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Mechanism of action of mammalian cystatins

studies of inhibition of cysteine endo- and exopeptidases by cystatins A and C

Pavlova, Alona (2003). Mechanism of action of mammalian cystatins. Diss. (sammanfattning/summary) Uppsala : Sveriges lantbruksuniv., Acta Universitatis agriculturae Sueciae. Veterinaria, 1401-6257 ; 154
ISBN 91-576-6379-3
[Doctoral thesis]

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Abstract

Mammalian cystatins are tight-binding, reversible inhibitors of potentially destructive cysteine proteases, both endogenous and exogenous. The N-terminal region residues, Ile2 and especially Pro3, and the second binding loop residues, Leu73 and to some extent Pro74, of the family 1 cystatin, cystatin A, were shown to be essential for effective inhibition of papain and cathepsins L and B. Both regions were found to act in endopeptidase inhibition primarily by anchoring the enzymes in long-lived complexes with the inhibitor. However, in inhibition of the exopeptidase, cathepsin B, the N-terminal region also contributes to the high association rate. As previously shown for cystatin C, stopped-flow kinetics demonstrated a two-step mechanism of cystatin A binding to cathepsin B, with a conformational change in the second, rate-limiting step. In contrast, binding of cystatins A and C to a cathepsin B variant with an increased mobility of the occluding loop that blocks the active-site cleft occurred in an apparent one-step reaction. The second step of cathepsin B binding by cystatins was therefore concluded to involve displacement of the occluding loop. Cystatin C was shown to be a much better displacer of this loop than cystatin A. New effective and specific inhibitors of cysteine proteases were engineered by grafting individual binding regions of more potent cystatins into cystatin A. Introduction of motifs from the second binding loops of cystatins B or C into the corresponding loop of cystatin A increased the affinity for papain by ~10-fold. Moreover, replacement of the N-terminal region of cystatin A by that of cystatin C resulted in the affinity for cathepsin B being increased ~15-fold, to a level higher than that of any natural cystatin known. The latter chimeric inhibitor was as effective as cystatin C in displacing the occluding loop of cathepsin B, the rate of this step being increased by ~100-fold. The N-terminal region of a cystatin was thus concluded to promote occluding loop displacement by appropriately positioning the binding loops of the inhibitor entering the active-site cleft of cathepsin B.

Authors/Creators:Pavlova, Alona
Title:Mechanism of action of mammalian cystatins
Subtitle:studies of inhibition of cysteine endo- and exopeptidases by cystatins A and C
Year of publishing :May 2003
Volume:154
Number of Pages:64
Papers/manuscripts:
NumberReferences
ALLI. Estrada, S., Pavlova, A., and Björk, I. (1999) The contribution of N-terminal region residues of cystatin A (stefin A) to the affinity and kinetics of inhibition of papain, cathepsin B, and cathepsin L. Biochemistry 38, 7339–7345. II. Pavlova, A., Estrada, S. and Björk, I. (2002) The role of the second binding loop of the cysteine protease inhibitor, cystatin A (stefin A), in stabilizing complexes with target proteases is exerted predominantly by Leu73. Eur. J. Biochem. 269, 5649–5658. III. Pavlova, A., Krupa, J. C., Mort, J. S., Abrahamson, M., Björk, I. (2000) Cystatin inhibition of cathepsin B requires dislocation of the proteinase occluding loop. Demonstration by release of loop anchoring through mutation of His110. FEBS Letters 487, 156–160. IV. Pavlova, A. and Björk, I. (2003) Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases. Manuscript.
Place of Publication:Uppsala
ISBN for printed version:91-576-6379-3
ISSN:1401-6257
Language:English
Publication Type:Doctoral thesis
Full Text Status:Public
Agris subject categories.:X Agricola extesions > X30 Life sciences
Subjects:Not in use, please see Agris categories
Agrovoc terms:molecular biology, inhibition, enzyme inhibitors
Keywords:cysteine protease, papain, cathepsin, inhibition, cystatin, affinity, binding kinetics, two-step reaction mechanism, occluding loop, binding loop
URN:NBN:urn:nbn:se:slu:epsilon-57
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-57
ID Code:300
Department:(VH) > Dept. of Molecular Biosciences (until 070101)
Deposited By: Staff Epsilon
Deposited On:03 Jun 2003 00:00
Metadata Last Modified:02 Dec 2014 10:03

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