title: Characteristics of C-terminal, β-amyloid peptide binding fragment of neuroprotective protease inhibitor, cystatin C.
abstract: Cystatin C originally identified as a cysteine proteases inhibitor has a broad spectrum of biological roles ranging from inhibition of extracellular cysteine protease activities, bone resorption, and modulation of inflammatory responses to stimulation of fibroblasts proliferation. There is an increasing number of evidence to suggest that human cystatin C (hCC) might play a protective role in the pathophysiology of sporadic Alzheimer's disease. In vivo and in vitro results well documented the association of hCC with Aβ and the hCC-induced inhibition of Aβ fibril formation. In our earlier work, using a combination of selective proteolytic methods and MS spectroscopy, C-terminal fragment hCC(101-117) was identified as the Aβ-binding region. The fragment of Aβ peptide responsible for the complex formation with hCC was found in the middle, highly hydrophobic part, Aβ(17-24). Structures and affinities of both Aβ and hCC binding sites were characterized by the enzyme-linked immunosorbent assay-like assay, by surface plasmon resonance, and by nano-ESI-FTICR MS of the hCC-Aβ-binding peptide complexes. In the in vitro inhibition studies, the binding cystatin sequence, hCC(101-117), revealed the highest relative inhibitory effect toward Aβ-fibril formation. Herein, we present further studies on molecular details of the hCC-Aβ complex. With Ala substitution, affinity experiments, and enzyme-linked immunosorbent assay-like assays for the Aβ-binding fragment, hCC(101-117), and its variants, the importance of individual amino acid residues for the protein interaction was evaluated. The results were analyzed using hCC(101-117) nuclear magnetic resonance structural data with molecular dynamics calculations and molecular modeling of the complexes. The results point to conformational requirements and special importance of some amino acid residues for the protein interaction. The obtained results might be helpful for the design of low molecular compounds modulating the biological role of both proteins. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Faculty of Chemistry, Department of Biomedical Chemistry, University of Gdansk, Gdansk, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Spodzieja
authors.affiliation: Faculty of Chemistry, Department of Biomedical Chemistry, University of Gdansk, Gdansk, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kalejta
authors.affiliation: Faculty of Chemistry, Department of Biomedical Chemistry, University of Gdansk, Gdansk, Poland.
authors.forename: Aleksandra S
authors.initials: AS
authors.lastname: Kołodziejczyk
authors.affiliation: Faculty of Chemistry, Department of Biomedical Chemistry, University of Gdansk, Gdansk, Poland.
authors.forename: Martyna
authors.initials: M
authors.lastname: Maszota-Zieleniak
authors.affiliation: Faculty of Chemistry, Department of Biomedical Chemistry, University of Gdansk, Gdansk, Poland.
authors.forename: Sylwia
authors.initials: S
authors.lastname: Rodziewicz-Motowidło
authors.affiliation: Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland.
authors.forename: Wioletta
authors.initials: W
authors.lastname: Żmudzińska
authors.affiliation: Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland.
authors.forename: Paulina
authors.initials: P
authors.lastname: Czaplewska
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D055316:Cystatin C / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D011485:Protein Binding; D059748:Proteolysis
chemical_list: D016229:Amyloid beta-Peptides; C525030:CST3 protein, human; D055316:Cystatin C
keywords: Alzheimer's disease; Aβ; amyloid; cerebral amyloidosis; cystatin C; neurodegeneration