Biochemical characterization and structural modeling of human cathepsin E variant 2 in comparison to the wild-type protein
-
Vida Puizdar
and
Vito Turk
Abstract
Cathepsin E splice variant 2 appears in a number of gastric carcinomas. Here we report detecting this variant in HeLa cells using polyclonal antibodies and biotinylated inhibitor pepstatin A. An overexpression of GFP fusion proteins of cathepsin E and its splice variant within HEK-293T cells was performed to show their localization. Their distribution under a fluorescence microscope showed that they are colocalized. We also expressed variants 1 and 2 of cathepsins E, with propeptide and without it, in Escherichia coli. After refolding from the inclusion bodies, the enzymatic activity and circular dichroism spectra of the splice variant 2 were compared to those of the wild-type mature active cathepsins E. While full-length cathepsin E variant 1 is activated at acid pH, the splice variant remains inactive. In contrast to the active cathepsin E, the splice variant 2 predominantly assumes β-sheet structure, prone to oligomerization, at least under in vitro conditions, as shown by atomic force microscopy as shallow disk-like particles. A comparative structure model of splice variant 2 was computed based on its alignment to the known structure of cathepsin E intermediate (Protein Data Bank code 1TZS) and used to rationalize its conformational properties and loss of activity.
©2012 by Walter de Gruyter Berlin Boston
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Articles in the same Issue
- Genes and Nucleic Acids
- The effect of cancer procoagulant on expression of metastatic and angiogenic markers in breast cancer and embryonic stem cell lines
- Characterization of RNA damage under oxidative stress in Escherichia coli
- Membranes, Lipids, Glycobiology
- Uncommon membrane distribution of Shiga toxin glycosphingolipid receptors in toxin-sensitive human glomerular microvascular endothelial cells
- Cell Biology and Signaling
- Nucleocytoplasmic shuttling of human inositol phosphate multikinase is influenced by CK2 phosphorylation
- Apolipoprotein A5 internalized by human adipocytes modulates cellular triglyceride content
- Mammalian carboxylesterase (CES) releases GPI-anchored proteins from the cell surface upon lipid raft fluidization
- Proteolysis
- Biochemical characterization and structural modeling of human cathepsin E variant 2 in comparison to the wild-type protein
- Novel Techniques
- A fluorescence correlation spectroscopy-based enzyme assay for human Dicer
- Development of a host blood meal database: de novo sequencing of hemoglobin from nine small mammals using mass spectrometry
- Prelims
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