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 |
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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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