Effects Of Different Surfactants On Protein Amyloidosis

Mounting researches have evidenced that a variety of human disease are related to the conformational alternations of proteins or peptides. The formation of protein amyloid fibrils is an extremely complex process. Oligomers, protofibrils, irregular aggregates and and many other intermediates may produced in this process. No matter the mature fibrils or other amyloid intermediates they will deposite in tissues or organs of the body, gather on the cell membranes, leading eventually to cell death and causing amyloidosis, inculding Parkinson’s disease, type Ⅱ diabetes and etc. Investigations have also found that other proteins, not correlated with disease, form amyloid fibrils under appropriate conditions. Therefore, amyloid formation is a common feature of protein backbones. All proteins in an organism play important roles in biological activities. It is of great significant to study the molecular mechanism of amyloid formation of a protein and to screen anti-amyloid medicinal candidates. Surfactants, a kind of flexible and versatile material, may affect amyloid fibrillation of a protein due to their capacities to alter protein conformations and to induce protein denaturation.In the present study, hen egg white lysozyme (referring as lysozyme) and bovine serum albumin (BSA) have been utilized as model proteins to explore the anti-amyloid properties of three surfactants, including zeitterionic, amphiphilic and cationic detergents phosphatidylcholine (PC), sodium dodecyl sulfonate (SDS) and dodecyl trimethyl ammonium chloride (DTAC) in the terms of alternations in the secondary structures, amyloid growth dynamics, fibrillar morphology and the fibrillar toxicity to human red blood cells.Methods and results1. Kinetics of amyloid fibrillation of lysozyme and BSAThT is a cationic fluorescent dye which can bind specifically to P-sheet of amyloid fibrillation. Therefore it can be used to detect the growth situation of protein fibrillation. The results showed that the growth curve of lysozyme amyloid fibrils appeared like a sigmoidal type, suggesting a typical dynamic characteristic of amyloid formation consisting of lag phase, elongation phase and saturation phase. Lysozyme form fibrils upon incubation, along with a decrease of a-helices and an increase of β-sheet structures. In contrast, the growth curve of BSA fibrillation didn’t appear a sigmoidal shape, implicating that amyloid formation proceded through without a lag phase. An increase in β-sheet structure ha sbeen observed in the process of BSA amyloid fibrillation.2. SDS-PAGE analysis of interactions between lysozyme fibrils and cell membranesErythrocyte membranes were separated and incubated with the fibrils produced in the absence and presence of detergents. SDS-PAGE results showed that aggregation of membrane proteins could be induced by lysozyme fibrils, suggesting a disruptive role on the membranes. Fibrils prepared in the presence of detergents demonstrated different roles on the membrane. PC inhibited aggregation of membrane proteins, while SDS showed a promoting effect. In contrast, DTAC inhibited membrane protein aggregation when applied at low concentration, while it promoted protein aggregation at high doses.3. Hemolysis and aggregation of red blood cellsHuman red blood cells are used as a model to detect the fibrillar toxicity to intact cells. The results showed that he molysis was induced by the fibrils prepared either in the presence or in the absence of detergents, although a weaker hemolytic role was observed when a detergent was applied.Cell aggregation induced by the fibrils was also detected under a microscope. Teating the cells with lysozyme fibrils resulted in cell aggregation and irregular cellular shapes. The fibrils prepared in the presence of a detergent showed a relative low disruptive role on the cells, suggesting a decreased fibrillar cytoxicity.4. Morphology of lysozyme fibrils and BSA fibrilsTransmission electron microscopy (TEM) is the most commonly used technology detecting the fibrils morphology. Mature lysozyme fibrils shows reticular and long filamentous shape. In the presence of surfactants, the resultane fibrils showed different morphologies. BSA mature fibrils exhibited vermicular shapes. SDS decreased significantly the fibril formation of BSA.5. Surface hydrophobicity of BSAANS is a kind of exogenous fluorescent substance to detect the surfacial hydrophobicity of protein and protein assemblies. In the process of BSA fibrillation, the fluorescence intensity of ANS decreased and the maximum emmission wavelength kept unchanged.ConclusionLysozyme and BSA are able to form amyloid fibrils under suitable experimental conditions. Amyloid fibrillation proceded with an increase of β-sheet structures and a exposure of hydrophobic core of the protein. The deffrence of amyloid growth kinetics between lysozyme and BSA is originated from their differences in molecular weights and structures. The present study explores the effects of three surfactants, PC, SDS and DTAC on amyloid formation of lysozyme and BSA. PC showed an inhibitory role on lysozyme fibrillation at a concentration lower than its critical micellar concentration (CMC), due probably to its binding to β-sheet structure of the amyloid species. SDS promoted amyloid formation of lysozyme, but showed a strong inhibitary role on BSA fibeillation in a dose-dependent manner. The acidity of the medium may play a determinant role on amyloid formation of a protein in the presence of SDS. We suggest that SDS inhibits amyloid fibrillation of a negatively charged protein and, for a positively charged protein, accelerates fibril growth. DTAC demonstrated a weak effect on amyloid formation of lysozyme and BSA in a dose-independent manner owing to a salt effect. The fibrils prepared in the presence of three surfactants showed different toxic effects on the cells. In most cases, decreased fibrillar cytotoxicity can be observed.