Neuronal β‐amyloid generation is independent of lipid raft association of β‐secretase BACE1: analysis with a palmitoylation‐deficient mutant

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.

Palmitoylation in Alzheimer⿿s disease and other neurodegenerative diseases

“…While these controversial findings on the role of BACE1 palmitoylation in A production were reported, two surprising reports were made on no influence of BACE1 palmitoylation on A production [151,155]. Palmitoylation-deficient BACE1 whose 4 cysteines (C474, C478, C482, and C485) were substituted by alanine residues (BACE1-CA4) exhibited non-raft localization and did not affected the BACE1 processing of APP or secretion of Aβ, indicating that palmitoylation of BACE1 is not required for APP processing and also even lipid rafts localization of BACE1 is not important for its cleavage activity of APP [151,155]. So far, the contribution of BACE1 palmitoylation to Aβ production and AD is still unclear, and it needs further investigation.…”

Progress toward Understanding Protein S-acylation: Prospective in Plants

“…Although, the direct mechanism of S-acylation on transmembrane proteins is not very clear it is thought that it plays multiple roles in altering signaling capacity (Merrick et al, 2011), reducing activity (Huang et al, 2010), trafficking modification (Abrami et al, 2008; Flannery et al, 2010) and changing stability of these proteins (Abrami et al, 2006; Maeda et al, 2010; Blaskovic et al, 2013). For example, S-acylation of transmembrane proteins, such as death receptor 4 (Oh et al, 2012), β-secretase BACE1 (Motoki et al, 2012), cannabinoid receptor (Oddi et al, 2012) and influenza virus M2 protein (Thaa et al, 2011), can promote their association with membrane lipid rafts. However, for some peripheral membrane proteins such as transferrin receptor and caveolin, their palmitoylation sites are localized to non-raft domains, therefore palmitoylation is not necessary for their raft localization (Alvarez et al, 1990; Dietzen et al, 1995; Charollais and Van Der Goot, 2009).…”

“…These proteins include the Arabidopsis α and γ subunits of heterotrimeric G protein (Adjobo-Hermans et al, 2006; Zeng et al, 2007), small GTPases (Deschenes et al, 1990; Bartels et al, 1999; Roth et al, 2006; Zhang et al, 2013); 3 for transmembrane proteins, the Cys residues are often situated in the cytoplasmic regions of membrane-spanning regions (Roth et al, 2006; Ohno et al, 2012). For instance, the S-acylation of C261-263 triplet in death receptor 4 (DR4) (Rossin et al, 2009) and C474 in β-secretase BACE1 (Motoki et al, 2012) promotes their association with lipid raft.…”

Can BACE1 Inhibition Mitigate Early Axonal Pathology in Neurological Diseases?

“…BACE1 may exist as a zymogen as well as various immature and mature forms that are enzymatically active [24, 27, 28]. BACE1 may undergo extensive posttranscriptional modulations, including glycosylation, acetylation, and palmitoylation and phosphorylation in Golgi apparatus and endosomes [23, 28-31], and may be trafficked into lipid rafts or non-raft membranous compartments and degraded in the lysosomal components [28, 31]. …”