Heterologous Protein On The Inhibition Of Insulin Amyloid Fibrosis And Its Cellular Toxicity

Protein amyloidogenesis, which is associated with more than 20 human diseases such as Alzheimer's and Huntington's disease, hemodialysis-related amyloid deposition and a number of systemic amyloidoses, has become a critical subject in scientific research and health concerns. In addition to the proteins related to diseases, it has been demonstrated that some proteins, peptides and poly-amino acids that are not related to disease can also form amyloid fibrils. Theoretically all proteins are able to rearrange their backbones to assemble into amyloid fibrils upon unfolding under certain circumstances. Therefore amyloid formation is recognized as a common property of proteins and peptides.Insulin is a predominantlyα-helical structured protein that can undergo amyloid fibril formation in destabilizing conditions. It is consisting of a 21-residue A chain and a 30-residue B chain which are linked together by two interchain disulfide bonds. The pharmaceutical preparation of insulin can form amyloid aggregates in the processes of production, delivery, storage and clinical application. These aggregates may contain immune epitopes and side-effects can be triggered. Therefore it is of great significance to elucidate the molecular mechanism and to screen inhibitors for insulin fibrillation. In this study, insulin has been used as a model molecule in order to explore the kinetics of fibrillation and the fibrillar cytotoxicity of a protein in the presence of heterogeneous proteins.Insulin tends to form amyloid fibrils under high temperature and low pH. It has been reported that some low molecular compounds such as amino acids are able to affect the fibrillation of insulin. It is well-established that the kinetics of fibrillation can be significantly accelerated by the addition of seed fibrils, which, at a sufficient concentration, will abolish the lag time completely. The seed fibrils serve as nucleation templates for insulin to assemble into amyloid fibrils. Whether heterogeneous protein or its amyloid fibrils affect the kinetics of insulin fibrillation? To address this question four proteins, hen egg white lysozyme (Lys), hemoglobin (Hem), horseradish peroxidase (HRP) and bovine serum albumin (BSA) have been selected to investigate the fibrillation kinetics and cytotoxicity of insulin in the presence of heterogeneous proteins.Experimental methods included ThT and ANS fluorescence to monitor the fibril growth and hydrophobic domain exposure, chaperone activity assay, characterization of the fibrillar structure by atomic force microscope and transmission microscope and evaluation of the fibrillar cytotoxicity by hemolytic assay. The results indicated that all the heterogeneous proteins inhibited insulin fibrillation in a dose range of 5% - 20%. The resultant insulin fibrils showed lower cytotoxicity than that of the control insulin fibrils. The inhibitory effect of lysozyme on insulin fibrillation was increased while lysozyme was applied in fibrillar forms. ANS fluorescence indicated that lysozyme fibrils promoted the exposure of hydrophobic domains of insulin. Chaperones activity assay revealed that HRP and BSA possessed chaperone activity to inhibit the B-chain aggregation of insulin. The fact that other two proteins Lys and Hem failed to inhibit insulin B-chain aggregation suggested that chaperone activity was not involved in the inhibitory effect of heterogeneous protein on insulin fibrillation. An alternative mechanism is suggested that the heterogeneous proteins provided a crowding environment that reduced the probability of insulin molecules assembled into aggregates.In conclusion, amyloid fibrillation and the fibrillar cytotoxicity of insulin were inhibited when insulin fibrils were prepared in the presence of heterogeneous proteins. Chaperone activity was not involved in this process. A crowding environment originated from the heterogeneous protein has been suggested to be the driving force in the inhibition of insulin fibrillation. The results of this study are of significance in elucidating amyloidogenesis of a protein and screening inhibitors for protein fibrillation.