| More than 20 human diseases, such as Alzheimer's disease, Parkinson disease, TypeⅡdiabetes, are associated with the conversion of proteins/polypeptides from their native state into highly ordered and P-sheet-enriched aggregates known as amyloid fibrils. We defined these human diseases as amyloidosis, which result from the extracellular deposition of normal proteins which are dissoluble in plasma convert into insoluble amyloid fibril morphology. This deposition can cause damage to almost of all the organs and tissues, therefore, amyloidosis have systematic characters. Amyloidosis can actually occur anywhere of human body and cause dysfunctions of cardiovascular system, nervous system, and urinary system. Challenges are still remained for scientists to develop novel methods and medications for the diagnosis and treatment of amyloidosis.Theoritically all of the proteins or peptides are potential to form amyloid fibrils through self-assembly under certain conditions. It has been found that many proteins, including some non-pathogenic proteins and short peptides can form amyloid fibrils. The list of amyloidogenic proteins and peptides includes lysozyme, Aβpeptide, insulin, prion protein, synuclein,β-micro-globulin etc. The formation of amyloid fibrils is a complex process, in which a series of intermediates with different size and morphology can be produced. The mature amyloid fibrils usually include oligomers, protofilaments, long fibrils and some amorphous aggragates. Although many attempts have been made to elucidate the molecular pathways of amyloidogenesis, the exact mechanism is still obscure and remains further explored.In the present study, hen egg white lysozyme was used as a model protein to produce amyloid fibrils and human erythrocytes were used as an in vitro model to investigate the fibrillar cytotoxicity. Investigations were performed in terms of the changes of molecular conformation and the cytotoxicity of lysozyme during the course of amyloid fibrillation, and the molecular pathway of erythrocyte damage caused by the amyloid fibrils. The results provide a noval insight into the amyloidosis of proteins/peptides both on the theoretical and experimental basis.Methods, results, and conclusions:1. Kinetics of lysozyme amyloid fibrillationThe kinetics of lysozyme amyloid fibrillation was monitored by using the fluorescent probes ThT and ANS, and circular dichroism. The results showed that the conformation of lysozyme was changed along with the incubation of the protein, including increases of the content ofβ-sheet structures and the surfacial hydrophobicity, and a decrease of the content of a-helices.2. The morphology of lysozyme fibrilsThe morphologies lysozyme fibrils were observed under a transmission electron microscopy and a polarizing microscopy after the Congo-red stainning. The results showed that lysozyme mature fibril demonstrated yellow-green under polarizing microscopy, which is a typical feature of amyloid fibrils. The transmission electron microscope image showed that the mature lysozyme fibrils were long fibrous structures; together with bundles and aggregates of the fibrillar assemblies. SDS-PAGE in tricine buffer showed that there was a small amount of the protein hydrolyzed during the process of amyloid fibrillation.3. Lysozyme fibril CytotoxicityThe cytotoxicity of lysozyme fibrils was investigated in two aspects. In the first assay, human red blood cells were separated from blood and incubated with lysozyme fibrls. Spectrophotometric data showed that hemoglobin was released from the cells upon incubation with the fibrils, indicating that the fibrils induced cell damage. The hemolytical effect of the fibrils increased along with the process of fibrillar maturation. Both the fibril-growth and the fibrllar cytotoxicity reached a plateau on the fourth day of the incubation. The pool of mature fibrils contained long fibrils and their large aggregates, oligomers, protofilaments, and some acid-hydrolyzed products, as demonstrated by high-speed centrifugation and ultracentrifugation. In a comparison with the fibrils and large aggregates, the oligomers and protofilaments showed higher cytotoxicity.4. Cellullar membrane damages by lysozyme fibrilsIn the second assay of the fibrillar cytotoxicity, membranes were separated from the cells and were incubated with lysozyme fibrils prior to SDS-PAGE. The results showed that some macromolecular aggregates appeared on the top of the gel lanes and the sample wells, and the bands of membrane proteins became weaker than the control. Western Blotting and mass spectrometry analyses showed that lysozyme molecules and/or its molecular species were incorporated into the high-molecular aggregates through an intermolecular disulfide cross-linking. This result indicates that the exchange of thiol-disulfide between the fibrilar species and membrane proteins plays a key role in the amyloid-induced cell damages.5. The exposure of disulfide bonds during amyloid fibrillationThe solvent-accessible disulfide bonds of lysozyme were determined by two methods. The first assay involved in the mBBr fluorescent labeling technology. In another method, the exposed disulfides of lysozyme were reduced into free thiol groups by DTT and then blocked by covalent binding to NEM. The resultant protein was digested and then analyzed by ES1-MS/MS mass spectrometry to identify the labeled thiol groups, by which the solvent-accessible disulfide bonds can be deduced. The data showed that the lysozyme fibrillation was associated with the exposure of internal disulfide bonds to the solvent; therefore, the cytotoxicity of the protein assemblies was increased. This study strongly supports the hypothesis that cellular membrane damage by amyloid fibrils involves intermolecular disulfide cross-linking between lysozyme and membrane proteins. |
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