Interactome analyses of mature γ-secretase complexes reveal distinct molecular environments of presenilin (PS) paralogs and preferential binding of signal peptide peptidase to PS2.

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid ß-peptides (Aß) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature γ-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the γ-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H(+)-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing γ-secretase complexes and was observed to influence Aß production.

TMP21 is a presenilin complex component that modulates gamma-secretase but not epsilon-secretase activity.

The presenilin proteins (PS1 and PS2) and their interacting partners nicastrin, aph-1 (refs 4, 5) and pen-2 (ref. 5) form a series of high-molecular-mass, membrane-bound protein complexes that are necessary for gamma-secretase and epsilon-secretase cleavage of selected type 1 transmembrane proteins, including the amyloid precursor protein, Notch and cadherins. Modest cleavage activity can be generated by reconstituting these four proteins in yeast and Spodoptera frugiperda (sf9) cells. However, a critical but unanswered question about the biology of the presenilin complexes is how their activity is modulated in terms of substrate specificity and/or relative activities at the gamma and epsilon sites. A corollary to this question is whether additional proteins in the presenilin complexes might subsume these putative regulatory functions. The hypothesis that additional proteins might exist in the presenilin complexes is supported by the fact that enzymatically active complexes have a mass that is much greater than predicted for a 1:1:1:1 stoichiometric complex (at least 650 kDa observed, compared with about 220 kDa predicted). To address these questions we undertook a search for presenilin-interacting proteins that differentially affected gamma- and epsilon-site cleavage events. Here we report that TMP21, a member of the p24 cargo protein family, is a component of presenilin complexes and differentially regulates gamma-secretase cleavage without affecting epsilon-secretase activity.

Presenilin 1 mutations activate gamma 42-secretase but reciprocally inhibit epsilon-secretase cleavage of amyloid precursor protein (APP) and S3-cleavage of notch.

The presenilin 1 (PS1) and presenilin 2 (PS2) proteins are necessary for proteolytic cleavage of the amyloid precursor protein (APP) within its transmembrane domain. One of these cleavage events (termed gamma-secretase) generates the C-terminal end of the Abeta-peptide by proteolysis near residue 710 or 712 of APP(770). Another event (termed gamma-like or epsilon-secretase cleavage) cleaves near residue 721 at approximately 2-5 residues inside the cytoplasmic membrane boundary to generate a series of stable, C-terminal APP fragments. This latter cleavage is analogous to S3-cleavage of Notch. We report here that specific mutations in the N terminus, loop, or C terminus of PS1 all increase the production of Abeta(42) but cause inhibition of both epsilon-secretase cleavage of APP and S3-cleavage of Notch. These data support the hypothesis that epsilon-cleavage of APP and S3-cleavage of Notch are similar events. They also argue that, although both the gamma-site and the epsilon-site cleavage of APP are presenilin-dependent, they are likely to be independent catalytic events.

The presenilin proteins are components of multiple membrane-bound complexes that have different biological activities.

Several lines of evidence have indicated that the presenilin proteins function within macromolecular complexes and are necessary for the regulated intramembranous proteolysis of certain type 1 transmembrane proteins, including the amyloid precursor protein, Notch, and p75. Data from multiple complementary experiments now suggest that there may be several distinct presenilin complexes. We show here that presenilin mutations and certain detergents affect the abundance and componentry of the presenilin complexes, and these structural effects correlate with their effects on gamma-secretase activity. Our data suggest that there are at least three complexes, including a approximately 150-kDa nicastrin-aph-1 complex (which is likely to be a precursor complex). There is a stable and abundant intermediate complex of approximately 440 kDa, which contains aph-1, pen-2, nicastrin, and PS1. However, it is the very low abundance, high mass (>/=670 kDa) heteromeric complexes that are associated with the highest gamma-secretase-specific activity.

APH-1 interacts with mature and immature forms of presenilins and nicastrin and may play a role in maturation of presenilin.nicastrin complexes.

APH-1 and PEN-2 genes modulate the function of nicastrin and the presenilins in Caenorhabditis elegans. Preliminary studies in transfected mammalian cells overexpressing tagged APH-1 proteins suggest that this genetic interaction is mediated by a direct physical interaction. Using the APH-1 protein encoded on human chromosome 1 (APH-1(1)L; also known as APH-1a) as an archetype, we report here that endogenous forms of APH-1 are predominantly expressed in intracellular membrane compartments, including the endoplasmic reticulum and cis-Golgi. APH-1 proteins directly interact with immature and mature forms of the presenilins and nicastrin within high molecular weight complexes that display gamma- and epsilon-secretase activity. Indeed APH-1 proteins can bind to the nicastrin delta312-369 loss of function mutant, which does not undergo glycosylation maturation and is not trafficking beyond the endoplasmic reticulum. The levels of expression of endogenous APH-1(1)L can be suppressed by overexpression of any other members of the APH-1 family, suggesting that their abundance is coordinately regulated. Finally, although the absence of APH-1 destabilizes the presenilins, in contrast to nicastrin and PEN-2, APH-1 itself is only modestly destabilized in cells lacking functional expression of presenilin 1 or presenilin 2. Taken together, our data suggest that APH-1 proteins, and APH-1(1) in particular, may have a role in the initial assembly and maturation of presenilin.nicastrin complexes.

Presenilin 1 and presenilin 2 have differential effects on the stability and maturation of nicastrin in Mammalian brain.

The presenilins and nicastrin form high molecular mass, multimeric protein complexes involved in the intramembranous proteolysis of several proteins. Post-translational glycosylation and trafficking of nicastrin is necessary for the activity of these complexes. We report here that although there are differences in the post-translational processing of nicastrin in neurons and glia, both of the presenilins are required for the physiological post-translational modification and for the correct subcellular distribution of nicastrin. Absence of presenilin 1 (PS1) is associated with dramatic reductions in the level of mature glycosylated nicastrin and with redistribution of nicastrin away from the cell surface. In contrast, absence of presenilin 2 (PS2) is associated with only modest reductions in the levels of immature nicastrin. It is notable that these differential effects parallel the differential effects of null mutations in PS1 and PS2 on APP and Notch processing. Our data therefore suggest that the differential interactions of PS1 and PS2 with nicastrin reflect different functions for the PS1 and PS2 complexes.