Study On The Effects Of Tyrosine Nitration On The Structure And Function Of Neuropeptide Y And Human Calcitonin

Protein tyrosine nitration is an important post-translational modification of protein.Nitration will increase the local steric hindrance,decrease the pKa value of the phenolic hydroxyl group,increase the negative charge density of the protein and ultimately affect the structure and function of protein.Protein nitration is ubiquitous in body and closely linked to the development of many diseases such as cardiovascular disease,diabetes and neurodegenerative diseases.Therefore,it is of great significance to study the nitration of important proteins in body for the understanding of pathogenesis,prevention and treatment of related diseases.However,due to the nitration of peptide caused by chemical methods,the oxidation of peptide is inevitable,which makes it difficult to study the effect of nitration on the structure and function of peptide.Herein,two peptides with well-defined function,ie neuropeptide Y?NPY?and human calcitonin?hCT?are selected to specifically study the effect of tyrosine nitration on their structure and function by site-specifically mutating tyrosine to 3-nitro-tyrosine in peptide sequence.NPY is a 36 amino acid active polypeptide that regulates multiple physiological functions in the central nervous system and has been shown to be closely associated with Alzheimer's disease?AD?.Due to the high level of oxidative and nitrative stress in AD,NPY,being rich in tyrosine,may undergo nitration,resulting in its dysfunction in AD.However,no work has been reported on its nitration and the effect of tyrosine nitration on NPY's structure and function.Human calcitonin?hCT?is an active peptide hormone known for its hypocalcaemic effect but has limited pharmaceutical potential due to a high tendency to aggregate.It has been shown that tyrosine nitration can inhibit the aggregation of amyloidogenic peptide in our previous study.Furthermore,it has been reported that the tyrosine residue plays a crucial role in hCT aggregation.Therefore,whether tyrosine nitration is a novel way to inhibit hCT aggregation and thereby enhance its activity is worth studying.In this paper,nitration of NPY and the effects of tyrosine nitration on the structure and function of NPY and hCT were studied in detail and the following results were obtained:?1?Heme could bind with NPY and catalyze NPY nitration,and the tyrosine nitration of NPY affected its structure and function:UV-visible spectroscopy,fluorescence spectroscopy and differential pulse voltammetry results revealed that NPY could bind with heme to form a complex,and this binding could significantly increase the peroxidase activity of free heme.Dot blot results indicated that in the presence of hydrogen peroxide and nitrite,NPY was highly susceptible to nitration after binding to heme.Moreover,LC-MS/MS results confirmed that Tyr36,an important resiude in NPY binding and activating neuropeptide receptors,was also nitrated under the nitration condition.Cell experiments using synthetic NPY?3N??the tyrosine at position 36 mutated to 3-nitro-tyrosine?showed that nitration would seriously damage its active monomer conformation and significantly impair its biological activity.These results suggest that NPY may be closely related to the development of AD via the interaction between heme and NPY,eliciting nitration and thereby counteracting its function in the nervous system.?2?Copper ions could bind with NPY and catalyze NPY nitration:Electrochemical,ESI-MS and spectroscopy results indicated that copper ions could bind with NPY to form a complex with low dissociation constant?Ka⁰.021?M?.Hydroxyl radicals assay showed that the combination of NPY with copper ions promoted the production of·OH and increased local oxidative stress.Dot blotting results suggested that NPY was also easily nitrated under the Cu²⁺-NO2^--H2O2 system.LC-MS/MS result indicated that all tyrosine residues in NPY were nitrated,which would cause the inactivation of NPY shown by our previous study.This study supports the hypothesis that copper has a close correlation with NPY and complicates the peptide in AD.?3?Tyrosine nitration significantly inhibited hCT aggregation and effectively maintained its biological activity:Fluorescence spectroscopy,fluorescence electron microscopy and transmission electron microscopy?TEM?showed that tyrosine nitration at position 12 significantly inhibited hCT aggregation.Moreover,the results of Nu-PAGE gel electrophoresis and circular dichroism?CD?spectroscopy showed that nitration could not only inhibit the formation of amyloid fibrils of hCT but also maintain its monomeric form for a long time.Furthermore,using chlorinated hCT as a contrast,it was found that the chlorination modification did not affect its aggregation.These results indicated that tyrosine nitration was most likely to inhibit hCT aggregation by increasing local steric hindrance to disrupt?-?stacking between hCT molecules.Finally,animal experiments demonstrated that tyrosine nitration did not have much effect on biological activity of wild-type hCT,and could maintain its hypocalcaemic potency for a long time.This study has an important guide significance for the design of highly homologous human calcitonin analog drugs.?4?Heme significantly inhibited hCT aggregation and effectively maintained its biological activity:UV-visible spectroscopy,differential pulse voltammetry and isothermal titration calorimetry results indicated that heme could bind to hCT with a moderate affinity.Moreover,such binding was reversible.Fluorescence probes,transmission electron microscopy,atomic force microscopy?AFM?,Nu-PAGE gel electrophoresis and CD spectroscopy results showed that heme could significantly inhibit hCT aggregation,which was most likely to be accomplished through the porphyrin macrocycle of heme blocking the?-?stacking of the core aggregation region of the hCT molecule.Finally,animal experiments showed that hCT coincubation with heme still maintained its biological activity in reducing blood calcium in mice after 24 h,while the hypocalcaemic potency of hCT alone after incubation for 24 h was significantly reduced due to its aggregation.This finding will be of great significance for the development of an optimal hCT preparation and for the clinical reuse of hCT.