Binding Epitopes and Interaction Structure of the Neuroprotective Protease Inhibitor Cystatin C with β-Amyloid Revealed by Proteolytic Excision Mass Spectrometry and Molecular Docking Simulation

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• 作者：Paulina Juszczyk ; Gabriela Paraschiv ; Aneta Szymanska ; Aneta S. Kolodziejczyk ; Sylwia Rodziewicz-Motowidlo ; Zbigniew Grzonka ; Michael Przybylski
• 刊名：Journal of Medicinal Chemistry
• 出版年：2009
• 出版时间：April 23, 2009
• 年：2009
• 卷：52
• 期：8
• 页码：2420-2428
• 全文大小：214K
• 年卷期：v.52,no.8(April 23, 2009)
• ISSN：1520-4804

文摘

Human cystatin C (HCC) is a protease inhibitor with a propensity to form β-amyloid (Aβ)-like fibrils and to coassociate with amyloidogenic proteins. Recently, a specific interaction between HCC and Aβ has been found. Here, we report the identification of the Aβ and HCC binding epitopes in the Aβ−HCC complex, using a combination of selective proteolytic excision and high resolution mass spectrometry. Proteolytic excision of Aβ(1−40) on sepharose-immobilized HCC and MALDI-MS identified the epitope Aβ(17−28). On immobilized Aβ(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−Aβ epitope peptide complexes. A structure model of the HCC−Aβ complex by molecular docking simulation showed full agreement with the identified Aβ and HCC epitopes. Inhibition studies in vitro revealed Aβ-fibril inhibiting activity of the HCC(101−117)-epitope. The Aβ−HCC interacting epitopes provide lead structures of neuroprotective inhibitors for AD and HCC amyloidosis therapy.