Palmitoylated APP Forms Dimers, Cleaved by BACE1

Abstract

A major rate-limiting step for Aβ generation and deposition in Alzheimer’s disease brains is BACE1-mediated cleavage (β-cleavage) of the amyloid precursor protein (APP). We previously reported that APP undergoes palmitoylation at two cysteine residues (Cys186 and Cys187) in the E1-ectodomain. 8–10% of total APP is palmitoylated in vitro and in vivo. Palmitoylated APP (palAPP) shows greater preference for β-cleavage than total APP in detergent resistant lipid rafts. Protein palmitoylation is known to promote protein dimerization. Since dimerization of APP at its E1-ectodomain results in elevated BACE1-mediated cleavage of APP, we have now investigated whether palmitoylation of APP affects its dimerization and whether this leads to elevated β-cleavage of the protein. Here we report that over 90% of palAPP is dimerized while only ~20% of total APP forms dimers. PalAPP-dimers are predominantly cis-oriented while total APP dimerizes in both cis- and trans-orientation. PalAPP forms dimers 4.5-times more efficiently than total APP. Overexpression of the palmitoylating enzymes DHHC7 and DHHC21 that increase palAPP levels and Aβ release, also increased APP dimerization in cells. Conversely, inhibition of APP palmitoylation by pharmacological inhibitors reduced APP-dimerization in coimmunoprecipitation and FLIM/FRET assays. Finally, in vitro BACE1-activity assays demonstrate that palmitoylation-dependent dimerization of APP promotes β-cleavage of APP in lipid-rich detergent resistant cell membranes (DRMs), when compared to total APP. Most importantly, generation of sAPPβ-sAPPβ dimers is dependent on APP-palmitoylation while total sAPPβ generation is not. Since BACE1 shows preference for palAPP dimers over total APP, palAPP dimers may serve as novel targets for effective β-cleavage inhibitors of APP as opposed to BACE1 inhibitors.

Insights Into Protein S-Palmitoylation in Synaptic Plasticity and Neurological Disorders: Potential and Limitations of Methods for Detection and Analysis

“…This process involves several enzymes, including BACE1 ( β -site A PP c leaving e nzyme) and β- and γ-secretases. Bhattacharyya et al (2013 , 2016 ) reported that two N-terminal cysteine residues of APP (Cys186 and Cys187) undergo S-PALM. S-PALM APP is enriched in membrane lipid rafts and forms dimers, leading to elevations of BACE1-mediated cleavage of the protein ( Bhattacharyya et al, 2013 ).…”

“…The PULSE-CHASE labeling method has been successfully applied for decades. This method allows determination of the half-lives of the S-PALM of various proteins, including ankyrin ( Staufenbiel, 1987 ), Gαi ( Degtyarev et al, 1994 ), α2A-adrenergic receptors ( Kennedy and Limbird, 1994 ), HTT ( Sanders and Hayden, 2015 ), BACE1 ( Bhattacharyya et al, 2016 ), H-Ras ( Baker et al, 2003 ), and N-Ras ( Magee et al, 1987 ). In this approach, cells are exposed to radioactively labeled palmitate for a short period of time (i.e., the PULSE phase), followed by a phase in which cells are incubated with unlabeled (non-radioactive) palmitate (i.e., the CHASE phase).…”

Palmitoylation is required for TNF-R1 signaling

“…Earlier reports showed that transmembrane TNF is palmitoylated which regulates its affinity to TNF-R1 [68, 69]. FasL and also the putative DR6 ligand APP require palmitoylation for their biological function [70–72]. We ruled out a possible role for endogenous TNF on TNF-R1 palmitoylation by co-culture with TNF targeting Fab.…”

The C99 domain of the amyloid precursor protein is a disordered membrane phase-preferring protein

“…However, these APP palmitoylation sites are somewhat unusual in that they are localized to the extracellular (luminal) domain, rather than in the cytoplasmic domain of the protein where most palmitoylation sites of transmembrane proteins are found (99). Palmitoylation has been suggested to regulate APP association with DRMs and processing in rafts, as well as its dimerization (99,100). Importantly however, the C99 domain of APP lacks any cysteines and can therefore cannot undergo palmitoylation, ruling out the possibility that this form of posttranslational modification determines whether C99 itself associates with rafts.…”