Interaction between Oligomers of Stefin B and Amyloid-β in Vitro and in Cells

Abstract

To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-␤-(1-40) peptide (A␤). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to A␤ is oligomer specific. The dimers and tetramers of stefin B, which bind A␤, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit A␤ fibril formation. When expressed in cultured cells, stefin B co-localizes with A␤ intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the A␤ epitope. Thus, stefin B is another APP/A␤-binding protein in vitro and likely in cells.Neurodegenerative diseases present a huge burden in the developed world's aging population. They are all in one way or another connected to aberrant protein folding and aggregation of the proteins involved (1). Various protein conformational disorders of the central and peripheral nervous system are known, which often appear sporadically but also run in families. These are among others: Parkinson and Alzheimer diseases, dementia with Lewy bodies, vascular and fronto-temporal dementia, and amyotrophic lateral sclerosis.The A␤ peptide implicated in Alzheimer disease pathology is a cleavage product of the membrane A␤ precursor protein (APP).3 It is the main constituent of extracellular amyloid plaques, however, together with its oligomers, it also resides intracellularly (2). It has been shown that A␤ oligomers prepared in vitro and those extracted from living cells exert cytotoxicity and cause symptoms of reversible memory loss in animal models (3). Amyloid protein oligomers have special structural properties, which are reflected in a common antioligomer antibody (4). This antibody not only binds the oligomers against which it was raised but also binds chaperones and some other proteins involved in disaggregating protein aggregates in cells (5). A␤-binding proteins, the so called "amateur chaperones," were suggested to have a potential in Alzheimer disease therapy (6, 7).It has been shown before that human cystatin C is an A␤-binding protein (8). Cystatins are single chain proteins that inhibit cysteine cathepsins (9). Human stefin B (also known as cystatin B) is a member of subfamily A of cystatins, classified as family I25 in the MEROPS scheme (10). Stefin B, a protein of 98 amino acid residues and 1 Cys, is predominantly intracellular, whereas cystatin C, a protein of 120 residues and 2 disulfide bonds, is a secretory protein. Three-dimensional structures of stefins and cystatin C have been determined, among others, the solution structure of stefin A (11) and cystatin C (12, 13).Human cystatin C has been found as a constituent of senile plaques of Alzheimer disease patients (14) and stefins A and B have also been reported to localize to a...

Structure and Function of Stefin B Oligomers – Important Role in Amyloidogenesis

“…ThT fluorescence and transmission electron microscopy (TEM) have shown that Y31 isoform completely inhibits Aβ peptide fibril formation ( Figure 9I). The direct interaction between those two proteins has also been shown by SPR measurements, where concentration dependent interaction has been reported and by ESI MS where the complex between dimer stB and monomer Aβ peptide has been detected ( Figure 9II) [38]. Furthermore, isolated oligomers of the wild type protein have been studied and it was shown that only the tetramer inhibits Aβ peptide fibril formation and that the higher oligomers show only a weak inhibition.…”

“…It exhibits nearly all features shared with other amyloid forming proteins: it forms mature fibrils under mildly acidic conditions or even at neutral pH at somewhat higher temperature, forms membrane pores and therefore promotes membrane leaking, binds copper ions and its' oligomers are toxic. It is not a model protein only, but also could be termed an "amateur chaperone" affecting Aβ peptide fibril formation both in vitro and in vivo [38].…”

“…Cysteine protease inhibitors reduce both Aβ peptide level in the brain and β-secretase activity in vivo [129]. Stefin B has an effect on production of Aβ peptides and furthermore it also inhibits Aβ peptide fibril formation [38].…”

“…Molecule/protein Type of interaction stefin B Inhibits Aβ peptide fibril formation, interaction in vitro and in cells [38].…”

“…Y31 isoform is predominantly dimeric [31], while Y31 P79S mutant is tetrameric [40]. All oligomers can be isolated as separate peaks by SEC and stay stable for weeks at pH 7.0 and 4 °C [38].…”