidase activator. The curve fits and 95% confidence intervals are shown. (B) Zinc ions added during the time-course blocked HINT1 isopeptidase activity activated by Ca 2+ -CaM, but failed when NO was the activator. HINT1 (2 μ M ), SNAP (100 μ M ), CaCl 2 (2.5 m M ), CaM (6 μ M ), sumoylated RGSZ2 (1 μ M ), and ZnCl 2 (300 μ M ). Data refer to the control group, which did not include HINT1 and was assigned an arbitrary value of 1; “*” for each group, Ca 2+ -CaM or SNAP indicates a significant difference with respect to the +15-min time interval. ANOVA, Holm-Sidak multiple comparisons, p < 0.05. Further details of immunoblot detection in “Materials and Methods” section and . DF, degrees of freedom; MS, mean square; SE, standard error. Color images are available online.

Article Snippet: All graphs and statistical analyses were generated and performed by using the Sigmaplot/SigmaStat v.14 package (SPSS Science Software; Erkrath).

Techniques: Activity Assay, Software, Western Blot

Journal: Antioxidants & Redox Signaling

Article Title: The Axonal Motor Neuropathy-Related HINT1 Protein Is a Zinc- and Calmodulin-Regulated Cysteine SUMO Protease

doi: 10.1089/ars.2019.7724

Figure Lengend Snippet: Regulation of HINT1 sumoylase activity by drugs affecting its adenylate hydrolase activity and third-partner interacting proteins. (A) The substrate TpAd, and the inhibitor TpGc of HINT1 adenylate hydrolase activity reduced the capacity of HINT1 to remove SUMO from RanGAP1 in a dose-dependent manner. The data were analyzed by nonlinear regression, competition at a single site (Sigmaplot/Sigmastat v 14.0; Systat Software, Inc.) and shown as curve fit and 95% confidence interval. TpGc (estimate ± SE): r = 0.93 ± 0.12, EC50 = 403.18 ± 12.20 n M , t = 16.13, DF total = 7, MS = 0.22, p < 0.05; TpAd (estimate ± SE): r = 0.97 ± 0.11, EC50 = 320.78 ± 15.85 n M , t = 19.31, DF total = 7, MS = 0.39, p < 0.05. (B) Effect of HINT1-interacting signaling proteins on HINT1 sumoylase activity: The σ1R long isoform reduced the sumoylase activity of HINT1 but not SENP2 in a concentration-dependent fashion. (C) The cytosolic C-terminal region C0–C1–C2 of the NMDAR NR1 subunit, but not the C-terminal region of MOR, reduced HINT1 isopeptidase activity. Further details of immunoblot detection in “Materials and Methods” section. MOR, mu-opioid receptor; NMDAR, N -methyl- d -aspartate receptor; TpAd, phosphoramidate tryptamine AMP; TpGc, guanosine-5′-tryptamine carbamate.

Article Snippet: All graphs and statistical analyses were generated and performed by using the Sigmaplot/SigmaStat v.14 package (SPSS Science Software; Erkrath).

Techniques: Activity Assay, Software, Concentration Assay, Western Blot