Two recent papers from the Journal of Biological Chemistry shed additional light on the roles of APH-1 and PEN-2, two proteins that were only last year identified in screens of the roundworm C. elegans and implicated as members of the presenilin (PS) complex (see ARF related news story). Through its γ-secretase activity this complex catalyzes the final proteolytic step in the release of the Aβ peptide from its precursor protein (AβPP), but the field’s understanding of both the make-up and function of the complex constituents remains patchy.
Working independently, two research groups-one led by Huaxi Xu at the Fisher Center for Alzheimer's Disease and Rockefeller University, New York, and the other by Peter St. George-Hyslop at the University of Toronto-strengthen the case for the involvement of APH-1 and PEN-2 in the γ-secretase complex.
First author Yongjun Gu and colleagues, working with St. George-Hyslop, used antibodies raised against the human analogue of APH-1 to identify where this protein resides in the cell and with what proteins it interacts. The antibody detected APH-1 in the endoplasmic reticulum and Golgi apparatus, two subcellular organelles where the presenilin complex assembles and matures. When the authors used the antibody to immunoprecipitate APH-1 from cell lysates, they found in the precipitate, PS1, PS2, N-terminal fragments of these two proteins, and nicastrin, all previously identified components of the complex, suggesting that APH-1 is a bona fide member of the presenilin club.
But what of its function? Interestingly, when Gu et al. immunoprecipitated APH-1 from cells expressing a mutant form of nicastrin that fails to mature, they detected immature nicastrin, but not PS1 in the precipitate, suggesting that mature nicastrin is required for APH-1 to bind the complex. Prior work on fruit flies has shown that loss of APH-1 reduces γ-secretase cleavage of AβPP (see Francis et al., 2002). St George-Hyslop and colleagues suggest that APH-1 may play a role in the maturation of the PS complex, perhaps by acting as a scaffold.
Under the direction of Huaxi Xu, Weijie Luo and colleagues investigated the role of the recently identified PEN-2, or presenilin enhancer-2 (see ARF related news story). When the authors used small interfering RNAs to silence PEN-2 expression in mammalian cells, they found a dramatic increase in the level of full-length PS1 and a reduction in the level of PS1 C-terminal fragments (PS1 C-terminal fragment); presenilin is normally proteolytically cleaved into smaller fragments that go on to form the γ-secretase complex. This result confirms a report from Christian Haas's laboratory, which showed that PEN-2 RNAi causes a dramatic reduction in levels of PS1 C-terminal fragment and a concomitant loss of the large PS1 complex (see Steiner et al., 2002).
Taken together, these experiments suggest that PEN-2 is necessary for proper processing of PS1. In support of this hypothesis, Luo et al. found that overexpression of PEN-2 enhances the proteolysis of full-length presenilin, leading to a dramatic increase of presenilin N-terminal fragments.
In a similar series of experiments the authors found that silencing aph-1 had little or no effect on PS1 processing. However, the accumulation of PS1 in cells lacking PEN-2 was reversed if aph-1 was also silenced. The authors suggest, therefore, that APH-1 stabilizes full length PS1, protecting it from rapid degradation. When Luo et al. overexpressed APH-1 they found it appeared to enhance the proteolytic effect of overexpressed PEN-2, and they comment that this may be explained by APH-1 also stabilizing the cleaved PS1 fragments.
Together the two papers seem to point to a scaffolding or protective role for APH-1, but a more active proteolytic role for PEN-2.—Tom Fagan
- New Playmates on the Presenilin/γ-Secretase Playground
- Stockholm: Aph-1 and Pen-2: New Names in the Presenilin Debate Complete the Complex?
- Francis R, McGrath G, Zhang J, Ruddy DA, Sym M, Apfeld J, Nicoll M, Maxwell M, Hai B, Ellis MC, Parks AL, Xu W, Li J, Gurney M, Myers RL, Himes CS, Hiebsch R, Ruble C, Nye JS, Curtis D. aph-1 and pen-2 are required for Notch pathway signaling, gamma-secretase cleavage of betaAPP, and presenilin protein accumulation. Dev Cell. 2002 Jul;3(1):85-97. PubMed.
- Steiner H, Winkler E, Edbauer D, Prokop S, Basset G, Yamasaki A, Kostka M, Haass C. PEN-2 is an integral component of the gamma-secretase complex required for coordinated expression of presenilin and nicastrin. J Biol Chem. 2002 Oct 18;277(42):39062-5. PubMed.
- Gu Y, Chen F, Sanjo N, Kawarai T, Hasegawa H, Duthie M, Li W, Ruan X, Luthra A, Mount HT, Tandon A, Fraser PE, St George-Hyslop P. APH-1 interacts with mature and immature forms of presenilins and nicastrin and may play a role in maturation of presenilin.nicastrin complexes. J Biol Chem. 2003 Feb 28;278(9):7374-80. PubMed.
- Luo WJ, Wang H, Li H, Kim BS, Shah S, Lee HJ, Thinakaran G, Kim TW, Yu G, Xu H. PEN-2 and APH-1 coordinately regulate proteolytic processing of presenilin 1. J Biol Chem. 2003 Mar 7;278(10):7850-4. Epub 2003 Jan 8 PubMed.