The Role of Various Aggregation Conditions in Structure and Toxicity of Type B Yeast Hexokinase Aggregates

Background and Formation of well-ordered fibrillar protein deposits is common to a large group of amyloid-associated disorders, including Alzheimer’s, Parkinson’s, type II diabetes and prion diseases. The nature of the pathogenic species and the mechanism by which the aggregation process results in cell damage are, however, not well established. In the present study, the propensity of hexokinase type B from Saccharomyces cerevisiae (YHKB) to form amyloid-like and amorphous aggregates is investigated under various conditions and their cytotoxicity is determined. Methods and Materials: In this experimental study, Amyloid-like aggregation was induced under three different conventional conditions including the preincubation of YHKB (1) in acidic pH, at 55 ºC and with agitation, (2) in acidic pH, room temperature, in presence of salt and without agitation, and (3) in the presence of trifluoroethanol (TFE). Types of aggregates in above conditions were compared for their capability to react with ThT (as a amyloid marker) and their morphology was analyzed by atomic force microscopy (AFM). Also, Cytotoxicity of the aggregates on human neuroblastoma (SH-SY5Y) were assayed by MTT reduction assay test. Aggregates produced under all three conditions had ThT binding ability but with different intensity. Atomic force microscopy indicated that aggregates morphologies in various conditions were completely different. Amorphous aggregates of the enzyme were also produced for comparing with ordered aggregates. Amorphous aggregates were not found toxic to human neuroblastoma cells, as indicated by the MTT reduction assay while those formed at acidic pH and in the presence of TFE indicated cytotoxicity. Based on the findings, differences in cytotoxicity can be attributed to the variations in the nature and morphology of the aggregates in the conditions tested.