Thiol Reagents To Study The Molecular Mechanisms Of Amyloid Fibrosis

Mounting investigations demonstrated that proteins and polypeptides, such as beta-amyloid peptide, lysozyme, insulin and a-synuclein were able to form amyloid fibrils which were associated with dozens of human diseases.The amyloidosis of a protein or polypeptide can lead to damage and dysfunction of an organ, causing eventually diseases including type II diabetes, Alzheimer’s and Parkinson’s diseases, and a series of systamic amyloidosis. In general, amyloid fibrils are deposited around the target cells, particularly gathered on the cell membrane, breaking the cellular structure and triggering programmed apoptosis or cell necrosis.The formation of amyloid fibrils is a complicated process, which involves the formation of a range of intermediates, such as oligomers, protofibrils and mature fibrils with different sizes, shapes and biological properties. A lot of studies showed that oligomers and protofilaments were the main cytotoxic substances that lead to amyloidosis. The exact molecular mechanism of amyloidosis is still kept unknown. Therefore it is of significance to explore the process of amyloid formation and the fibrillar toxicity to cells.In the present study egg white lysozyme and bovine serum albumin (BSA) have been chosen for investigating the influence of thiol reagents on amyloidosis. The growth kinetics, morphology, physical and biological properties of amyloid fibrils were analyzed to evaluate the role of a thiol chemical on protein fibrillation. Electrophoresis of cell membranes was performed for unveiling the role of amyloid fibrils on cytoskeleton proteins. The results showed that1,4-dithiothreitol (DTT) has a strong inhibitory effect on lysozyme fibrillation. The thiol-structure is a prerequisite for the anti-amyloid property of DTT. Thiol-disulfide exchange reaction is suggested to be involved in the inhibitory role of DTT on amyloid formation. In contrast, thiol reagents, including DTT, cysteine and glutathione showed only weak effect on BSA amyloid fibrillation owing probably to high molecular weight and specific structure of the protein.Methods and results:1. Growth kietics of lysozyme amyloid fibrilsThT is a fluorescent probe to monitor amyloid formation based on its specific binding to the β-sheet riched amyloid structures. The fluorescent profile of ThT bound to fibrillar species of lysozyme was characterized by a lag phase which was followed by a sigmoid-like elongation phase to a saturation phase, showing as an "S" type curve. The growth curve corresponds to different stages of fibril formation including oligomerization, neucleation, fibril assembly and fibril maturation.2. Influence of thiol reagents on lysozyme fibrillationThe influence of DTT, cysteine and glutathione on amyloid formation was analyzed by incubating lysozyme with the thiol chemicals at same concentration. The results showed that DTT had a strong inhibitory role on lysozyme fibrillation. In contrast cysteine and glutathione played a weak inhibitory effect on amyloid formation.3. Inhibition of lysozyme fibrillation by DTT is dose-dependentThT data indicated that DTT played an inhibitory role of on lysozyme fibrillation in a dose-dependent manner.1mM DTT can completely inhibit the growth of lysozyme fibrils. Native-PAGE results indicated that high-molecular weight assemblies were significantly reduced upon co-incubating lysozyme with0.5mM and1mM DTT, suggesting the inhibitory role of DTT on lysozyme assembly. Furthermore, lysozyme fibrils can cause aggregation of cell membrane proteins. DTT attenuated the fibril-induced formation of protein aggregates.4. The role of DTT at different stages of amyloid formationDTT was added to lysozyme at different stages of amyloid formation (0-9d) and ThT assay, CD and electrophoresis analysis were performed for monitoring the fibril growth. The results showed that the inhibition of lysozyme fibrillation by DTT occurred at early stages of incubation. In contrast, addition of DTT in7-9d only resulted in weak inhibition. This fact suggested that DTT played inhibitory role on lysozyme fibrillation mainly through suppressing oligomerization and nucleation of protein molecules.5. Influence of DTT on mature fibrillar structuresTo explore the influence of DTT on fibrillar structures, mature fibrils were incubated with DTT prior to ThT assay. The experimental results showed that DTT was able to disrupt the fibrillar structure, inducing the fibrils transformed into a structure with less β-sheets. Cysteine and glutathione showed no obvious effect on the mature fibrils.6. Kietics of BSA amyloid fibrillation and the influence of thiol reagents.ThT profile of BSA amyloid fibrillation did not present an "S" type curve as lysozyme. Instead the fibril growth did not undergo a lag phase and amyloid formation started when incubation began. In other words, BSA fibrillation did not involve nucleation stage. Thiol reagents, including DTT, cysteine and glutathione, affect weakly on amyloid fibrillation of BSA probably because of its high molecular weight.ConclusionBoth lysozyme and BSA are able to form amyloid fibirs upon incubation. However, these two proteins do not share same pathway in amyloid formation due to the structural difference between the proteins. BSA fibrillation did not undergo a nucleation phase as lysozyme did. Thiol reagent DTT demonstrated a strong inhibitory role on amyloid fibrillation of lysozyme. During lysozyme amyloid fibrillation, sulfhydryl groups generated upon the disulfide bonds breaking down. Therefore thiol reagents were able to affect the process of fibril formation by thiol-disulfide exchanges. In other words, the thiol-structure is a prerequisite for the anti-amyloid property of DTT. Electrophoresis experiment showed that DTT inhibited the fibril-induced aggregation of membrane proteins, suggesting that thiol chemicals could be served as a medicinal candidate for treating amyloidosis.