Ubiquilin regulates presenilin endoproteolysis and modulates gamma-secretase components, Pen-2 and nicastrin.
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 gamma-secretase, which is responsible for cleaving beta-amyloid precursor protein to release Abeta. Thus, studying factors that modulate PS fragment levels could provide important information about gamma-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 gamma-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 gamma-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.
Massey
Leann K
LK
Molecular and Cell Biology Graduate Program, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA.
Mah
Alex L
AL
Monteiro
Mervyn J
MJ
D048868:Adaptor Proteins, Signal Transducing; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D053764:Presenilin-1; D053766:Presenilin-2; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D034622:RNA Interference; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C508853:PSEN1 protein, human; C508861:PSEN2 protein, human; C469349:PSENEN protein, human; D010446:Peptide Fragments; D053764:Presenilin-1; D053766:Presenilin-2; C107792:UBQLN1 protein, human; C405662:UBQLN2 protein, human; D014452:Ubiquitins; C413604:nicastrin protein; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D046988:Proteasome Endopeptidase Complex