Home Life Sciences Influence of partial unfolding and aggregation of human stefin B (cystatin B) EPM1 mutants G50E and Q71P on selective cleavages by cathepsins B and S
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Influence of partial unfolding and aggregation of human stefin B (cystatin B) EPM1 mutants G50E and Q71P on selective cleavages by cathepsins B and S

  • Mira Polajnar EMAIL logo , Robert Vidmar , Matej Vizovišek , Marko Fonović , Nataša Kopitar-Jerala and Eva Žerovnik
Published/Copyright: January 28, 2013

Abstract

Human stefins and cystatins are physiologically important cysteine proteinase inhibitors, acting as a first line of defense against undesirable proteolysis. Mutations in the cystatin B gene cause a rare form of epilepsy EPM1. Its two missense mutants, G50E and Q71P, lack the inhibitory activity and are partially unfolded, which leads to changes in their aggregation behavior, both in vitro and in the cell. SDS-PAGE and MALDI-TOF mass spectrometry were used to follow the hydrolysis of human stefin B wild type, G50E and Q71P, by cathepsins B and S in vitro. Cathepsin S was found to degrade both mutants, with Q71P being degraded faster. This correlates with the openness of the protein structure, Q71P having more exposed hydrophobic surfaces. Cathepsin B acted more selectively, degrading G50E into smaller fragments, while still leaving a portion of the full-length protein intact. Q71P was cleaved only at the exposed N-terminal end. The co-localization of stefin B wild type and EPM1 mutants with cathepsins showed that cathepsins accumulate around the aggregates formed by the EPM1 mutants. We hypothesize that the aggregation of both full-length mutants prevents the cathepsin molecule from accessing the substrate protein’s core, whereas the cleaved fragments would be expected to aggregate stronger.


Corresponding author: Mira Polajnar, Jožef Stefan Institute, Department of Biochemistry, Molecular and Structural Biology, Jamova 39, 1000 Ljubljana, Slovenia

We thank prof. R.H. Pain (IJS) for some final corrections in English usage.

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Received: 2012-8-30
Accepted: 2013-1-20
Published Online: 2013-01-28
Published in Print: 2013-06-01

©2013 by Walter de Gruyter Berlin Boston

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