Requirement of PEN-2 for stabilization of the presenilin N-/C-terminal fragment heterodimer within the gamma-secretase complex.
Gamma-secretase is a protease complex composed of presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, which catalyzes intramembrane cleavage of several type I transmembrane proteins including the Alzheimer's disease-associated beta-amyloid precursor protein. We generated stable RNA interference-mediated PEN-2 knockdown cells to probe mutant PEN-2 variants for functional activity. Knockdown of PEN-2 was associated with impaired NCT maturation and deficient PS1 endoproteolysis, which was efficiently rescued by wild type or N-terminally tagged PEN-2 but not by C-terminally tagged PEN-2 or by the C-terminally truncated PEN-2-DeltaC mutant. Although the latter mutants rescued the PS1 holoprotein accumulation associated with the PEN-2 knockdown, they failed to restore normal levels of the PS1 N- and C-terminal fragments and to maturate NCT. PEN-2-DeltaC was highly unstable and rapidly turned over by proteasomal degradation consistent with its failure to become stably incorporated into the gamma-secretase complex. In addition, expression of PEN-2-DeltaC caused a selective instability of the PS1 N-/C-terminal fragment heterodimer that underwent proteasomal degradation, whereas NCT and APH-1 were stable. Interestingly, when we knocked down PEN-2 in the background of the endoproteolysis-deficient PS1 Deltaexon9 mutant, immature NCT still accumulated, demonstrating that PEN-2 is also required for gamma-secretase complex maturation when PS endoproteolysis cannot occur. Taken together, our data suggest that PEN-2 is required for the stabilization of the PS fragment heterodimer within the gamma-secretase complex following PS endoproteolysis. This function critically depends on the PEN-2 C terminus. Moreover, our data show that PEN-2 is generally required for gamma-secretase complex maturation independent of its activity in PS1 endoproteolysis.
Prokop
Stefan
S
Adolf-Butenandt-Institute, Department of Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease Research, Schillerstrasse 44, Ludwig-Maximilians-University, 80336 Munich, Germany.
Shirotani
Keiro
K
Edbauer
Dieter
D
Haass
Christian
C
Steiner
Harald
H
D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002460:Cell Line; D006224:Cricetinae; D019281:Dimerization; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D053764:Presenilin-1; D013329:Structure-Activity Relationship
D008565:Membrane Proteins; C508853:PSEN1 protein, human; C469349:PSENEN protein, human; D010446:Peptide Fragments; D053764:Presenilin-1; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human