The carbonylation and covalent dimerization of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity is inhibited by the radical scavenger tempol

Abstract

Tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of various diseases via mechanisms that are not completely understood. Recently, we reported that high doses of tempol moderately increased survival in a rat model of ALS (amyotrophic lateral sclerosis) while decreasing the levels of oxidized hSOD1 (human Cu,Zn-superoxide dismutase) in spinal cord tissues. To better understand such a protective effect in vivo, we studied the effects of tempol on hSOD1 oxidation in vitro. The chosen oxidizing system was the bicarbonate-dependent peroxidase activity of hSOD1 that consumes H2O2 to produce carbonate radical, which oxidizes the enzyme. Most of the experiments were performed with 30 μM hSOD1, 25 mM bicarbonate, 1 mM H2O2, 0.1 mM DTPA (diethylenetriaminepenta-acetic acid) and 50 mM phosphate buffer at a final pH of 7.4. The results showed that tempol (5-75 μM) does not inhibit hSOD1 turnover, but decreases its resulting oxidation to carbonylated and covalently dimerized forms. Tempol acted by scavenging the carbonate radical produced and by recombining with hSOD1-derived radicals. As a result, tempol was consumed nearly stoichiometrically with hSOD1 monomers. MS analyses of turned-over hSOD1 and of a related peptide oxidized by the carbonate radical indicated the formation of a relatively unstable adduct between tempol and hSOD1-Trp32•. Tempol consumption by the bicarbonate-dependent peroxidase activity of hSOD1 may be one of the reasons why high doses of tempol were required to afford protection in an ALS rat model. Overall, the results of the present study confirm that tempol can protect against protein oxidation and the ensuing consequences.

Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme

“…In parallel, similar amounts of hSOD1 were inactivated (ϳ50%) but the amount of copper(I) ion released was slightly higher for the enzyme incubated in the absence than in the presence of bicarbonate (ϳ30 and 20%, respectively, of the initial concentration of copper ions in hSOD1 (50 M)). All of these results are in line with previously reported data for hSOD1 WT (6,36,43). Previous studies have also shown that hSOD1 WT residues, in particular Trp 32 and His residues, are targets of the carbonate radical produced during the bicarbonate-dependent peroxidase activity, being oxidized to the corresponding hSOD1-Trp 32 ⅐ and hSOD1-His ⅐ radicals.…”

“…The hydrolysates were analyzed by LC-MS on a UHR-ESI-Q-TOF Bruker Daltonics MaXis 3G system with Captive Spray source in the positive mode coupled on line to a nano-UPLC system. The hydrolysates were subjected to reverse-phase chromatography using a ZORBAX 300SB-C18 (150 mm ϫ 75 m, particle size 3.5 m) (Agilent Technologies) (36). The interface conditions for the nano captive source were as follows: capillary, 1.8 kV; dry heater, 150°C; dry gas, 6 liters/min.…”

“…Analysis of hSOD1 Carbonylation-Protein carbonyl contents were determined as previously described (36). After 1 h of incubation, the reactions were stopped by the addition of catalase (2 units/ml).…”

Infliximab Modulates Cisplatin-Induced Hepatotoxicity in Rats

“…If the CPS-1 activity is decreased, this may raise serum bicarbonate levels (15,33). The bicarbonate-dependent peroxidase activity of the scavenger enzyme superoxide dismutase converts hydrogen peroxide to carbonate radicals, which oxidize the enzyme (34). Elevated CA-II enzyme activity accompanied by low CPS-1 enzyme activity may lead to increased levels of bicarbonate and peroxynitrite radicals, thus inducing tissue damage.…”

Ditryptophan Cross-Links as Novel Products of Protein Oxidation

“…• and inhibited the covalent dimerization of hSOD1 in vitro 50 and also apparently, in vivo. 51 Additionally, we confirmed that mutation of Trp32 to Phe32 inhibited the covalent dimerization and non-amyloid aggregation of hSOD1.…”