title: Interaction of human stefin B in the prefibrillar oligomeric form with membranes. Correlation with cellular toxicity.
abstract: Protein aggregation is central to most neurodegenerative diseases, as shown by familial case studies and by animal models. A modified 'amyloid cascade' hypothesis for Alzheimer's disease states that prefibrillar oligomers, also called amyloid-beta-derived diffusible ligands or globular oligomers, are the responsible toxic agent. It has been proposed that these oligomeric species, as shown for amyloid-beta, beta2-microglobulin or prion fragments, exert toxicity by forming pores in membranes, initiating a cascade of detrimental events for the cell. Interaction of granular aggregates and globular oligomers of an amyloidogenic protein, human stefin B, with model lipid membranes and monolayers was studied. Prefibrillar oligomers/aggregates of stefin B are shown to cause concentration-dependent membrane leaking, in contrast to the homologous stefin A. Prefibrillar oligomers/aggregates of stefin B also increase the surface pressure at an air-water interface, i.e. they have amphipathic character and are surface seeking. In addition, they show stronger interaction with 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] monolayers than native stefin A or nonaggregated stefin B. Prefibrillar aggregates interact predominantly with acidic phospholipids, such as dioleoylphosphatidylglycerol or dipalmitoylphosphatidylserine, as shown by calcein release experiments and surface plasmon resonance. The same preparations are toxic to neuroblastoma cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, again in contrast to the homologue stefin A, which does not aggregate under any of the conditions studied. This study is aimed to contribute to the general model of cellular toxicity induced by prefibrillar oligomers of amyloidogenic proteins, not necessarily involved in pathology.
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia.
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.forename: Ion
authors.initials: I
authors.lastname: Gutierrez-Aguirre
authors.forename: Sabina
authors.initials: S
authors.lastname: Rabzelj
authors.forename: Slavko
authors.initials: S
authors.lastname: Ceru
authors.forename: Natasa
authors.initials: N
authors.lastname: Kopitar-Jerala
authors.forename: Peter
authors.initials: P
authors.lastname: Macek
authors.forename: Vito
authors.initials: V
authors.lastname: Turk
authors.forename: Eva
authors.initials: E
authors.lastname: Zerovnik
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D055312:Cystatin A; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D019636:Neurodegenerative Diseases / Q000503:physiopathology; D016874:Neurofibrillary Tangles / Q000378:metabolism*; D010539:Permeability; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods; D014407:Tumor Cells, Cultured
chemical_list: C524994:CSTB protein, human; D055312:Cystatin A; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D005452:Fluoresceins; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D013778:Tetrazolium Salts; D013844:Thiazoles; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; C026407:CSTA protein, human; D055313:Cystatin B; C017251:1,2-oleoylphosphatidylcholine; C007740:fluorexon