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.

References:
Gu Y, Chen F, Sanjo B, Kawarai T, Hasegawa H, Duthie M, Li W, Ruan X, Luthra A, Mount HTJ, Tandon A, Fraser PE, St George-Hyslop P. APH-1 interacts with mature and immature forms of presenilin and nicastrin and may play a role in maturation of presenilin-nicastrin complexes. J. Biological Chem. 2002 Dec 5.Abstract

Luo W, Wang H, Li H, Kim BS, Shah S, Lee H-J, Thinakaran G, Kim T-W, Yu G, Xu H. PEN-2 and APH-1 coordinately regulate proteolytic processing of presenilin 1. J. Biological Chem. 2003 January 8.Abstract

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  1. It has only been a few months that APH-1 and PEN-2 were discovered as major players in γ-secretase function in the Notch pathway in C. elegans. Together with recent papers from Haass and Yu, the two present papers demonstrate compelling biochemical evidence that γ-secretase is a complex of four proteins: presenilin, nicastrin, PEN-2 and APH-1. The final proof, however, which would be the reconstitution of an active γ-secretase, either in vitro, or in a cell type that lacks endogenous γ-secretase activity (e.g., yeast), is still missing. The current discoveries may be useful in the design and optimization of γ-secretase inhibitors, which remain a major direction of Alzheimer's disease drug discovery despite the problems with Notch-related toxicity. A better understanding of the molecular mechanism of the γ-secretase protease complex could guide the design of drugs that influence the specificity of the protease for the different substrates, finally leading to a drug that would inhibit AβPP cleavage but not, or to a lesser extent, the cleavage of Notch and other substrates.

  2. The elegant data from both labs are quite consistent with the notion that APH-1 and PEN2 have important roles in stabilizing the PS complex and in promoting endoproteolysis. Indeed, we presented data in Orlando that fully supported these findings (our work is presently under review). There is still a bit of work to figure out where in the cell these events occur (PEN-2 appears to reside in the Golgi) and the subcellular destination/distribution of presenilin and its associated factors. Ultimately, it would be important to establish that overexpression of PS and the complex has an impact on promoting γ-secretase processing of APP-CTF, Notch S2/NEXT, or other substrates.

Comments on Primary Papers for this Article

  1. One of several studies that show that APH-1 and PEN-2 function together in the gamma-secretase complex

    View all comments by Eddie Koo

References

News Citations

  1. New Playmates on the Presenilin/γ-Secretase Playground
  2. Stockholm: Aph-1 and Pen-2: New Names in the Presenilin Debate Complete the Complex?

Paper Citations

  1. . 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.
  2. . 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.
  3. . 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.
  4. . PEN-2 and APH-1 coordinately regulate proteolytic processing of presenilin 1. J Biol Chem. 2003 Mar 7;278(10):7850-4. PubMed.

Further Reading

Papers

  1. . 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.
  2. . PEN-2 and APH-1 coordinately regulate proteolytic processing of presenilin 1. J Biol Chem. 2003 Mar 7;278(10):7850-4. PubMed.

News

  1. Budding RNAi Therapies, APP Protein Interaction Map Impress at Meeting

Primary Papers

  1. . 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.
  2. . PEN-2 and APH-1 coordinately regulate proteolytic processing of presenilin 1. J Biol Chem. 2003 Mar 7;278(10):7850-4. PubMed.