Intracellular pathways of folded and misfolded amyloid precursor protein degradation

doi:10.1016/j.abb.2006.05.002

Archives of Biochemistry and Biophysics

Volume 451, Issue 1, 1 July 2006, Pages 79-90

https://doi.org/10.1016/j.abb.2006.05.002 Get rights and content

Abstract

A number of studies suggest that early events in the maturation of amyloid precursor protein (APP) are important in determining its entry into one of several alternative processing pathways, one of which leads to the toxic protein β-amyloid (Aβ). In pulse-labeled APP expressing CHO cells two proteolytic systems can degrade newly translated APP: the proteosome and a cysteine protease. When N-glycosylation was inhibited by tunicamycin, the former system is the dominant mechanism of APP degradation. Without tunicamycin present, the cysteine protease is operational: cysteine protease inhibitors completely inhibit APP turnover in cells in which the secretory pathway is interrupted with brefeldin A or when α-secretase and endosomal degradation are also pharmacologically blocked. APP immunoprecipitated from cells extracted under mild conditions and labeled in the presence of tunicamycin exhibited greater sensitivity to endoproteinase glu-C (V8) or lys-C than from cells without drug. The V8 fragment missing in tunicamyin treated cells encompassed the KPI inhibitor insertion site but was distinct from the site of N-glycosylation. It is concluded that a conformational change caused by interrupted N-glycosylation shunts newly translated APP into the proteasomal degradation pathway. Pulse-labeled and chased cells showed an additional V8 fragment that was not present in pulsed-labeled cells and was not due to glycosylation since it was also present in cells labeled in the presence of brefeldin. This latter result indicates that an additional, delayed conformational alteration occurs in the endoplasmic reticulum.

Section snippets

Cell lines, labeling, and drug treatments

SV695, a Chinese hamster ovary (CHO) cell line stably transfected with APP695 (a gift from Dr. S. Sisodia, U. of Chicago), was grown in Delbecco’s modified Eagle’s media (DMEM) supplemented with 5% fetal bovine serum, 200–400 μg/ml G418, 100 μg/ml penicillin, and 100 μg/ml streptomycin. An untransfected CHO cell line was grown under the same conditions. A CHO cell line stably transfected with APP750 containing the Swedish mutation was obtained from Dr. N.K. Robakis, Mount Sinai School of Medicine,

Intracellular APP traffics to different compartments in over-expressing CHO cells

Newly translated APP becomes N-glycosylated by ER localized enzymes and a fraction of these molecules leave the ER to become O-glycosylated by Golgi localized enzymes. To assess whether transfected, overexpressing CHO cells represent a viable experimental system to study APP turnover and folding, the efficiency by which newly synthesized APP was able to reach the secretory pathway was measured by the appearance of APPs in the media and the rate of appearance of intracellular P83. Because P83

Discussion

As is the case with most secreted proteins, newly translated APP has several alternative fates. It can proceed on to the secretory pathway by leaving the ER, becoming further chemically modified in the Golgi, and localizing to the cell membrane where it is shed into the media by the action of α-secretase. Alternatively, cell surface localized protein can internalize and become degraded within the endocytic pathway. The third possibility is that it can be degraded prior to accessing

Acknowledgments

This work was supported by the Alzheimer’s Research Alliance of Oregon and the OHSU Alzheimer’s Disease Center (P30 AG60817).

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Intracellular pathways of folded and misfolded amyloid precursor protein degradation

Abstract

Section snippets

Cell lines, labeling, and drug treatments

Intracellular APP traffics to different compartments in over-expressing CHO cells

Discussion

Acknowledgments

Trends Cell Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Neurobiol. Aging

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Cell

J. Biol. Chem.

Cell

Trends Cell Biol.

Trends Cell Biol.

Trends Cell Biol.

Biochim. Biphys. Acta

Biophys. J.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Trends Mol. Med.

Science

Nature

Proc. Natl. Acad. Sci. USA

Biochemistry

J. Biol. Chem.

Proc. Natl. Acad. Sci. USA