Home Cystatins as Calpain Inhibitors: Engineered Chicken Cystatin- and Stefin B-Kininogen Domain 2 Hybrids Support a Cystatin-Like Mode of Interaction with the Catalytic Subunit of µ-Calpain
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Cystatins as Calpain Inhibitors: Engineered Chicken Cystatin- and Stefin B-Kininogen Domain 2 Hybrids Support a Cystatin-Like Mode of Interaction with the Catalytic Subunit of µ-Calpain

  • Beatriz García Díaz , Stefan Gross , Irmgard Assfalg-Machleidt , Dietmar Pfeiler , Nicole Gollmitzer , Dusica Gabrijelcic-Geiger , Milton T. Stubbs , Hans Fritz , Ennes A. Auerswald and Werner Machleidt
Published/Copyright: July 5, 2005
Biological Chemistry
From the journal Volume 382 Issue 1

Abstract

Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit µ- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and ?L110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefinB by the corresponding KD2 sequence resulted in a calpain inhibitor of KI = 188 nM. Deletion of L110, which forms a ?-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of µ-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substratelike cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatintype inhibitors do not bind to the calmodulinlike domain of the small subunit of calpain, and their interaction with the µ-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatinlike mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.

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Published Online: 2005-07-05
Published in Print: 2001-01-06

Copyright © 2001 by Walter de Gruyter GmbH & Co. KG

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  15. Altered Neutrophil Homeostasis in Kinin B1 Receptor-Deficient Mice
  16. Cystatins as Calpain Inhibitors: Engineered Chicken Cystatin- and Stefin B-Kininogen Domain 2 Hybrids Support a Cystatin-Like Mode of Interaction with the Catalytic Subunit of µ-Calpain
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