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Intracellular trafficking of the amyloid β-protein precursor (APP) regulated by novel function of X11-like.

Saito Y, Akiyama M, Araki Y, Sumioka A, Shiono M, Taru H, Nakaya T, Yamamoto T, Suzuki T - PLoS ONE (2011)

Bottom Line: With this novel function, X11L suppresses overall APP metabolism and results in further suppression of Aβ generation.Trafficking of imAPP to plasma membrane is observed in other X11 family proteins, X11 and X11L2, but not in other APP-binding partners such as FE65 and JIP1.It is herein clear that respective functional domains of X11L regulate APP metabolism at multiple steps in intracellular protein secretory pathways.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroscience, Graduate School of Pharmaceutical Science, Hokkaido University, Sapporo, Japan.

ABSTRACT

Background: Amyloid β (Aβ), a causative peptide of Alzheimer's disease, is generated by intracellular metabolism of amyloid β-protein precursor (APP). In general, mature APP (mAPP, N- and O-glycosylated form) is subject to successive cleavages by α- or β-, and γ-secretases in the late protein secretory pathway and/or at plasma membrane, while immature APP (imAPP, N-glycosylated form) locates in the early secretory pathway such as endoplasmic reticulum or cis-Golgi, in which imAPP is not subject to metabolic cleavages. X11-like (X11L) is a neural adaptor protein composed of a phosphotyrosine-binding (PTB) and two C-terminal PDZ domains. X11L suppresses amyloidogenic cleavage of mAPP by direct binding of X11L through its PTB domain, thereby generation of Aβ lowers. X11L expresses another function in the regulation of intracellular APP trafficking.

Methodology: In order to analyze novel function of X11L in intracellular trafficking of APP, we performed a functional dissection of X11L. Using cells expressing various domain-deleted X11L mutants, intracellular APP trafficking was examined along with analysis of APP metabolism including maturation (O-glycosylation), processing and localization of APP.

Conclusions: X11L accumulates imAPP into the early secretory pathway by mediation of its C-terminal PDZ domains, without being bound to imAPP directly. With this novel function, X11L suppresses overall APP metabolism and results in further suppression of Aβ generation. Interestingly some of the accumulated imAPP in the early secretory pathway are likely to appear on plasma membrane by unidentified mechanism. Trafficking of imAPP to plasma membrane is observed in other X11 family proteins, X11 and X11L2, but not in other APP-binding partners such as FE65 and JIP1. It is herein clear that respective functional domains of X11L regulate APP metabolism at multiple steps in intracellular protein secretory pathways.

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Cell surface expression of imAPP induced by X11L mutant proteins.(A) Effect of X11L mutant proteins. N2a cells (∼2×105) were transiently transfected with pcDNA3-FLAG-APP (0.8 µg) in the presence of various cDNA plasmids encoding EGFP-X11L proteins (a: 0.18 µg, b: 0.6 µg, c: 0.6 µg, d: 0.06 µg, e: 0.36 µg, f: 0.18 µg, g: 0.08 µg, h: 0.15 µg, i: 0.6 µg, and j: 0.54 µg). To standardize the plasmid amount, empty vector was added to yield 1.4 µg of plasmid in total. Cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect the biotinylated proteins. The cell lysates and biotinylated proteins (Cell surface) were analyzed by immunoblotting with an anti-FLAG antibody to detect APP, an anti-EGFP antibody to detect X11L proteins, and an anti-actin antibody to detect actin. Lanes a to j correspond to the constructs described in Figure 1A. (B) Specificity of X11s function. N2a cells (∼2×105) were transiently cotransfected with pcDNA3-FLAG-APP695 (0.6 µg) and 0.2 µg of the following plasmids: pcDNA3.1-HA-X11 (lane 3), pcDNA3.1-HA-X11L (lane 4), pcDNA3.1-HA-X11L2 (lane 5), pcDNA3.1-HA-FE65 (lane 6), or pcDNA3.1-HA-JIP1b (lane 7). Cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect biotinylated proteins. The cell lysate and biotinylated proteins (Cell surface) were analyzed by immunoblotting with an anti-FLAG antibody to detect APP, an anti-HA antibody to detect HA-binding proteins, and an anti-actin antibody to detect actin.
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pone-0022108-g003: Cell surface expression of imAPP induced by X11L mutant proteins.(A) Effect of X11L mutant proteins. N2a cells (∼2×105) were transiently transfected with pcDNA3-FLAG-APP (0.8 µg) in the presence of various cDNA plasmids encoding EGFP-X11L proteins (a: 0.18 µg, b: 0.6 µg, c: 0.6 µg, d: 0.06 µg, e: 0.36 µg, f: 0.18 µg, g: 0.08 µg, h: 0.15 µg, i: 0.6 µg, and j: 0.54 µg). To standardize the plasmid amount, empty vector was added to yield 1.4 µg of plasmid in total. Cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect the biotinylated proteins. The cell lysates and biotinylated proteins (Cell surface) were analyzed by immunoblotting with an anti-FLAG antibody to detect APP, an anti-EGFP antibody to detect X11L proteins, and an anti-actin antibody to detect actin. Lanes a to j correspond to the constructs described in Figure 1A. (B) Specificity of X11s function. N2a cells (∼2×105) were transiently cotransfected with pcDNA3-FLAG-APP695 (0.6 µg) and 0.2 µg of the following plasmids: pcDNA3.1-HA-X11 (lane 3), pcDNA3.1-HA-X11L (lane 4), pcDNA3.1-HA-X11L2 (lane 5), pcDNA3.1-HA-FE65 (lane 6), or pcDNA3.1-HA-JIP1b (lane 7). Cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect biotinylated proteins. The cell lysate and biotinylated proteins (Cell surface) were analyzed by immunoblotting with an anti-FLAG antibody to detect APP, an anti-HA antibody to detect HA-binding proteins, and an anti-actin antibody to detect actin.

Mentions: (A) Schematic representation of the structures of amino-terminal tagged X11L proteins. a: human X11L, b: X11L-PTB+C (368 to 749), c: X11L-N+PTB (1 to 555), d: X11L-ΔPTB (Δ369 to 555), e: X11L-ΔPDZ1 (Δ561 to 651), f: X11L-ΔPDZ2 (Δ661 to 749), g: X11L-N (1 to 368), h: X11L-C (556 to 749), i: X11L-PTB (369 to 555), j: X11L F520V, k: human X11, and l: human X11L2. PTB, phosphotyrosine binding domain; PDZ, PDZ domain. Oval indicates FLAG (Fig. 2) or EGFP (Fig. 1, 3, 6) tag. Numbers indicate amino acid positions. (B to D) Alteration of APP maturation and proteolytic metabolism in cells expressing EGFP-X11L mutant proteins, X11 and X11L2. N2a cells (∼2×105) were transiently transfected with pcDNA3-FLAG-APP695 (0.8 µg) in the presence of various X11L cDNA plasmids, pcDNA3.1-EGFP-X11L and pcDNA3.1-EGFP-X11L2 (a: 0.18 µg, b: 0.6 µg, c: 0.6 µg, d: 0.06 µg, e: 0.36 µg, f: 0.18 µg, g: 0.08 µg, h: 0.15 µg, i: 0.6 µg, j: 0.54 µg, k: 0.09 µg, and l: 0.12 µg). Plasmid amounts were adjusted to produce a similar level of protein expression, and empty vector was added to yield a total of 1.4 µg of plasmid to standardize the amount of plasmid. The cell lysates were analyzed by immunoblotting with an anti-FLAG M2 antibody to detect APP, an anti-EGFP antibody to detect X11L derivatives, an anti-actin antibody to detect actin, and an anti-APP/C antibody to detect APP-CTFα/β (B). (C) The relative ratio of imAPP/mAPP is quantified. The relative ratio in cells in the absence of X11L was set as 1.0 (column 2). (D) Aβ40 and Aβ42 secreted into the medium of N2a cells were quantified with sELISA. Concentrations of Aβ40 (left) and Aβ42 (right) are shown as means ± s.e. (n = 4). Data were analyzed using the Student's t-test (*, P<0.05; **, P<0.01; ***, P<0.001). Proteins showing a significant decrease (P<0.01 and P<0.001) in Aβ generation are shown as closed columns.


Intracellular trafficking of the amyloid β-protein precursor (APP) regulated by novel function of X11-like.

Saito Y, Akiyama M, Araki Y, Sumioka A, Shiono M, Taru H, Nakaya T, Yamamoto T, Suzuki T - PLoS ONE (2011)

Cell surface expression of imAPP induced by X11L mutant proteins.(A) Effect of X11L mutant proteins. N2a cells (∼2×105) were transiently transfected with pcDNA3-FLAG-APP (0.8 µg) in the presence of various cDNA plasmids encoding EGFP-X11L proteins (a: 0.18 µg, b: 0.6 µg, c: 0.6 µg, d: 0.06 µg, e: 0.36 µg, f: 0.18 µg, g: 0.08 µg, h: 0.15 µg, i: 0.6 µg, and j: 0.54 µg). To standardize the plasmid amount, empty vector was added to yield 1.4 µg of plasmid in total. Cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect the biotinylated proteins. The cell lysates and biotinylated proteins (Cell surface) were analyzed by immunoblotting with an anti-FLAG antibody to detect APP, an anti-EGFP antibody to detect X11L proteins, and an anti-actin antibody to detect actin. Lanes a to j correspond to the constructs described in Figure 1A. (B) Specificity of X11s function. N2a cells (∼2×105) were transiently cotransfected with pcDNA3-FLAG-APP695 (0.6 µg) and 0.2 µg of the following plasmids: pcDNA3.1-HA-X11 (lane 3), pcDNA3.1-HA-X11L (lane 4), pcDNA3.1-HA-X11L2 (lane 5), pcDNA3.1-HA-FE65 (lane 6), or pcDNA3.1-HA-JIP1b (lane 7). Cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect biotinylated proteins. The cell lysate and biotinylated proteins (Cell surface) were analyzed by immunoblotting with an anti-FLAG antibody to detect APP, an anti-HA antibody to detect HA-binding proteins, and an anti-actin antibody to detect actin.
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Related In: Results  -  Collection

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pone-0022108-g003: Cell surface expression of imAPP induced by X11L mutant proteins.(A) Effect of X11L mutant proteins. N2a cells (∼2×105) were transiently transfected with pcDNA3-FLAG-APP (0.8 µg) in the presence of various cDNA plasmids encoding EGFP-X11L proteins (a: 0.18 µg, b: 0.6 µg, c: 0.6 µg, d: 0.06 µg, e: 0.36 µg, f: 0.18 µg, g: 0.08 µg, h: 0.15 µg, i: 0.6 µg, and j: 0.54 µg). To standardize the plasmid amount, empty vector was added to yield 1.4 µg of plasmid in total. Cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect the biotinylated proteins. The cell lysates and biotinylated proteins (Cell surface) were analyzed by immunoblotting with an anti-FLAG antibody to detect APP, an anti-EGFP antibody to detect X11L proteins, and an anti-actin antibody to detect actin. Lanes a to j correspond to the constructs described in Figure 1A. (B) Specificity of X11s function. N2a cells (∼2×105) were transiently cotransfected with pcDNA3-FLAG-APP695 (0.6 µg) and 0.2 µg of the following plasmids: pcDNA3.1-HA-X11 (lane 3), pcDNA3.1-HA-X11L (lane 4), pcDNA3.1-HA-X11L2 (lane 5), pcDNA3.1-HA-FE65 (lane 6), or pcDNA3.1-HA-JIP1b (lane 7). Cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect biotinylated proteins. The cell lysate and biotinylated proteins (Cell surface) were analyzed by immunoblotting with an anti-FLAG antibody to detect APP, an anti-HA antibody to detect HA-binding proteins, and an anti-actin antibody to detect actin.
Mentions: (A) Schematic representation of the structures of amino-terminal tagged X11L proteins. a: human X11L, b: X11L-PTB+C (368 to 749), c: X11L-N+PTB (1 to 555), d: X11L-ΔPTB (Δ369 to 555), e: X11L-ΔPDZ1 (Δ561 to 651), f: X11L-ΔPDZ2 (Δ661 to 749), g: X11L-N (1 to 368), h: X11L-C (556 to 749), i: X11L-PTB (369 to 555), j: X11L F520V, k: human X11, and l: human X11L2. PTB, phosphotyrosine binding domain; PDZ, PDZ domain. Oval indicates FLAG (Fig. 2) or EGFP (Fig. 1, 3, 6) tag. Numbers indicate amino acid positions. (B to D) Alteration of APP maturation and proteolytic metabolism in cells expressing EGFP-X11L mutant proteins, X11 and X11L2. N2a cells (∼2×105) were transiently transfected with pcDNA3-FLAG-APP695 (0.8 µg) in the presence of various X11L cDNA plasmids, pcDNA3.1-EGFP-X11L and pcDNA3.1-EGFP-X11L2 (a: 0.18 µg, b: 0.6 µg, c: 0.6 µg, d: 0.06 µg, e: 0.36 µg, f: 0.18 µg, g: 0.08 µg, h: 0.15 µg, i: 0.6 µg, j: 0.54 µg, k: 0.09 µg, and l: 0.12 µg). Plasmid amounts were adjusted to produce a similar level of protein expression, and empty vector was added to yield a total of 1.4 µg of plasmid to standardize the amount of plasmid. The cell lysates were analyzed by immunoblotting with an anti-FLAG M2 antibody to detect APP, an anti-EGFP antibody to detect X11L derivatives, an anti-actin antibody to detect actin, and an anti-APP/C antibody to detect APP-CTFα/β (B). (C) The relative ratio of imAPP/mAPP is quantified. The relative ratio in cells in the absence of X11L was set as 1.0 (column 2). (D) Aβ40 and Aβ42 secreted into the medium of N2a cells were quantified with sELISA. Concentrations of Aβ40 (left) and Aβ42 (right) are shown as means ± s.e. (n = 4). Data were analyzed using the Student's t-test (*, P<0.05; **, P<0.01; ***, P<0.001). Proteins showing a significant decrease (P<0.01 and P<0.001) in Aβ generation are shown as closed columns.

Bottom Line: With this novel function, X11L suppresses overall APP metabolism and results in further suppression of Aβ generation.Trafficking of imAPP to plasma membrane is observed in other X11 family proteins, X11 and X11L2, but not in other APP-binding partners such as FE65 and JIP1.It is herein clear that respective functional domains of X11L regulate APP metabolism at multiple steps in intracellular protein secretory pathways.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroscience, Graduate School of Pharmaceutical Science, Hokkaido University, Sapporo, Japan.

ABSTRACT

Background: Amyloid β (Aβ), a causative peptide of Alzheimer's disease, is generated by intracellular metabolism of amyloid β-protein precursor (APP). In general, mature APP (mAPP, N- and O-glycosylated form) is subject to successive cleavages by α- or β-, and γ-secretases in the late protein secretory pathway and/or at plasma membrane, while immature APP (imAPP, N-glycosylated form) locates in the early secretory pathway such as endoplasmic reticulum or cis-Golgi, in which imAPP is not subject to metabolic cleavages. X11-like (X11L) is a neural adaptor protein composed of a phosphotyrosine-binding (PTB) and two C-terminal PDZ domains. X11L suppresses amyloidogenic cleavage of mAPP by direct binding of X11L through its PTB domain, thereby generation of Aβ lowers. X11L expresses another function in the regulation of intracellular APP trafficking.

Methodology: In order to analyze novel function of X11L in intracellular trafficking of APP, we performed a functional dissection of X11L. Using cells expressing various domain-deleted X11L mutants, intracellular APP trafficking was examined along with analysis of APP metabolism including maturation (O-glycosylation), processing and localization of APP.

Conclusions: X11L accumulates imAPP into the early secretory pathway by mediation of its C-terminal PDZ domains, without being bound to imAPP directly. With this novel function, X11L suppresses overall APP metabolism and results in further suppression of Aβ generation. Interestingly some of the accumulated imAPP in the early secretory pathway are likely to appear on plasma membrane by unidentified mechanism. Trafficking of imAPP to plasma membrane is observed in other X11 family proteins, X11 and X11L2, but not in other APP-binding partners such as FE65 and JIP1. It is herein clear that respective functional domains of X11L regulate APP metabolism at multiple steps in intracellular protein secretory pathways.

Show MeSH
Related in: MedlinePlus