Biochem. J. (2005) 391
(513–525) (Printed in Great Britain)
Ubiquilin regulates presenilin endoproteolysis and modulates g-secretase components, Pen-2 and nicastrin
Leann K. MASSEY*, Alex L. MAH* and Mervyn J. MONTEIRO*†1
*Molecular and Cell Biology Graduate Program, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, U.S.A., and †Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, U.S.A.
Mutations in presenilin proteins (PS1 and PS2) lead to early-onset Alzheimer's disease. PS proteins are endoproteolytically cleaved into two main fragments: the NTF (PS N-terminal fragment) and the CTF (PS C-terminal fragment). The two fragments are believed to constitute the core catalytic enzyme activity called g-secretase, which is responsible for cleaving b-amyloid precursor protein to release Ab. Thus, studying factors that modulate PS fragment levels could provide important information about g-secretase. Previously, we demonstrated that the protein, ubiquilin-1, interacts both in vivo and in vitro with PS and that overexpression of ubiquilin-1 or -2 leads to increased accumulation of full-length PS proteins. Using wild-type HEK-293 cells (human embryonic kidney 293 cells) and PS-inducible cells, we now show that overexpression of either ubiquilin-1 or -2 decreases the PS NTF and CTF levels. Conversely, siRNA (small interfering RNA)-mediated knockdown of ubiquilin-1 and -2 proteins increased the PS NTF and CTF levels. We considered that ubiquilin might alter PS fragment accumulation by acting as a shuttle factor escorting PS fragments to the proteasome for degradation. However, through proteasome inhibition studies, we show that this does not occur. Instead, our results suggest that ubiquilin regulates PS fragment production. We also examined whether other components of the g-secretase complex are affected by ubiquilin expression. Interestingly, overexpression of ubiquilin resulted in a decrease in Pen-2 and nicastrin levels, two essential components of the g-secretase complex. In contrast, knockdown of ubiquilin-1 and -2 protein expression by RNAi (RNA interference) increased Pen-2 and nicastrin levels. Finally, we show that inhibition of the proteasome results in decreased PS fragment production and that reversal of proteasome inhibition restores PS fragment production, suggesting that the proteasome may be involved in PS endoproteolysis. These studies implicate ubiquilin as an important factor in regulating PS biogenesis and metabolism.
Key words: Alzheimer's disease, presenilin, proteasome, g-secretase, small interfering RNA, ubiquilin.
Abbreviations used: AD, Alzheimer's disease; ALLN, N-acetyl-Leu-Leu-Nle-CHO; APP, b-amyloid precursor protein; PS, presenilin; CTF, PS C-terminal fragment; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; FBS, fetal bovine serum; FL, full-length; GST, glutathione S-transferase; HEK-293 cells, human embryonic kidney 293 cells; NTF, PS N-terminal fragment; PonA, ponasterone A; RNAi, RNA interference; RT, reverse transcriptase; siRNA, small interfering RNA; SEC, siRNA expression cassette.
1To whom correspondence should be addressed (email monteiro@umbi.umd.edu).
Received 23 March 2005/13 June 2005; accepted 24 June 2005
Published as BJ Immediate Publication 24 June 2005, doi:10.1042/BJ20050491
The Biochemical Society, London ©2005
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