Protective Effects of Carnosine and N-Acetylcarnosine on Salsolinol-mediated Cu,Zn-superoxide Dismutase Inactivation

Abstract

The oxidation of cellular proteins is implicated in the development of many human disease and aging. The vulnerability of various amino acid residues of protein to oxidation varies with reactive oxygen species (ROS) used. A group of metalloproteins known as Cu,Zn-superoxide dismutase (SOD) catalyzes the dismutation of two superoxide anions into one oxygen and one hydrogen peroxide and is thus involved in protecting the cell from oxygen toxicity. Previous reports showed that the reaction of Cu,Zn-SOD with H2O2 resulted in the oxidation of histidine 1 and the generation of protein fragmentation. However, oxidation of Cu,Zn-SOD by the thiol/Fe(III)/O2 mixed-function oxidation system did not lead to the oxidation of histidine. The modification of Cu,Zn-SOD by the lipid peroxidation product, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE), lead to the exclusive modification of histidine residues and the generation of protein-protein cross-linked derivatives. Finally the modification of proteins by glycation led to the modification of lysine. It is therefore evident that each form of ROS elicits a different pattern of protein oxidation. Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) an endogenous neurotoxin, is known to be involved in the pathogenesis of Parkinsons disease (PD). Salsolinol has been detected in cerebrospinal fluid of PD patients. Salsolinol was also found in urine of PD patients administered with L-DOPA. The properties of salsolinol, as a neurotoxin, are intensively studied. Salsolinol has a molecular structure similar to 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) and also to 6-hydroxy dopamine, which are known to induce loss of catecholaminergic cells. Salsolinol and its methylated dervatives have been suggested to act as endogenous dopaminergic neurotoxins, inducing selective neuronal cell death and eliciting symptoms almost identical to idiopathic Parkinson's disease. It has been reported that salsolinol in conjugation with cupric ion or ferric ion undergoes redox cycling to produce ROS such as hydroxyl radicals that cause DNA strand scission and cell death. Although neurotoxicologic effects of salsolinol have been extensively investigated, little is known about the modification of proteins mediated by salsolinol. Carnosine (β-alanyl-L-histidine) is a potent antioxidant, antiglycating, pH-buffer and metal chelating agent. Carnosine is accumulated in excitible tissues (brain, heart and skeletal muscles) of vertebrates in large amounts (up to 20 mM in humans). The N-acetyl derivatives of histidine and carnosine exist in brain and heart muscle. These compounds also demonstrate antioxidant, proton buffering or metal chelating abilities but their biological function has not been clarified. In the present study, we investigated the protective effects of carnosine and N-acetylcarnosine on the inactivation of Cu,Zn-SOD by salsolinol. We showed that exposure of Cu,Zn-SOD to salsolinol led to the protein fragmentation and enzyme inactivation. Carnosine and Nacetylcarnosine effectively inhibited the fragmentation and inactivation of salsolinol-treated Cu,Zn-SOD.