Dimerization of beta-site beta-amyloid precursor protein-cleaving enzyme.
Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.
Adolf Butenandt Institute, Department of Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease Research, Schillerstrasse 44, Ludwig Maximilians University, 80336 Munich, Germany.
Gil G
GG
Westmeyer
Michael
M
Willem
Stefan F
SF
Lichtenthaler
Glenn
G
Lurman
Gerd
G
Multhaup
Irmgard
I
Assfalg-Machleidt
Karina
K
Reiss
Paul
P
Saftig
Christian
C
Haass
D020816:Amino Acid Motifs; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000502:physiology*; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014162:Transfection; D019076:Transgenes
D000939:Epitopes; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse