PS (presenilin)-dependent γ-secretase occurs as a high-molecular-mass complex composed of either PS1 or PS2 associated with Nct (nicastrin), PEN2 (presenilin enhancer 2 homologue) and APH1 (anterior pharynx defective 1 homologue). Numerous reports have documented the very complicated physical and functional cross-talk between these proteins that ultimately governs the biological activity of the γ-secretase, but very few studies examined the fate of the components of the complex. We show that, in both HEK-293 cells and the TSM1 neuronal cell line, the immunoreactivities of overexpressed myc-tagged-APH1a and -PEN2 were enhanced by the proteasome inhibitors ZIE and lactacystin, whereas a broad range of protease inhibitors had no effect. By contrast, proteasome inhibitors were totally unable to affect the cellular expression of endogenous APH1aL and PEN2 in HEK-293 cells, TSM1 and primary cultured cortical neurons. To explain this apparent discrepancy, we examined the degradation of myc-tagged-APH1a and -PEN2, in vitro, by cell extracts containing endogenous proteasome and by purified 20S proteasome. Strikingly, myc-tagged-APH1a and -PEN2 resist proteolysis by endogenous proteasome and purified 20S proteasome. We also show that endogenous PEN2 expression was drastically higher in wild-type than in PS- and Nct-deficient fibroblasts and was enhanced by proteasome inhibitors only in the two deficient cell systems. However, here again, purified 20S proteasome appeared unable to cleave endogenous PEN2 present in PS-deficient fibroblasts. The levels of endogenous APH1aL-like immunoreactivity were not modified by proteasome inhibitors and were unaffected by PS deficiency. Altogether, our results indicate that endogenous PEN2 and APH1aL do not undergo proteasomal degradation under physiological conditions in HEK-293 cells, TSM1 cells and fibroblasts and that the clearance of PEN2 in PS- and Nct-deficient fibroblasts is not mediated by 20S proteasome. Whether the 26S proteasome participates to PEN2 proteolysis in deficient fibroblasts remains to be established.
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Research Article|
February 10 2006
Catabolism of endogenous and overexpressed APH1a and PEN2: evidence for artifactual involvement of the proteasome in the degradation of overexpressed proteins
Julie Dunys;
Julie Dunys
*Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Valbonne, France
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Toshitaka Kawarai;
Toshitaka Kawarai
†Center for Research in Neurodegenerative Diseases, Department of Medicine, University of Toronto and University Health Network, 6 Queen's Park Crescent, Toronto, Ontario, Canada, M5S 3H2
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Sherwin Wilk;
Sherwin Wilk
‡Mount Sinai School of Medicine, New York, NY 10029, U.S.A.
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Peter St. George-Hyslop;
Peter St. George-Hyslop
†Center for Research in Neurodegenerative Diseases, Department of Medicine, University of Toronto and University Health Network, 6 Queen's Park Crescent, Toronto, Ontario, Canada, M5S 3H2
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Cristine Alves Da Costa;
Cristine Alves Da Costa
*Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Valbonne, France
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Frédéric Checler
Frédéric Checler
1
*Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Valbonne, France
1To whom correspondence should be addressed (email checler@ipmc.cnrs.fr).
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Publisher: Portland Press Ltd
Received:
July 25 2005
Revision Received:
November 16 2005
Accepted:
November 22 2005
Accepted Manuscript online:
November 22 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 394 (2): 501–509.
Article history
Received:
July 25 2005
Revision Received:
November 16 2005
Accepted:
November 22 2005
Accepted Manuscript online:
November 22 2005
Citation
Julie Dunys, Toshitaka Kawarai, Sherwin Wilk, Peter St. George-Hyslop, Cristine Alves Da Costa, Frédéric Checler; Catabolism of endogenous and overexpressed APH1a and PEN2: evidence for artifactual involvement of the proteasome in the degradation of overexpressed proteins. Biochem J 1 March 2006; 394 (2): 501–509. doi: https://doi.org/10.1042/BJ20051197
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