Biochemical characterization and structural modeling of human cathepsin E variant 2 in comparison to the wild-type protein.
Biol Chem
; 393(3): 177-86, 2012 Mar.
Article
en En
| MEDLINE
| ID: mdl-22718633
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 ß-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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Catepsina E
Límite:
Humans
Idioma:
En
Revista:
Biol Chem
Asunto de la revista:
BIOQUIMICA
Año:
2012
Tipo del documento:
Article
País de afiliación:
Eslovenia