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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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APPβ is generated from APPFL that is concentrated in the ER. NT2N  cultures of ∼1 × 106 cells  were infected with recombinant SFV containing either wild-type APP695 or  APP695ΔKK constructs. The  dilysine motif concentrates  APPFL to the ER by an efficient retrieval mechanism.  Duplicate cultures infected  with wild-type APP695 were  treated with 20 μg/ml BFA  for comparison. Under these  conditions, the cells were  metabolically labeled with  [35S]methionine for 16 h. Radiolabeled cell lysates and  media were then immunoprecipitated with antibody 53  (for APPβ, A) and Karen  (for APPFL and APPα/β, B).  Radiolabeled immunoprecipitates were used to expose PhosphorImager plates (72 h) for visualization of bands. Unlike APPβ produced under BFA inhibition,  APPβ derived from APP695ΔKK was modified and secreted into the medium.
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Figure 10: APPβ is generated from APPFL that is concentrated in the ER. NT2N cultures of ∼1 × 106 cells were infected with recombinant SFV containing either wild-type APP695 or APP695ΔKK constructs. The dilysine motif concentrates APPFL to the ER by an efficient retrieval mechanism. Duplicate cultures infected with wild-type APP695 were treated with 20 μg/ml BFA for comparison. Under these conditions, the cells were metabolically labeled with [35S]methionine for 16 h. Radiolabeled cell lysates and media were then immunoprecipitated with antibody 53 (for APPβ, A) and Karen (for APPFL and APPα/β, B). Radiolabeled immunoprecipitates were used to expose PhosphorImager plates (72 h) for visualization of bands. Unlike APPβ produced under BFA inhibition, APPβ derived from APP695ΔKK was modified and secreted into the medium.

Mentions: To determine whether or not APPβ can be produced from APP695ΔKK, wild-type APP695 and APP695ΔKK were separately expressed in NT2N neurons by infection with SFV vectors bearing these constructs. After infection, duplicate wells containing wild-type APP695-infected cells were also treated with 20 μg/ml BFA. The [35S]methionine- labeled cell lysates and the media were then sequentially immunoprecipitated with the antibodies 53 and Karen. Only the immature form of APPFL was detected from cells expressing APP695ΔKK (Fig. 10 b, compare lanes 1 and 3). Significantly, intracellular production and secretion of APPβ was not affected by genetic targeting of APP to the ER (Fig. 10 a, lanes 3 and 6). Furthermore, we found that unlike inhibition with BFA that eliminates transport of all proteins from the ER to the Golgi, specific retrieval of full length APP695ΔKK to the ER allowed the APPβ fragment generated in the ER/IC to be transported to the Golgi complex for modification before secretion (Fig. 10 a, compare lanes 2 and 3 and lanes 5 and 6). This suggests that once the ER retention motif is cleaved from the APPβ fragment, it can then be transported to the Golgi complex for further maturation and subsequent secretion.


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)

APPβ is generated from APPFL that is concentrated in the ER. NT2N  cultures of ∼1 × 106 cells  were infected with recombinant SFV containing either wild-type APP695 or  APP695ΔKK constructs. The  dilysine motif concentrates  APPFL to the ER by an efficient retrieval mechanism.  Duplicate cultures infected  with wild-type APP695 were  treated with 20 μg/ml BFA  for comparison. Under these  conditions, the cells were  metabolically labeled with  [35S]methionine for 16 h. Radiolabeled cell lysates and  media were then immunoprecipitated with antibody 53  (for APPβ, A) and Karen  (for APPFL and APPα/β, B).  Radiolabeled immunoprecipitates were used to expose PhosphorImager plates (72 h) for visualization of bands. Unlike APPβ produced under BFA inhibition,  APPβ derived from APP695ΔKK was modified and secreted into the medium.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141643&req=5

Figure 10: APPβ is generated from APPFL that is concentrated in the ER. NT2N cultures of ∼1 × 106 cells were infected with recombinant SFV containing either wild-type APP695 or APP695ΔKK constructs. The dilysine motif concentrates APPFL to the ER by an efficient retrieval mechanism. Duplicate cultures infected with wild-type APP695 were treated with 20 μg/ml BFA for comparison. Under these conditions, the cells were metabolically labeled with [35S]methionine for 16 h. Radiolabeled cell lysates and media were then immunoprecipitated with antibody 53 (for APPβ, A) and Karen (for APPFL and APPα/β, B). Radiolabeled immunoprecipitates were used to expose PhosphorImager plates (72 h) for visualization of bands. Unlike APPβ produced under BFA inhibition, APPβ derived from APP695ΔKK was modified and secreted into the medium.
Mentions: To determine whether or not APPβ can be produced from APP695ΔKK, wild-type APP695 and APP695ΔKK were separately expressed in NT2N neurons by infection with SFV vectors bearing these constructs. After infection, duplicate wells containing wild-type APP695-infected cells were also treated with 20 μg/ml BFA. The [35S]methionine- labeled cell lysates and the media were then sequentially immunoprecipitated with the antibodies 53 and Karen. Only the immature form of APPFL was detected from cells expressing APP695ΔKK (Fig. 10 b, compare lanes 1 and 3). Significantly, intracellular production and secretion of APPβ was not affected by genetic targeting of APP to the ER (Fig. 10 a, lanes 3 and 6). Furthermore, we found that unlike inhibition with BFA that eliminates transport of all proteins from the ER to the Golgi, specific retrieval of full length APP695ΔKK to the ER allowed the APPβ fragment generated in the ER/IC to be transported to the Golgi complex for modification before secretion (Fig. 10 a, compare lanes 2 and 3 and lanes 5 and 6). This suggests that once the ER retention motif is cleaved from the APPβ fragment, it can then be transported to the Golgi complex for further maturation and subsequent secretion.

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