Lys(203) and Lys(382) are essential for the proteasomal degradation of BACE1.
Amyloid β protein (Aβ) is the primary component of senile plaques in Alzheimer's disease brains and its aggregate form is neurotoxic. Aβ is generated through proteolysis of β-amyloid precursor protein (APP) by two proteases: β-secretase and γ-secretase. BACE1, the β-secretase in vivo and the key rate-limiting enzyme that initiates the formation of Aβ, is an attractive drug target for AD therapy. Our previous study demonstrated that BACE1 is ubiquitinated and its degradation and effect on APP cleaving process are mediated by the ubiquitin-proteasome pathway. However, the specific underlying mechanism is still not well defined. In present study, we determined the specific binding sites responsible for the proteasomal degradation of BACE1. Ten fragments of human BACE1 cDNA with each of them containing 1 to 3 Lys codons were cloned, and HEK293 cells transfected with these recombinant plasmids were treated with specific proteasome inhibitor lactacystin. The protein levels of fragment-3 (Pro(149)-Leu(180)), -4 (IIe(179)-Ser(230)) and -8 (Met(349)-Arg(400)) were significantly increased by lactacystin treatment, and immunocytochemical staining results showed that fragment-3, -4 and -8 proteins were colocalized with ubiquitin. Site-directed mutagenesis at Lys(203) and Lys(382) of BACE1 abolished the proteasomal degradation of BACE1 and affected APP processing at β site and Aβ production. Taken together, our study demonstrated that BACE1 Lys(203) and Lys(382) are essential for its proteasomal degradation, and the results may advance our understanding of regulation of BACE1 and APP processing by the ubiquitin proteasome system in AD pathogenesis and shed new insights on its pharmaceutical potential.
School of Life Science and Technology, Beijing Institute of Technology, Haidian District, Beijing 100081, China.
Rong
R
Wang
Zhengxin
Z
Ying
Juan
J
Zhao
Yanyan
Y
Zhang
Ran
R
Wang
Hong
H
Lu
Yulin
Y
Deng
Weihong
W
Song
Hong
H
Qing
D000544:Alzheimer Disease / Q000201:enzymology*; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D003062:Codon / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D054875:Ubiquitination / Q000502:physiology*
D016229:Amyloid beta-Peptides; D003062:Codon; D018076:DNA, Complementary; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D008239:Lysine