Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity?
In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57 degrees C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease.
Malisauskas
Mantas
M
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå 90187, Sweden.
Ostman
Johan
J
Darinskas
Adas
A
Zamotin
Vladimir
V
Liutkevicius
Evaldas
E
Lundgren
Erik
E
Morozova-Roche
Ludmilla A
LA
D000682:Amyloid / Q000378:metabolism*; D000686:Amyloidosis / Q000209:etiology / Q000473:pathology; D000818:Animals; D016923:Cell Death; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D019281:Dimerization; D005347:Fibroblasts / Q000473:pathology; D006736:Horses; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008807:Mice, Inbred BALB C; D018625:Microscopy, Atomic Force; D009113:Muramidase / Q000378:metabolism*; D009447:Neuroblastoma / Q000473:pathology; D009474:Neurons / Q000473:pathology*
D000682:Amyloid; D009113:Muramidase