The Molecular Mechanism Of Transhyretin Tryosine Nitration Participating In Amyloidosis With FAP Patients

Objective Familial amyloid polyneuropathy(FAP)is an autosomal dominant disorder characterized by the extracellular deposition of mutated Transthyretin(TTR)amyloid fibrils.TTR is synthesized mainly by the liver and the choroid plexus of the brain,existing in the blood and cerebrospinal fluid with homotetrameric form under physiological conditions.It was reported that the products of peroxidation were presenting in amyloid deposition in FAP,such as Thiobarbituric acid reactive substances(TBARS)which was the marker of lipid peroxidation,and Carbonyl which indicated the protein oxidation.In addition,S-Nitrosylation of TTR induced structural changes,leading to promote the fibril formation in a low-pH environment.Therefore,Oxidative modification may be involved in the formation of TTR amyloid fibril.The aim of this study is to investigate the relationship between the tyrosine nitration of TTR protein molecule and the formation of amyloid fibril in patients with FAP,to provide the evidence for the analysis of the molecular mechanism of FAP amyloid formation.Methods1.DNA was extracted from peripheral blood cells of 5 cases of patients with FAP(3male,2 female,aged 58-68 years).To determine the TTR gene mutation,we designed the specific primers of TTR exon and analyzed the DNA sequence after PCR amplification.2.The heart,kidney and cerebral tissue of the patients were collected and embedded in paraffin,manufacturing the pathological tissue sections.Tissue sections were stained with Congo red(CR)via observation under polarized light to determine the presence of amyloid deposition in patients with FAP.Immunohistochemistry was performed using anti-human transthyretin antibodies to identify the type of amyloid.3.To detect the expression of MPO,Immunohistochemistry using anti MPO antibody was performed on the same tissues examined by Congo red staining.4.In vitro,oxidative reactions of cardiac tissue sections from patients with FAP were carried out in 10 mM phosphate buffer(pH 7.4)supplemented with 100 μM DTPA,100 μM H2O2,10 μM sodium nitrite,and 10 μM N-acetyl-tyrosine at 37℃ for 12 h.And then immunohistochemistry using anti 3-nitrotyrosine antibody was performed.5.Serum total protein(TP)was quantified between 65.2-83.5g/L from patients with FAP.Oxidation reactions were carried out in 10 mM phosphate buffer(pH 7.4)supplemented with 200μg serum protein,100 μM DTPA,150 mIU MPO,100 μM H2O2,10 μM sodium nitrite and the other was the control group which only contained200μg serum protein at 37℃ for 2h.6.After serum exposure to the MPO/H2O2/NO2-system,the expression of TTR was analyzed using two-dimensional electrophoresis(2-DE)and Western Blot.Results1.TTR mutation in all of patients with FAP was a substitution of a methionine for a valine at position 30 of the polypeptide chain(V30M)by PCR amplification and DNA sequencing.2.By CR staining combined with polarized light microscopy,it is found that the amyloid deposition was presenting in heart and kidney in FAP patients.The anti-TTR antibody immunohistochemical reaction colocalized with amyloid deposition,revealing sedimentary amyloid type for TTR.3.Amyloid depositin was found in the site of tunica intima and tunica adventitia in vessel from patients with FAP.4.The positive reaction of the anti-MPO antibody colocalized with amyloid deposition in the heart,kidney,and aorta of patients with FAP,suggesting that the MPO molecular existed in amyloid deposition.5.Anti-3-nitro-L-tyrosine immunoreaction co-localized with amyloid deposition in FAP patients,suggesting that TTR amyloid deposition were nitrated when exposed to the MPO/H2O2/NO2-system.6.2-DE and Western Blot results showed that after treated with MPO/H2O2/NO2-system,serum TTR changed in the isoelectric point and showed protein aggregation.Conclusions1.The distribution characteristics of amyloid deposition in patients with FAP were analyzed.It is reported that amyloid deposition appeared in the region of tunica intima and tunica adventitia in the vessel of patients with FAP,filling in theinformation about the organization and organ involved in the amyloid deposition.2.It is firstly found that MPO was presenting in amyloid deposition with patients with FAP.And when reacted with the MPO/H2O2/NO2-system,the protein of amyloid deposition in patients with FAP could be modified by tyrosine nitration.3.After treated with MPO/H2O2/NO2-system,serum TTR was modified by tyrosine nitration and TTR aggregation was markedly increased.The results suggest that the TTR tyrosine nitration in patients with FAP may promote TTR aggregation,which is beneficial to the formation of amyloid deposition.