Dimerization of -Site -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


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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    • "However, the physiological significance of this BACE1 shedding remains to be established. In addition, BACE1 appears to exist as a homodimer in the native state (Westmeyer et al. 2004; Schmechel et al. 2004). We are yet to determine whether BACE1 shedding and dimerization are affected by its palmitoylation. "
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    ABSTRACT: β-Secretase, BACE1 is a neuron-specific membrane-associated protease that cleaves amyloid precursor protein (APP) to generate β-amyloid protein (Aβ). BACE1 is partially localized in lipid rafts. We investigated whether lipid raft localization of BACE1 affects Aβ production in neurons using a palmitoylation-deficient mutant and further analyzed the relationship between palmitoylation of BACE1 and its shedding and dimerization. We initially confirmed that BACE1 is mainly palmitoylated at four C-terminal cysteine residues in stably transfected neuroblastoma cells. We found that raft localization of mutant BACE1 lacking the palmitoylation modification was markedly reduced in comparison to wild-type BACE1 in neuroblastoma cells as well as rat primary cortical neurons expressing BACE1 via recombinant adenoviruses. In primary neurons, expression of wild-type and mutant BACE1 enhanced production of Aβ from endogenous or overexpressed APP to similar extents with the β-C-terminal fragment (β-CTF) of APP mainly distributed in nonraft fractions. Similarly, β-CTF was recovered mainly in nonraft fractions of neurons expressing Swedish mutant APP only. These results show that raft association of BACE1 does not influence β-cleavage of APP and Aβ production in neurons, and support the view that BACE1 cleaves APP mainly in nonraft domains. Thus, we propose a model of neuronal Aβ generation involving mobilization of β-CTF from nonraft to raft domains. Additionally, we obtained data indicating that palmitoylation plays a role in BACE1 shedding but not dimerization.
    Full-text · Article · May 2012 · Brain and Behavior
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    • "The following plasmids were used for transfections: pCMV2-HA-NRG1FL, pCMV2-HA-NRG1GIEF, and pcDNA3.1-BACE1 (Westmeyer et al.,2004). Protease inhibitor β-secretase inhibitor IV (2 μM; Calbiochem) was added 2 h after transfection. "
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    ABSTRACT: Myelin sheath thickness is precisely adjusted to axon caliber, and in the peripheral nervous system, neuregulin 1 (NRG1) type III is a key regulator of this process. It has been proposed that the protease BACE1 activates NRG1 dependent myelination. Here, we characterize the predicted product of BACE1-mediated NRG1 type III processing in transgenic mice. Neuronal overexpression of a NRG1 type III-variant, designed to mimic prior cleavage in the juxtamembrane stalk region, induces hypermyelination in vivo and is sufficient to restore myelination of NRG1 type III-deficient neurons. This observation implies that the NRG1 cytoplasmic domain is dispensable and that processed NRG1 type III is sufficient for all steps of myelination. Surprisingly, transgenic neuronal overexpression of full-length NRG1 type III promotes hypermyelination also in BACE1 null mutant mice. Moreover, NRG1 processing is impaired but not abolished in BACE1 null mutants. Thus, BACE1 is not essential for the activation of NRG1 type III to promote myelination. Taken together, these findings suggest that multiple neuronal proteases collectively regulate NRG1 processing.
    Full-text · Article · Feb 2012 · Glia
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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: J. Neurochem. (2012) 120, 869–880. 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.
    Full-text · Article · Dec 2011 · Journal of Neurochemistry
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