The Effect Of Oxidation On Protein Structure And Its Amyloid Fibrosis Behavior

Protein molecules are essential for physiological activity of cells.A correct spatial structure is a prerequisite for a protein performing a regular biological function.Under some circumstances,a protein may undergo mis-folding and subsequently form amyloid fibrils.Amyloid deposition is one of common features of many human diseases,such as Huntington’s disease,Diabetes,Parkinson and alzheimer’s diseases.Mounting studies demonstrate that amyloid fibrils formed by different proteins possess highly similar structure,despite of their variations in sequence,length,folding state and structure of precursor protein.Therefore,the formation of amyloid fibrils is a common property of all protein backbones.Investigations suggest that three phases are involved in protein amyloid fibrillation,including nucleation,elongation and maturation,corresponding to lag phase,growth phase and finally plateau phase.There are many factors,such as temperature,acidity,ion strength,metal ions and some small molecules can affect protein amyloid fibrillation.Oxidation is able to bring changes to the structure and property of a protein,resulting in an alternation of fibrillation process.In the present study,utilizing bovine insulin and hen egg white lysozyme as in vitro models,the effects of oxidation on molecular structure and amyloid fibrillation of a protein are determined.The molecular mechanism of action as well as fibrillar cytotoxicity is also explored.Methods and results1.Oxidative role of hydrogen peroxide on proteinsTo investigate the role of oxidation on the molecular structure and amyloid fibrillation of lysozyme,H2O2 was used as oxidative reagent for treatment of the protein.The UV data demonstrate that aromatic amino acids of lysozyme were not affected by hydrogen peroxide.In contrast,the intrinsic fluorescence was changed upon H2O2-treating,suggesting an alternation in three-dimensional structure.H2O2 affected lysozyme structure in a dose-dependent manner.A denaturation assay indicated that oxidized lysozyme was more susceptible to the denaturant than the non-oxidized form.The denaturation of oxidized lysozyme belongs to a fashion of polymorphic state,in contrast to the fashion of three state of non-oxidized protein.In comparison with lysozyme,the UV spectra and intrinsic fluorescence of bovine insulin were kept unchanged after oxidation by H2O2.Analysis of mass spectrometry suggested that oxygenation occurred in the oxidation of insulin by H2O2.2.Amyloid fibrillation of the H2O2-treated proteinsThT and ANS fluorescence,Congo red and circular dichroism have been adopted to track amyloid fibrillation of proteins.The growth curve of amyloid fibrils of a non-oxidized protein appeared as a sigmoidal shape,accompanied with a decrease in a-helix and increases in β-sheet and surface hydrophobicity.In comparison,the tendency of amyloid fibrillation of an oxidized protein descended obviously.TEM images showed that mature fibrils of a non-oxidized protein had a typical amyloid morphology characterized by long and dense fibrils.Both the density and length of the fibrils were decreased when the protein was in an oxidized form.These facts indicated that,after treating by H2O2,the process of protein amyloid fibrillation was inhibited.3.Amyloid fibrillation of oxidized protein induced by AAPHAAPH is usually used as a source of water-soluble free radicals.As an alternative oxidative reagent,AAPH was added into protein solution prior to incubation at 37℃for 24 h.Non-reacted AAPH was removed by extensive dialysis.The results of intrinsic fluorescent and ultraviolet spectrum showed that aromatic amino acids of proteins were oxidized by AAPH.After oxidation,the tendency of amyloid fibrillation of proteins decreased obviously.After incubation of the oxidized protein for 6 days,aggregate particles and fibrils with low density were observed under TEM,suggesting that amyloid fibrillation was inhibited.The AAPH-oxidized protein was subject to a native-PAGE analysis.The results demonstrated that AAPH induced crosslinking of protein monomers.These crosslinked protein aggregates hindered the assembly of protein molecules,and thus amyloid fibrillation was inhibited.4.Cytotoxicity of amyloid fibrilsHuman erythrocytes were used as an in vitro model to explore the cytotoxicity of aggregates formed from oxidized and non-oxidized proteins.The results indicated that intense hemolysis was induced by amyloid fibrils of non-oxidized insulin and lysozyme.In contrast,the assemblies formed from oxidized proteins showed a decreased hemolytic effect.These results were further supported by the observations of cell morphology under an optical microscope.ConclusionUpon oxidation by hydrogen peroxide and free radicals,the molecular structures of insulin and lysozyme were changed;this in turn altered the process of amyloid fibrillation.These alternations are mainly embodied in extending nucleation phase,reducing surface hydrophobicity of aggregates,and lowering fibrillar toxicity to erythrocytes.Despite different mechanism of protein oxidation induced by hydrogen peroxide and free radicals,the oxidized proteins exhibited similar properties,including an interruption of amyloid fibrillation.The hindrance of amyloid formation can be attributed to the alternations in protein conformation and formation of crosslinked aggregates.The results of this study are of great significance in exploring the molecular mechanism of protein amyloid fibrillation under oxidative stress.