title: Binding epitopes and interaction structure of the neuroprotective protease inhibitor cystatin C with beta-amyloid revealed by proteolytic excision mass spectrometry and molecular docking simulation.
abstract: Human cystatin C (HCC) is a protease inhibitor with a propensity to form beta-amyloid (Abeta)-like fibrils and to coassociate with amyloidogenic proteins. Recently, a specific interaction between HCC and Abeta has been found. Here, we report the identification of the Abeta and HCC binding epitopes in the Abeta-HCC complex, using a combination of selective proteolytic excision and high resolution mass spectrometry. Proteolytic excision of Abeta(1-40) on sepharose-immobilized HCC and MALDI-MS identified the epitope Abeta(17-28). On immobilized Abeta(1-40), affinity MS of HCC fragments identified a specific C-terminal epitope, HCC(101-117). Binding specificities of both epitopes were ascertained by ELISA and surface plasmon resonance and by direct electrospray MS of the HCC-Abeta epitope peptide complexes. A structure model of the HCC-Abeta complex by molecular docking simulation showed full agreement with the identified Abeta and HCC epitopes. Inhibition studies in vitro revealed Abeta-fibril inhibiting activity of the HCC(101-117)-epitope. The Abeta-HCC interacting epitopes provide lead structures of neuroprotective inhibitors for AD and HCC amyloidosis therapy.
authors.affiliation: Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany.
authors.forename: Paulina
authors.initials: P
authors.lastname: Juszczyk
authors.forename: Gabriela
authors.initials: G
authors.lastname: Paraschiv
authors.forename: Aneta
authors.initials: A
authors.lastname: Szymanska
authors.forename: Aneta S
authors.initials: AS
authors.lastname: Kolodziejczyk
authors.forename: Sylwia
authors.initials: S
authors.lastname: Rodziewicz-Motowidlo
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Grzonka
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski