Study On Tyrosine Nitration Of Vasoactive Intestinal Peptide Catalyzed By Heme And Copper Ions

Protein tyrosine nitration is an important post-translational modification of protein in vivo.Due to the the introduction of a large volume of the electron-withdrawing group nitro on the ortho-position of the phenolic hydroxyl group of the tyrosine residue of the protein yields 3-nitrotyrosine（3-NT）,thereby altering the structure of the protein and possibly affecting its function.Abnormal levels of 3-NT have been found in some diseases（such as neurodegenerative diseases,cardiovascular disease and diabetes mellitus,etc.）,and the formation of 3-NT is believed to be related to the occurrence and development of these diseases,but the specific influencing mechanism is not clear.Vasoactive intestinal peptide（VIP）is an important and conserved active peptide in vivo,which plays an important role in maintaining nerve cell function,and its tyrosine residues are closely related to maintaining its activity.In neurodegenerative diseases,heme and Cu²⁺metabolisms are disturbed,and the oxidative stress in these disease states is intensified,leading to increased levels of hydrogen peroxide（H₂O₂）and nitrite（NO₂^-）in vivo.Thus,heme or Cu²⁺catalysis of protein tyrosine nitration is considered to be one of the causes of increased protein tyrosine nitration in these disease states.Will VIP have tyrosine nitration in this environment?What is the effect of tyrosine nitration of VIP on its structure and function?It has not been reported yet.In this paper,the possibility of heme and Cu²⁺catalyzing the tyrosine nitration of VIP in the presence of H₂O₂ and NO₂^-was studied,and the effects of tyrosine nitration on structure and function of VIP were studied.The results of this study may provide a possibility to elucidate the mechanism of the occurrence and development of neurodegenerative diseases.The main findings are as follows:（1）UV-visible absorption spectroscopy,fluorescence quenching,and ¹H NMR spectra proved that VIP and heme could bind to form heme-VIP complex,and the binding site of heme is near the tyrosine residues of VIP.Fluorescence quenching,electrospray ionization mass spectrometry and ¹H NMR spectra proved that VIP and Cu²⁺could bind to form Cu²⁺-VIP complex,and the binding site of Cu²⁺is histidine residues and near tyrosine residues of VIP.（2）It was found that the efficiency of the heme-VIP complex catalyzed H₂O₂ oxidation of L-tyrosine to form dityrosine was significantly higher than of free heme,which indicated that the complex of VIP and heme had higher catalytic oxidation activity.Dot blotting confirmed that VIP had undergone tyrosine nitration in heme-H₂O₂-NO₂^-system.Three tyrosine nitration of VIP products（VIP（10）（Y10 is nitrated to 3-NT）,VIP（22）（Y22 is nitrated to 3-NT）and VIP（10,22）（Y10 and Y22 are nitrated to 3-NT））were detected by liquid mass spectrometry,which indicated that both of the two tyrosine residues of VIP could be nitrated under this system.It was found that Cu²⁺-VIP complex catalyzed hydroxyl radical generation from H₂O₂ more effectively than Cu²⁺,which indicated that the complex of VIP and Cu²⁺promoted the ability of Cu²⁺to catalyze hydroxyl radical generation.Dot blotting confirmed that VIP had undergone tyrosine nitration in Cu²⁺-H₂O₂-NO₂^-system.These results strongly suggest that VIP can bind with heme or with Cu²⁺,and the complex increases the catalytic activity of heme/Cu²⁺and improves the probability of in situ tyrosine nitration of VIP.（3）The circular dichromatic spectrometric method showed that the structure of VIP monomer was changed by tyrosine nitration.Cell experiment results showed that tyrosine nitration weakened the ability of VIP to promote c AMP production,which indicated that tyrosine nitration would weaken the biological activity of VIP.VIP tyrosine nitration leads to changes its function,which may be related to the occurrence and development of neurodegenerative diseases.