Detection of sequence-specific tyrosine nitration of manganese SOD and SERCA in cardiovascular disease and aging

Abstract

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.

Cisplatin-induced Ototoxicity Is Mediated by Nitroxidative Modification of Cochlear Proteins Characterized by Nitration of Lmo4

“…Background-corrected bands (NIH Image J software) were normalized against actin bands obtained by stripping the membrane with 25 mM glycine (Bio-Rad) (pH 2.0), 1% lauryl sulfate (Fisher) and reprobing with an anti-actin monoclonal antibody (Millipore, Bellerica, MA). For negative controls, the membranes were incubated with sodium dithionite (100 mM in 100 mM sodium borate (pH 9.0) for 5 min) to reduce nitrotyrosine to aminotyrosine before immunoblotting (22). Differences in intensity were analyzed by one-tailed t tests.…”

Proteomic analysis of age dependent nitration of rat cardiac proteins by solution isoelectric focusing coupled to nanoHPLC tandem mass spectrometry

“…An illustrative example is the mitochondrial manganese superoxide dismutase (Mn-SOD), which catalyzes the disproportionation of superoxide to O 2 and H 2 O 2 . Mn-SOD was found to undergo almost complete inhibition when nitrated at Tyr 34 (MacMillan-Crow and Thompson, 1999;Quint et al, 2006;Xu et al, 2006). The crystal structures of native Mn-SOD and nitrated Mn-SOD were found to be closely superimposable; however, the nitration of Tyr 34 disrupts the Hbonding network at the active site, which may be the reason for protein inactivation (Quint et al, 2006).…”

Peroxynitrite, a Stealthy Biological Oxidant

“…with manganese is hindered by both steric constraints and electrostatic repulsion (63,64). The nitration of Mn-SOD Tyr-34 is strongly supportive of the intramitochondrial peroxynitrite formation (65,66). Other oxidizing and nitrating species tend to react with tyrosines other than Tyr-34 and do not lead to enzyme inactivation.…”