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
, Robert Vidmar
, Matej Vizovišek , Marko Fonović , Nataša Kopitar-Jerala and Eva Žerovnik
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.
We thank prof. R.H. Pain (IJS) for some final corrections in English usage.
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Articles in the same Issue
- Masthead
- Masthead
- Reviews
- The unique activity of bone morphogenetic proteins in bone: a critical role of the Smad signaling pathway
- The formate/nitrite transporter family of anion channels
- The human Ah receptor: hints from dioxin toxicities to deregulated target genes and physiological functions
- Heparan sulfate: a key regulator of embryonic stem cell fate
- Research Articles/Short Communications
- Genes and Nucleic Acids
- Expression and translation of the COX-1b gene in human cells – no evidence of generation of COX-1b protein
- Protein Structure and Function
- Evaluation of the metal binding sites in a recombinant coagulation factor VIII identifies two sites with unique metal binding properties
- Hydrolysis of dipeptide derivatives reveals the diversity in the M49 family
- Molecular Medicine
- Betulinic acid suppresses NGAL-induced epithelial-to-mesenchymal transition in melanoma
- Proteolysis
- 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
- The Staphylococcus aureus leucine aminopeptidase is localized to the bacterial cytosol and demonstrates a broad substrate range that extends beyond leucine
- Book Review
- Extracellular Matrix: Pathobiology and Signaling