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Dimerization of β-Site β-Amyloid Precursor Protein-cleaving Enzyme

Freie Universität Berlin, Berlín, Berlin, Germany
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2005; 279(51):53205-12. DOI: 10.1074/jbc.M410378200
Source: PubMed

ABSTRACT 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.

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    • " of two BACE1 constructs , each mutated at a single and different catalytic aspartate , can partially rescue BACE1 activity in BACE1 - / - mouse embryo fibroblasts ( Jin et al . 2010 ) . The dimeric form of BACE1 is more active in cleaving a synthetic APP substrate than monomeric soluble BACE1 lacking both the transmembrane and cytosolic domains ( Westmeyer et al . 2004 ) , suggesting that this is the optimal form of the enzyme in vivo ."
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    ABSTRACT: β-Site APP-cleaving enzyme (BACE1) cleaves the amyloid precursor protein (APP) at the β-secretase site to initiate the production of Aβ peptides. These accumulate to form toxic oligomers and the amyloid plaques associated with Alzheimer's disease (AD). An increase of BACE1 levels in the brain of AD patients has been mostly attributed to alterations of its intracellular trafficking. Golgi-associated adaptor proteins, GGA sort BACE1 for export to the endosomal compartment, which is the major cellular site of BACE1 activity. BACE1 undergoes recycling between endosome, trans-Golgi network (TGN), and the plasma membrane, from where it is endocytosed and either further recycled or retrieved to the endosome. Phosphorylation of Ser498 facilitates BACE1 recognition by GGA1 for retrieval to the endosome. Ubiquitination of BACE1 C-terminal Lys501 signals GGA3 for exporting BACE1 to the lysosome for degradation. In addition, the retromer mediates the retrograde transport of BACE1 from endosome to TGN. Decreased levels of GGA proteins and increased levels of retromer-associated sortilin have been associated with AD. Both would promote the co-localization of BACE1 and the amyloid precursor protein in the TGN and endosomes. Decreased levels of GGA3 also impair BACE1 degradation. Further understanding of BACE1 trafficking and its regulation may offer new therapeutic approaches for the treatment of Alzheimer's disease.
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    • "Prior to pro-peptide cleavage and subsequent maturation, BACE1 is processed between Leu228 and Ala229 to generate stable N-and Cterminal fragments which remain covalently associated via a disulfide bond (Huse et al. 2003). This dimerization may facilitate binding and cleavage of BACE1 substrates (Schmechel et al. 2004; Westmeyer et al. 2004). The b-secretase activity of BACE1 is dependent on the extent of N-glycosylation at Asn -153, -172, -223 and -354 (Capell et al. 2000; Haniu et al. 2000; Huse and Doms 2000; Charlwood et al. 2001), and mature N-glycosylated moieties Fig. 1 Generation of Ab from APP by cleavage of b and c-secretase. "
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    • "Upon trafficking through the secretory pathway, the propeptide is cleaved by furin, leading to the active (mature) BACE1 protease (Bennett et al. 2000; Capell et al. 2000; Huse et al. 2000; Creemers et al. 2001). BACE1 forms dimers, which show a higher activity than the monomers (Schmechel et al. 2004; Westmeyer et al. 2004). BACE1 is ubiquitously expressed, with higher expression levels observed in brain and in neurons (Vassar et al. 1999), which explains why neurons are particularly vulnerable in AD because of the increased BACE1-mediated Ab generation . "
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