Effects Of Tyrosine Nitration On The Structure And Functions Of Aβ

Alzheimer‘s disease（AD）is a devastating age-related neurodegenerative disorder that is characterized by the aggregation of amyloidβpeptide（Aβ）into amyloid fibrils and oxidative stress.It has been reported that the interactions between Aβand metal ions would enhance the production of reactive oxygen species（ROS）and play a crucial role in AD.The complex Aβ-Cu²⁺and Aβ-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 Aβpeptide,which might be prone to nitration.However,although numerous studies have focused attention on the relationship between metal ions and Aβin AD,the effect of tyrosine nitration on the structure and functions of Aβremains unclear.In this study,the effects of tyrosine nitration on the structure and functions of Aβwere studied and the results are as follows:（1）Effect of tyrosine nitration on Aβ_1-40‘s aggregation and neurotoxicity.Aβ_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 Aβ_1-40 to heme and the peroxidase activity of Aβ_1-40-heme complex.Interestingly,we found tyrosine nitration could significantly inhibit the aggregation of Aβ_1-40 and reduce its neurotoxicity.These results suggest that tyrosine nitration may be a compensatory reaction against oxidative stress and Aβaggregation.（2）Effect of tyrosine nitration on Aβ_1-42‘s structure and functions.Dot blotting result indicated that Aβ_1-42 could be nitrated in the presence of hydrogen peroxide,nitrite and heme.CD spectroscopy showed an increase ofβ-sheet structure of Aβ_1-42 and its mutants,indicating tyrosine nitration has no effect on the conformational change of Aβ_1-42 after incubating.Thioflavin T（ThT）flourescence assay revealed that tyrosine nitration could significantly inhibited Aβ_1-42 aggregation.TEM and AFM observations of Aβpeptide aggregates further confirmed that tyrosine nitration inhibited Aβ_1-42 fibril formation.The cytotoxicity study of native and nitrated Aβ_1-42 on SH-SY5Y cells revealed that tyrosine nitration remarkably reduced the neurotoxicity of Aβ_1-42.In addition,chlorinated Aβ_1-42was used as a contrast and found tyrosine chlorination also could inhibit Aβ_1-42 aggregation.On the basis of these results,we hypothesize that tyrosine nitration may disturb intermolecular interactions of Aβ_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 Aβ.（3）Effect of tyrosine nitration on Cu²⁺-induced Aβ_1-42 oligomerization and neurotoxicity.We found that Aβ_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 Aβ_1-42 to Cu²⁺and catalase activity of Aβ_1-42-Cu²⁺complex.Circular dichroism studies also revealed significant conformational change of Aβ_1-42 and Aβ_1-42NT when interacting with Cu²⁺.Interestingly,Cu²⁺seemed lost its exacerbation effect on the aggregation of Aβ_1-42NT as it acts on the wild type Aβ_1-42.These phenomena might mainly because that tyrosine nitration can significantly disturb intermolecular interactions of Aβ_1-42,while Cu²⁺only has influence on conformational changes of Aβ_1-42.Cell study also showed that Cu²⁺failed in enhancing neurotoxicity of Aβ_1-42NT.Moreover,nitrated Aβ_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 Aβand deserve more attention in AD drug development.