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Novel beta-secretase cleavage of beta-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells.

Chyung AS, Greenberg BD, Cook DG, Doms RW, Lee VM - J. Cell Biol. (1997)

Bottom Line: Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments.In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates.Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

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

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Intracellular β and γ cleavages occur in a pre-Golgi  compartment in NT2N neurons. Cultures of NT2N cells were first  preincubated with 20 μg/ml BFA for 1 h before radiolabeling with  [35S]methionine for 16 h in the continuous presence of 20 μg/ml  BFA. Control cultures were processed similarly, except that BFA  was absent in the medium. Radiolabeled proteins from BFA-treated and untreated cell lysates and media were immunoprecipitated with Karen (for APPFL in the cell lysates and APPα/β in  the media as shown in A), with antibody 53 (for APPβ in B), and  with the mAb 6E10 (for Aβ in C). Note that APPβ and Aβ were  recovered in the cell lysate but not in the medium of BFA-treated  cells. (M, mature APPFL; I, immature APPFL.
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Figure 7: Intracellular β and γ cleavages occur in a pre-Golgi compartment in NT2N neurons. Cultures of NT2N cells were first preincubated with 20 μg/ml BFA for 1 h before radiolabeling with [35S]methionine for 16 h in the continuous presence of 20 μg/ml BFA. Control cultures were processed similarly, except that BFA was absent in the medium. Radiolabeled proteins from BFA-treated and untreated cell lysates and media were immunoprecipitated with Karen (for APPFL in the cell lysates and APPα/β in the media as shown in A), with antibody 53 (for APPβ in B), and with the mAb 6E10 (for Aβ in C). Note that APPβ and Aβ were recovered in the cell lysate but not in the medium of BFA-treated cells. (M, mature APPFL; I, immature APPFL.

Mentions: Since APPβ is produced in an intracellular compartment in NT2N neurons, we sought to identify the subcellular site(s) of β-secretase cleavage. Therefore, NT2N neurons were metabolically labeled with [35S]methionine in the presence or absence of 20 μg/ml BFA (Fig. 7). BFA is a pharmacological agent that causes a redistribution of the Golgi into the ER (Doms et al., 1989; Lippincott-Schwartz, 1989; Pelham, 1991). In the absence of BFA, APPFL, APPβ, and Aβ were recovered from the cell lysates, while APPα, APPβ, and Aβ were detected in the media of NT2N neurons (Fig. 7, a–c, lanes 1 and 3). Surprisingly, in the presence of BFA, not only APPFL but also APPβ and Aβ continued to be recovered from NT2N cell lysates (Fig. 7, a–c, lane 2). The effectiveness of BFA was verified by the fact that the secretion of APPα, APPβ, and Aβ into the medium was completely abolished in its presence (Fig. 7, a–c, lane 4). Furthermore, we found that APPβ recovered from BFA-treated cells (Fig. 7 b, lane 2) migrate with an accelerated electrophoretic mobility compared to APPβ from nontreated cells (Fig. 7 b, lane 1), suggesting that this fragment may have been derived from immature APP. Indeed, the faster mobility of mature APPFL in the presence of BFA (Fig. 7 a, compare M of lanes 1 and 2) indicates that this agent blocks APP from acquiring at least some of the posttranslational modifications. Thus, Aβ may be generated from immature as well as mature forms of APP.


Novel beta-secretase cleavage of beta-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells.

Chyung AS, Greenberg BD, Cook DG, Doms RW, Lee VM - J. Cell Biol. (1997)

Intracellular β and γ cleavages occur in a pre-Golgi  compartment in NT2N neurons. Cultures of NT2N cells were first  preincubated with 20 μg/ml BFA for 1 h before radiolabeling with  [35S]methionine for 16 h in the continuous presence of 20 μg/ml  BFA. Control cultures were processed similarly, except that BFA  was absent in the medium. Radiolabeled proteins from BFA-treated and untreated cell lysates and media were immunoprecipitated with Karen (for APPFL in the cell lysates and APPα/β in  the media as shown in A), with antibody 53 (for APPβ in B), and  with the mAb 6E10 (for Aβ in C). Note that APPβ and Aβ were  recovered in the cell lysate but not in the medium of BFA-treated  cells. (M, mature APPFL; I, immature APPFL.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2141643&req=5

Figure 7: Intracellular β and γ cleavages occur in a pre-Golgi compartment in NT2N neurons. Cultures of NT2N cells were first preincubated with 20 μg/ml BFA for 1 h before radiolabeling with [35S]methionine for 16 h in the continuous presence of 20 μg/ml BFA. Control cultures were processed similarly, except that BFA was absent in the medium. Radiolabeled proteins from BFA-treated and untreated cell lysates and media were immunoprecipitated with Karen (for APPFL in the cell lysates and APPα/β in the media as shown in A), with antibody 53 (for APPβ in B), and with the mAb 6E10 (for Aβ in C). Note that APPβ and Aβ were recovered in the cell lysate but not in the medium of BFA-treated cells. (M, mature APPFL; I, immature APPFL.
Mentions: Since APPβ is produced in an intracellular compartment in NT2N neurons, we sought to identify the subcellular site(s) of β-secretase cleavage. Therefore, NT2N neurons were metabolically labeled with [35S]methionine in the presence or absence of 20 μg/ml BFA (Fig. 7). BFA is a pharmacological agent that causes a redistribution of the Golgi into the ER (Doms et al., 1989; Lippincott-Schwartz, 1989; Pelham, 1991). In the absence of BFA, APPFL, APPβ, and Aβ were recovered from the cell lysates, while APPα, APPβ, and Aβ were detected in the media of NT2N neurons (Fig. 7, a–c, lanes 1 and 3). Surprisingly, in the presence of BFA, not only APPFL but also APPβ and Aβ continued to be recovered from NT2N cell lysates (Fig. 7, a–c, lane 2). The effectiveness of BFA was verified by the fact that the secretion of APPα, APPβ, and Aβ into the medium was completely abolished in its presence (Fig. 7, a–c, lane 4). Furthermore, we found that APPβ recovered from BFA-treated cells (Fig. 7 b, lane 2) migrate with an accelerated electrophoretic mobility compared to APPβ from nontreated cells (Fig. 7 b, lane 1), suggesting that this fragment may have been derived from immature APP. Indeed, the faster mobility of mature APPFL in the presence of BFA (Fig. 7 a, compare M of lanes 1 and 2) indicates that this agent blocks APP from acquiring at least some of the posttranslational modifications. Thus, Aβ may be generated from immature as well as mature forms of APP.

Bottom Line: Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments.In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates.Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

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

Show MeSH
Related in: MedlinePlus