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Nicastrin is dispensable for gamma-secretase protease activity in the presence of specific presenilin mutations.

ABSTRACT: gamma-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial gamma-secretase substrates containing amyloid precursor protein or Notch fragments. The gamma-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4p release from the membrane-bound substrates, i.e. growth of yeast on histidine and adenine, or beta-galactosidase assay. We screened and evaluated gamma-secretase mutants using this reconstitution system in yeast, which does not possess endogenous gamma-secretase activity. When we introduced familial Alzheimer mutants of PS1 in this system, their activities were shown to be loss of function. Although the protease activity of wild type PS1 depends on the other three subunits introduced, we obtained 15 new PS1 mutants, which are active in the absence of NCT. They possessed a S438P mutation at the ninth transmembrane domain (TM9) together with one missense mutation distributed through transmembrane and loop regions. These mutations were not related to familial Alzheimer mutations of PS1 as identified so far. The S438P mutant was partially active but required other mutations for full activation. Results of the beta-galactosidase assay suggested that they have wild type protease activities, which were further confirmed by the endoproteolysis of PS1, amyloid beta peptides, and Notch intracellular domain production in mammalian cells. These results suggest that NCT is dispensable for the protease activity of gamma-secretase.

SUBMITTER: Futai E

PROVIDER: S-EPMC2676034 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Nicastrin is dispensable for gamma-secretase protease activity in the presence of specific presenilin mutations.

Futai Eugene E Yagishita Sosuke S Ishiura Shoichi S

The Journal of biological chemistry 20090302 19

gamma-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial gamma-secretase substrates containing amyloid precursor protein or Notch fragments. The gamma-secretase activities were evaluated by transcriptional activation ...[more]

PMID: 19254953

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Project description: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.

Dataset Information

Nicastrin is dispensable for gamma-secretase protease activity in the presence of specific presenilin mutations.

Publications

Nicastrin is dispensable for gamma-secretase protease activity in the presence of specific presenilin mutations.

Similar Datasets

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