APH-1 Interacts with Mature and Immature Forms of Presenilins and Nicastrin and May Play a Role in Maturation of Presenilin·Nicastrin Complexes

Abstract

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 ␥-and ⑀-secretase activity. Indeed APH-1 proteins can bind to the nicastrin ⌬312-369 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 presenilin⅐nicastrin complexes.Presenilin 1 (PS1) 1 (1), presenilin 2 (PS2) (2), and nicastrin (3) are components of high molecular weight protein complexes that are required for the intramembranous proteolysis of some type 1 transmembrane proteins, including the ␤-amyloid precursor protein (APP) (4), Notch (5-11), and ErbB-4 (12). Genetic screens in Caenorhabditis elegans have identified two additional proteins, APH-1 (13) and PEN-2 (14), in which loss of function mutations phenotypically modulate Notch signaling in a manner similar to that of null mutants in nicastrin and the presenilins. Preliminary studies in transfected cells overexpressing tagged APH-1 proteins suggest that the genetic interaction between APH-1 and the presenilin-dependent cleavage of Notch and APP is mediated by a direct physical interaction between APH-1 and nicastrin or the presenilins (14). We report here that both the APH-1 homologue on human chromosome 1 (termed APH-1 a in Ref. 14, but here referred to as APH-1 1 for clarity to avoid confusion with labeling of alternate splice forms) and the APH-1 homologue on chromosome 15 (previously referred to as APH-1 b ; here termed APH-1 15 ) are widely expressed in multiple tissues and that the APH-1 1 transcript is present as several different alternatively spliced forms (data not shown). We also report that endogenous APH-1 1 directly interacts with both immature and mature forms of the presenilins and nicastrin and that high molecular weight complexes containing these proteins display ␥-and ⑀-secretase activity. Overexpression of any one member of the APH-1 family...

Ontogenic reduction of Aph-1b mRNA and γ-secretase activity in rats with a complex neurodevelopmental phenotype

“…[15][16][17] Similar to the results obtained with reduced Aph-1a expression, knockdown or knockout of either of the g-secretase components PS1, Nct or PEN-2 in the mouse led to affected levels of most of the nonsilenced g-secretase subunits. [18][19][20][21][22][23][24] In contrast, ablation of mouse PS2 expression had little effect on the expression levels of the other g-secretase components, 20,24,25 comparable to what we have observed in the I/I, II/II and III/III rat lines concerning the effect of reduced Aph-1b expression. 7 These findings suggest that PS2 and Aph-1b are somehow related.…”

Prostaglandin I 2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

“…Indeed, APH‐1 often combines with PEN‐2, nicastrin, and PS to generate an active form of γ‐secretase complex, which is responsible for the cleavage of β‐APP and for the deposition of Aβ (De Strooper, 2003). Once APH‐1 was found in C. elegans (Francis et al ., 2002), the APH‐1 complex was confirmed in several experimental models (Gu et al ., 2003; Luo et al ., 2003; Hansson et al ., 2004). Along these lines, APH‐1α/1β might also be involved in regulating the deposition of Aβ 1–42 in response to PGI 2 stimulation.…”

Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations

“…It is ideal for analysis and partial isolation of membrane Carbonate washed SY5Y membranes (500 lg) were run on BN/PAGE, proteins electroeluted from the PS complex region, treated with reducing sample buffer and analysed on 10% Tris/tricine gels using mini Biorad apparatus. PS1 NTF and full-length PS1 were detected with PS1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] antibody, PS1 loop antibody detected PS1 CTF, NCT was detected at 140 kDa, PEN-2 at 10 kDa and PS2 CTF detected with PS2 CTF antibody. Parallel poly(vinylidene difluoride) strips were also probed with APH 1a and 1b antibodies but APH-1 was not detected.…”

“…PEN-2 is a small double membrane-spanning protein with N-and C-termini facing the lumen [14]. APH-1 contains seven putative transmembrane domains and has a variety of isoforms with differing C-terminal domains; in humans, APH-1a long and short forms are encoded by a gene on chromosome 1 and APH-1b by chromosome 15 [15,16].…”