The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain

Autor: Kuo-Chang Yin, Theresa Wood, Sang Na, Jules A. Shafer, Xiao-Ping Shi, Ming-Tain Lai, R. Bruce Register, Michael Platchek, Victor M. Garsky, Mohinder K. Sardana, Yue-Ming Li, Stephen J. Gardell, Elizabeth Chen, James Thiebeau, Mei-Jy Tang
Rok vydání: 2001
Předmět:
Protein Denaturation
Protein Folding
DNA
Complementary
Insecta
Time Factors
medicine.medical_treatment
Immunoblotting
Molecular Sequence Data
Peptide
Biochemistry
Catalysis
Cell Line
Zymogen
Endopeptidases
medicine
Amyloid precursor protein
Concanavalin A
Animals
Aspartic Acid Endopeptidases
Humans
Amino Acid Sequence
Subtilisins
Protein Precursors
Molecular Biology
Furin
Guanidine
chemistry.chemical_classification
Protease
Binding Sites
biology
Dose-Response Relationship
Drug
Chemistry
Reverse Transcriptase Polymerase Chain Reaction
Cell Biology
Precipitin Tests
Transmembrane protein
Recombinant Proteins
Protein Structure
Tertiary
N-terminus
Transmembrane domain
Kinetics
Culture Media
Conditioned
biology.protein
Electrophoresis
Polyacrylamide Gel
Amyloid Precursor Protein Secretases
Baculoviridae
Zdroj: The Journal of biological chemistry. 276(13)
ISSN: 0021-9258
Popis: beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.
Databáze: OpenAIRE
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