Essential Role of Proline Isomerization in Stefin B Tetramer Formation

Limited Proteolysis Reveals That Amyloids from the 3D Domain-Swapping Cystatin B Have a Non-Native β-Sheet Topology

“…Cystatin tetramers have been identified for cystatin B [32] and chicken cystatin [33] where this 20 amino acid strand is maintained. This additional oligomerisation occurs through "loop II" between strands 3 and 4 of molecules (Fig 1c).…”

“…The cystatin B proteinase binding motif is composed primarily of a constrained loop linking β-strand 2 and 3 [40], which is inactivated by the straightening and extension of the β-strands in the 3D domain swapped dimer and tetramer [25,32] and is also a feature observed within the fibril [27]. The cystatin B monomer is therefore the only protein isoform capable of inhibition in vitro and typically constitutes 10% of the protein population with the remaining protein being dimer and <5% is tetramer.…”

Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation

“…Stefins are intracellular proteins present in the cytosol [14,15], including stefins A and B in humans [16] and stefins, A, B and C in bovidae [17,18]. Human stefin B [19,20,21,22,23], chimeric stefins [24] and cystatin C [25,26] have been used as suitable model proteins to study protein folding and amyloid fibril formation. Human stefin B is a small globular protein consisting of 98 amino acids with no disulfide bonds; its native sequence possesses a free Cys residue at position 3.…”

Human Stefin B Role in Cell's Response to Misfolded Proteins and Autophagy

“…Domain-swapped dimers are believed to constitute the building blocks for fibril formation in vitro [11]. Accordingly, tetramers are formed by association of two domain-swapped dimers [12]. [13] [10], [12]Isolated higher-order oligomers bind to lipid monolayers effectively and are internalized through the plasma membrane via endocytosis, which results in decreased cell viability [8], [9], [10], [14].…”

“…Of interest, stefin B interacted with Aβ in oligomer dependent way, namely, stefin B tetramers and a dimeric Y31 variant exhibited complete inhibition of amyloid formation by Aβ in vitro [13], [18]. Using electron-spray ionization mass spectrometry (ESI MS), it was shown that Aβ and stefin B Y31 variant dimer bind in a ratio of 1∶2, which means that one molecule of Aβ binds to a stefin B domain-swapped dimer and two molecules of Aβ to a stefin B tetramer [12] composed of two such dimers. Both cystatins might thus be neuroprotective by an amateur chaperone action.…”