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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 appearance of imAPP by X11L expression in cells suffered from reduced temperature.N2a cells (∼1×106) transferred transiently with 0.6 µg of pcDNA3-FLAG-APP with (+) or without (−) 0.2 µg of pcDNA3.1-HA-X11L were cultured for indicated time (h) at 20°C. To standardize the plasmid amount, empty vector was added to yield 0.8 µg of plasmid in total. The cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect biotinylated proteins. (A) The cell lysates and biotinylated proteins (Cell surface) were subjected to immunoblot analysis with anti-FLAG antibody to detect APP, anti-HA antibody to detect X11L, and anti-α-tubulin antibody to detect α tubulin. (B) The relative ratio of cell surface mAPP in cells without X11L expression (first row of left panel; lanes 3 to 7 in A) and that of cell surface imAPP in cells with X11L expression (first row of right panel; lanes 8 to 12 in A) are quantified. The relative ratio in cells at time 0 (lanes 3 and 8) was set as 1.0. Data were analyzed using the Student's t-test with standard error (n = 4; *, P<0.05).
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pone-0022108-g004: Cell surface appearance of imAPP by X11L expression in cells suffered from reduced temperature.N2a cells (∼1×106) transferred transiently with 0.6 µg of pcDNA3-FLAG-APP with (+) or without (−) 0.2 µg of pcDNA3.1-HA-X11L were cultured for indicated time (h) at 20°C. To standardize the plasmid amount, empty vector was added to yield 0.8 µg of plasmid in total. The cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect biotinylated proteins. (A) The cell lysates and biotinylated proteins (Cell surface) were subjected to immunoblot analysis with anti-FLAG antibody to detect APP, anti-HA antibody to detect X11L, and anti-α-tubulin antibody to detect α tubulin. (B) The relative ratio of cell surface mAPP in cells without X11L expression (first row of left panel; lanes 3 to 7 in A) and that of cell surface imAPP in cells with X11L expression (first row of right panel; lanes 8 to 12 in A) are quantified. The relative ratio in cells at time 0 (lanes 3 and 8) was set as 1.0. Data were analyzed using the Student's t-test with standard error (n = 4; *, P<0.05).

Mentions: We further and carefully performed studies to verify the transport of imAPP. It is known that the reduced temperature prevents transfer of membrane glycoproteins to cell surface [26], [27]. Cells with or without expression of X11L were cultured at 20°C for 0 to 12 h and APP exposed on the outer surface of cells was analyzed by immunoblotting along with total cellular APP (Fig. 4). In the absence of X11L expression, cell surface-localized mAPP decreased depending on culture time and no surface mAPP was detected at 8 h (Fig. 4A, lanes 3 to 7), while in the presence of X11L, cell surface-localized imAPP first decreased by 2–4 h but re-increased at 8 to 12 h (Fig. 4A, lanes 8 to 12). The quantitative analysis of APP exposed on the outer surface of cells was also shown in Fig 4 B (left, mAPP on cell surface of cells without X11L expression; right, imAPP on cell surface of cells with X11L expression). This study supports our idea that X11L mediates imAPP transfer onto plasma membrane by unidentified mechanism but not through the conventional Golgi mediated secretory pathway.


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 appearance of imAPP by X11L expression in cells suffered from reduced temperature.N2a cells (∼1×106) transferred transiently with 0.6 µg of pcDNA3-FLAG-APP with (+) or without (−) 0.2 µg of pcDNA3.1-HA-X11L were cultured for indicated time (h) at 20°C. To standardize the plasmid amount, empty vector was added to yield 0.8 µg of plasmid in total. The cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect biotinylated proteins. (A) The cell lysates and biotinylated proteins (Cell surface) were subjected to immunoblot analysis with anti-FLAG antibody to detect APP, anti-HA antibody to detect X11L, and anti-α-tubulin antibody to detect α tubulin. (B) The relative ratio of cell surface mAPP in cells without X11L expression (first row of left panel; lanes 3 to 7 in A) and that of cell surface imAPP in cells with X11L expression (first row of right panel; lanes 8 to 12 in A) are quantified. The relative ratio in cells at time 0 (lanes 3 and 8) was set as 1.0. Data were analyzed using the Student's t-test with standard error (n = 4; *, P<0.05).
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Related In: Results  -  Collection

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pone-0022108-g004: Cell surface appearance of imAPP by X11L expression in cells suffered from reduced temperature.N2a cells (∼1×106) transferred transiently with 0.6 µg of pcDNA3-FLAG-APP with (+) or without (−) 0.2 µg of pcDNA3.1-HA-X11L were cultured for indicated time (h) at 20°C. To standardize the plasmid amount, empty vector was added to yield 0.8 µg of plasmid in total. The cells were labeled with sulfo-NHS-LC-biotin and NeutrAvidin was used to collect biotinylated proteins. (A) The cell lysates and biotinylated proteins (Cell surface) were subjected to immunoblot analysis with anti-FLAG antibody to detect APP, anti-HA antibody to detect X11L, and anti-α-tubulin antibody to detect α tubulin. (B) The relative ratio of cell surface mAPP in cells without X11L expression (first row of left panel; lanes 3 to 7 in A) and that of cell surface imAPP in cells with X11L expression (first row of right panel; lanes 8 to 12 in A) are quantified. The relative ratio in cells at time 0 (lanes 3 and 8) was set as 1.0. Data were analyzed using the Student's t-test with standard error (n = 4; *, P<0.05).
Mentions: We further and carefully performed studies to verify the transport of imAPP. It is known that the reduced temperature prevents transfer of membrane glycoproteins to cell surface [26], [27]. Cells with or without expression of X11L were cultured at 20°C for 0 to 12 h and APP exposed on the outer surface of cells was analyzed by immunoblotting along with total cellular APP (Fig. 4). In the absence of X11L expression, cell surface-localized mAPP decreased depending on culture time and no surface mAPP was detected at 8 h (Fig. 4A, lanes 3 to 7), while in the presence of X11L, cell surface-localized imAPP first decreased by 2–4 h but re-increased at 8 to 12 h (Fig. 4A, lanes 8 to 12). The quantitative analysis of APP exposed on the outer surface of cells was also shown in Fig 4 B (left, mAPP on cell surface of cells without X11L expression; right, imAPP on cell surface of cells with X11L expression). This study supports our idea that X11L mediates imAPP transfer onto plasma membrane by unidentified mechanism but not through the conventional Golgi mediated secretory pathway.

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