scholarly journals Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

2012 ◽  
Vol 23 (12) ◽  
pp. 2339-2351 ◽  
Author(s):  
Yogikala Prabhu ◽  
Patricia V. Burgos ◽  
Christina Schindler ◽  
Ginny G. Farías ◽  
Javier G. Magadán ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amyloid Precursor Protein ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Proteolytic Processing ◽  
Precursor Protein ◽  
Amyloid Precursor Protein Processing ◽  
Intermediate Compartment ◽  
Golgi Network

The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway.

scholarly journals The trans-Golgi network is a major site for α-secretase processing of amyloid precursor protein in primary neurons

2018 ◽  
Vol 294 (5) ◽  
pp. 1618-1631 ◽  
Author(s):  
Jing Zhi A. Tan ◽  
Paul A. Gleeson
Keyword(s):  
Amyloid Precursor Protein ◽  
Hela Cells ◽  
Secretory Pathway ◽  
Adaptor Protein ◽  
Precursor Protein ◽  
Primary Neurons ◽  
Major Site ◽  
App Processing ◽  
Band Size ◽  
Golgi Network

Amyloid precursor protein (APP) is processed along the amyloidogenic pathway by the β-secretase, BACE1, generating β-amyloid (Aβ), or along the nonamyloidogenic pathway by α-secretase, precluding Aβ production. The plasma membrane is considered the major site for α-secretase–mediated APP cleavage, but other cellular locations have not been rigorously investigated. Here, we report that APP is processed by endogenous α-secretase at the trans-Golgi network (TGN) of both transfected HeLa cells and mouse primary neurons. We have previously shown the adaptor protein complex, AP-4, and small G protein ADP-ribosylation factor–like GTPase 5b (Arl5b) are required for efficient post-Golgi transport of APP to endosomes. We found here that AP-4 or Arl5b depletion results in Golgi accumulation of APP and increased secretion of the soluble α-secretase cleavage product sAPPα. Moreover, inhibition of γ-secretase following APP accumulation in the TGN increases the levels of the membrane-bound C-terminal fragments of APP from both α-secretase cleavage (α-CTF, named C83 according to its band size) and BACE1 cleavage (β-CTF/C99). The level of C83 was ∼4 times higher than that of C99, indicating that α-secretase processing is the major pathway and that BACE1 processing is the minor pathway in the TGN. AP-4 silencing in mouse primary neurons also resulted in the accumulation of endogenous APP in the TGN and enhanced α-secretase processing. These findings identify the TGN as a major site for α-secretase processing in HeLa cells and primary neurons and indicate that both APP processing pathways can occur within the TGN compartment along the secretory pathway.

scholarly journals The discrepancy between presenilin subcellular localization and γ-secretase processing of amyloid precursor protein

2001 ◽  
Vol 154 (4) ◽  
pp. 731-740 ◽  
Author(s):  
Philippe Cupers ◽  
Mustapha Bentahir ◽  
Katleen Craessaerts ◽  
Isabelle Orlans ◽  
Hugo Vanderstichele ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Amyloid Precursor Protein ◽  
Primary Cultures ◽  
Brefeldin A ◽  
Precursor Protein ◽  
Subcellular Compartment ◽  
Marker Proteins ◽  
Intermediate Compartment ◽  
The Relationship

We investigated the relationship between PS1 and γ-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent γ-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no γ-secretase processing was observed when holo-APP or APP-C99, a direct substrate for γ-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent γ-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that γ-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the γ-cleavage of APP-C99. In agreement, we found that intracellular γ-secretase processing of APP-C99-KK both at the γ40 and the γ42 site could be restored partially after brefeldin A treatment. Our data confirm the “spatial paradox” and raise several questions regarding the PS1 is γ-secretase hypothesis.

scholarly journals FE65: Roles beyond amyloid precursor protein processing

2015 ◽  
Vol 20 (1) ◽  
Author(s):  
Wan Ning Vanessa Chow ◽  
Hei Nga Maggie Cheung ◽  
Wen Li ◽  
Kwok-Fai Lau
Keyword(s):  
Amyloid Precursor Protein ◽  
Synapse Formation ◽  
Adaptor Protein ◽  
Ubiquitin Proteasome System ◽  
Precursor Protein ◽  
Amyloid Precursor Protein Processing ◽  
Developmentally Regulated ◽  
App Processing ◽  
Neuronal Processes ◽  
Initial Work

AbstractFE65 is a brain-enriched, developmentally regulated adaptor protein that was first identified as a binding partner of amyloid precursor protein (APP), an important molecule in Alzheimer’s disease. FE65 possesses three protein interaction domains, including an N-terminal WW domain and two C-terminal phosphotyrosine-binding (PTB) domains. It is capable of mediating the assembly of multimolecular complexes. Although initial work reveals its roles in APP processing and gene transactivation, increasing evidence suggests that FE65 participates in more diverse biological processes than originally anticipated. This article discusses the role of FE65 in signal transduction during cell stress and protein turnover through the ubiquitin-proteasome system and in various neuronal processes, including neurogenesis, neuronal migration and positioning, neurite outgrowth, synapse formation and synaptic plasticity, learning, and memory.

scholarly journals Secretory processing of amyloid precursor protein is inhibited by increase in cellular cholesterol content

1997 ◽  
Vol 322 (3) ◽  
pp. 893-896 ◽  
Author(s):  
Marco RACCHI ◽  
Roberta BAETTA ◽  
Nathalie SALVIETTI ◽  
Paola IANNA ◽  
Guido FRANCESCHINI ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amyloid Precursor Protein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Indirect Evidence ◽  
Proteolytic Processing ◽  
Precursor Protein ◽  
Low Density ◽  
Membrane Composition ◽  
Simultaneous Treatment

Plasma-membrane composition plays a crucial role in most of the cellular functions that depend on membrane processes. In virtually all cell types the proteolytic processing of Alzheimer amyloid precursor protein (APP) to generate soluble APP (sAPP) is believed to occur at the plasma membrane or in its immediate proximity. Alteration of this metabolic pathway has been linked to the pathogenesis of Alzheimer's disease. We analysed the effect of membrane cholesterol enrichment on APP metabolism. Incubation of COS cells with increasing concentrations of non-esterified cholesterol carried by rabbit β-very low-density lipoprotein caused a dose-dependent inhibition of sAPP release: 70% inhibition with 10 μg/ml non-esterified cholesterol. A less pronounced inhibitory effect was observed on treatment with human low-density lipoprotein. Inhibition of sAPP release was independent of receptor-mediated lipoprotein metabolism since simultaneous treatment with chloroquine did not modify the effect of lipoprotein treatment. In addition, treatment with cholesterol dissolved in either ethanol or methyl-β-cyclodextrin elicited the same effect. Excess non-esterified cholesterol did not cause cell toxicity. Cell cholesterol mass inversely correlated with sAPP release. Progesterone, which inhibits shuttling of non-esterified cholesterol between the plasma membrane and intracellular pools, had no effect on the inhibition of sAPP release from cholesterol-loaded cells, providing indirect evidence that cholesterol may act at the plasma membrane.

scholarly journals Cholesterol decreases secretion of the secreted form of amyloid precursor protein by interfering with glycosylation in the protein secretory pathway

2000 ◽  
Vol 348 (2) ◽  
pp. 307-313 ◽  
Author(s):  
Josè Luis GALBETE ◽  
Teresa RODRIGUEZ-MARTIN ◽  
Elisa PERESSINI ◽  
Piergiorgio MODENA ◽  
Roberto BIANCHI ◽  
...  
Keyword(s):  
Amyloid Precursor Protein ◽  
Secretory Pathway ◽  
Protein A ◽  
Brefeldin A ◽  
Immunoblot Analysis ◽  
Protein Glycosylation ◽  
Precursor Protein ◽  
Cell Surface Biotinylation ◽  
Time Courses ◽  
Proteolytic Cleavages

Cerebral deposits of β-amyloid (βA) are a major feature in Alzheimer's disease. βA is derived from amyloid precursor protein (APP). APP is subject to N- and O-glycosylation and undergoes a series of proteolytic cleavages that lead to the release of βA or of a non-amyloidogenic secreted form of APP (APPs). We used primary neuronal and glial cultures to investigate how cholesterol affects the production and secretion of APPs. Exposure to cholesterol for 2 h did not change the neuronal release of APPs; after 6 h APPs release was slightly lower, whereas 24 h of exposure decreased APPs in the medium by approx. 60%. The time courses were similar in astrocytes and microglia preparations. To verify whether the effect of cholesterol was a consequence of membrane rigidification we tested the activity of ganglioside GM1 and prion protein fragment PrP 106-126, which affect membrane fluidity similarly to cholesterol, on APPs secretion. Neither altered the production of APPs. APP mRNA and the total amount of APP in the cells were slightly decreased by cholesterol after 2 and 24 h respectively. Immunoblot analysis of APP associated with neuronal cells and astrocytes indicated that cholesterol progressively decreased the glycosylated forms of the protein; a similar tendency was noted in cells treated with brefeldin A and monensin, two substances that interfere with protein glycosylation. The cell-surface biotinylation method showed that in cholesterol-treated cells APP reached the plasma membrane. Our results indicate that cholesterol decreases the secretion of APPs by interfering with APP maturation and inhibiting glycosylation of the protein; although APP is inserted in the membrane it is not cleaved by α-secretase.

scholarly journals Novel β-Secretase Cleavage of β-Amyloid Precursor Protein in the Endoplasmic Reticulum/Intermediate Compartment of NT2N Cells

1997 ◽  
Vol 138 (3) ◽  
pp. 671-680 ◽  
Author(s):  
Abraham S.C. Chyung ◽  
Barry D. Greenberg ◽  
David G. Cook ◽  
Robert W. Doms ◽  
Virginia M.-Y. Lee
Keyword(s):  
Amyloid Precursor Protein ◽  
Amyloid Β ◽  
Neuronal Cell ◽  
Brefeldin A ◽  
Precursor Protein ◽  
Embryonal Carcinoma Cell ◽  
Amyloid Β Peptide ◽  
Wild Type ◽  
Intermediate Compartment ◽  
Β Amyloid

Previous studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type β-amyloid precursor protein (APP) to amyloid β peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by β-secretase cleavage (APPβ) is indeed produced from the endogenous full length APP (APPFL). Pulse–chase studies demonstrated a precursor–product relationship between APPFL and APPβ as well as intracellular and secreted APPβ fragments. In addition, trypsin digestion of intact NT2N cells at 4°C did not abolish APPβ recovered from the cell lysates. Furthermore, the production of intracellular APPβ from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPβ was not detected in several non-neuronal cell lines. Significantly, production of APPβ occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15°C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane

2013 ◽  
Vol 450 (1) ◽  
pp. 73-83 ◽  
Author(s):  
Sonja Huser ◽  
Gregor Suri ◽  
Pascal Crottet ◽  
Martin Spiess
Keyword(s):  
Plasma Membrane ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Trans Golgi Network ◽  
Src Homology 3 ◽  
Amphiphysin 1 ◽  
Src Homology ◽  
Golgi Network

The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)–GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.

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