| Alzheimer‘s disease?AD?is a devastating age-related neurodegenerative disorder that is characterized by the aggregation of amyloid?peptide?A??into amyloid fibrils and oxidative stress.It has been reported that the interactions between A?and metal ions would enhance the production of reactive oxygen species?ROS?and play a crucial role in AD.The complex A?-Cu2+and A?-heme are able to catalyze nitration in the presence of hydrogen peroxide and nitrite.Protein tyrosine nitration?PTN?is a stable post-translational modification,which can significantly affect the function of some important proteins.Interestingly,there is a tyrosine residue,Y10,in the sequence of A?peptide,which might be prone to nitration.However,although numerous studies have focused attention on the relationship between metal ions and A?in AD,the effect of tyrosine nitration on the structure and functions of A?remains unclear.In this study,the effects of tyrosine nitration on the structure and functions of A?were studied and the results are as follows:?1?Effect of tyrosine nitration on A?1-40‘s aggregation and neurotoxicity.A?1-40 can be nitrated by binding to heme when hydrogen peroxide and nitrite are present.Tyrosine nitration has little effect on the binding of A?1-40 to heme and the peroxidase activity of A?1-40-heme complex.Interestingly,we found tyrosine nitration could significantly inhibit the aggregation of A?1-40 and reduce its neurotoxicity.These results suggest that tyrosine nitration may be a compensatory reaction against oxidative stress and A?aggregation.?2?Effect of tyrosine nitration on A?1-42‘s structure and functions.Dot blotting result indicated that A?1-42 could be nitrated in the presence of hydrogen peroxide,nitrite and heme.CD spectroscopy showed an increase of?-sheet structure of A?1-42 and its mutants,indicating tyrosine nitration has no effect on the conformational change of A?1-42 after incubating.Thioflavin T?ThT?flourescence assay revealed that tyrosine nitration could significantly inhibited A?1-42 aggregation.TEM and AFM observations of A?peptide aggregates further confirmed that tyrosine nitration inhibited A?1-42 fibril formation.The cytotoxicity study of native and nitrated A?1-42 on SH-SY5Y cells revealed that tyrosine nitration remarkably reduced the neurotoxicity of A?1-42.In addition,chlorinated A?1-42was used as a contrast and found tyrosine chlorination also could inhibit A?1-42 aggregation.On the basis of these results,we hypothesize that tyrosine nitration may disturb intermolecular interactions of A?1-42,as a result,it can inhibit its aggregation.Thus,this result further confirms that tyrosine nitration may be an important protective mechanism for the normal functions of A?.?3?Effect of tyrosine nitration on Cu2+-induced A?1-42 oligomerization and neurotoxicity.We found that A?1-42 could be nitrated by Cu2+in the presence of hydrogen peroxide and nitrite.Tyrosine nitration exhibited little impact on the binding o f A?1-42 to Cu2+and catalase activity of A?1-42-Cu2+complex.Circular dichroism studies also revealed significant conformational change of A?1-42 and A?1-42NT when interacting with Cu2+.Interestingly,Cu2+seemed lost its exacerbation effect on the aggregation of A?1-42NT as it acts on the wild type A?1-42.These phenomena might mainly because that tyrosine nitration can significantly disturb intermolecular interactions of A?1-42,while Cu2+only has influence on conformational changes of A?1-42.Cell study also showed that Cu2+failed in enhancing neurotoxicity of A?1-42NT.Moreover,nitrated A?1-42 may be able to protect neurons against copper ions toxicity through chelating copper ions and reacting with reactive oxygen species.We are more and more convinced that tyrosine nitration may be an important protective mechanism for normal function of A?and deserve more attention in AD drug development. |
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