Recasting Nicastrin as Substrate Snare

Nicastrin is one of four proteins in the γ-secretase complex but other than that, precious little is known about exactly what it does. Some studies claim that its sole job is to promote formation of the complex. Others suggest that in the secretase’s processing of amyloid precursor protein (APP) and other transmembrane fodder, nicastrin binds substrates. Now, new evidence lends support to the substrate-binding argument. In the May 14 Proceedings of the National Academy of Sciences, Sangram Sisodia, University of Chicago, Illinois, and colleagues describe how they stumbled upon a nicastrin domain that seems to control substrate binding, which would make two found so far.

"There hasn't been any synthesis of opinion in the field about what nicastrin does," said Sisodia. While his results do not settle the matter, "people might be swayed by the evidence in favor of a role of nicastrin in substrate binding," he told Alzforum.

γ-Secretase comprises nicastrin, presenilin (PS1 or PS2), presenilin enhancer 2 (better known as Pen-2), and anterior pharynx-defective 1 (Aph-1). The complex cuts APP C-terminal fragments into Aβ. It also processes a slew of other proteins, including Notch, which is essential for development. How γ-secretase recognizes its substrates remains unclear, though in 2005, Gang Yu, University of Texas Southwestern Medical Center, Dallas, and colleagues suggested that was nicastrin's role (see ARF related news story). For this proposed function, the group identified a nicastrin ectodomain region they named the DAP domain. Alas, later studies implied that nicastrin plays no part in substrate recognition (see ARF related news story on Zhao et al., 2010), suggesting instead that nicastrin helped assemble the γ-secretase complex (see Chávez-Gutiérrez et al., 2008).

To settle the matter, first author Xulun Zhang and colleagues raised antibodies that would bind and stabilize nicastrin for crystallization. Some fit the bill. One, Fab2, bound to the nicastrin ectodomain region that lies on the C-terminal side of the DAP domain. Fab2 blocked γ-secretase activity in a test tube, which the authors took to mean that the bound area was necessary for γ-secretase function. Using a database search, Zhang found that the Fab2 binding site is homologous to the previously described tetratricopeptide repeat (TPR) domain that is important for other protein-protein interactions (see D'Andrea and Regan, 2003). "That gave us even more incentive to pursue this domain, because we thought we could identify important residues needed for substrate binding," said Sisodia.

To see if this TPR domain bound γ-secretase substrates, the researchers mutated two amino acids within the region predicted to be important for protein binding. One of those mutations suppressed Notch processing. Several others also disrupted cleavage, but none prevented γ-secretase complex formation, confirming for the authors that the TPR-like region functions in peptide binding.

"As a whole, this is very interesting—a new motif in nicastrin has been identified that may be important for substrate recognition," said Michael Wolfe, Brigham and Women's Hospital, Boston, Massachusetts. There are other possible explanations for the results that give him pause and provide grounds for further study, Wolfe said. For instance, rather than binding to and specifically interfering with a domain necessary for substrate recognition, Fab2's size and position could have blocked γ-secretase activity. Sisodia acknowledged that it is a fair point, but noted that a similarly sized Fab12 antibody also bound nearby and did not block the enzyme's action.

The search for binding domains in nicastrin is important, because they could have different affinities for the complex’s various substrates, opening up new possibilities of differentially regulating those sites, said Yu. That could point to new therapeutic targets for AD that change the processing of APP while leaving Notch and other proteins alone. Several γ-secretase modulators, which are designed to do just that, are in various stages of clinical development (see ARF related news story). To date, a lot of effort has been put into manipulating presenilin, which does the actual cleaving, while nicastrin has taken a backseat, Yu said. "Nicastrin deserves to be looked at," he suggested. "I would argue that the best way to manage γ-secretase is to manipulate the mechanism that differentially recognizes all its different substrates."

Sisodia and his group are currently looking for more antibodies that bind nicastrin. They have found some that bind in the same domain as Fab2, but do not overlap with its epitope. "I would predict that there would be other synthetic antibodies that can bind to different domains of nicastrin," he said, some of which "could affect processing of one substrate versus another.”—Gwyneth Dickey Zakaib

Comments

  1. We read with great interest the manuscript of Zhang et al. They provide preliminary evidence for a predicted novel peptide-binding region in the ectodomain of nicastrin (Nct), which could have a potential role in substrate recruitment to the γ-secretase complex.

    We would like to comment on a few specific points in the manuscript:

    1. While the inhibitory effect of the anti-Nct antibody and the mutagenesis data provide evidence for the involvement of Nct ectodomain in γ-secretase activity and suggest a possible role for Nct in substrate recruitment, alternative interpretations, such as steric hindrance caused by the binding of the antibody or effects on the overall conformation of the complex, are also possible, and have not been excluded or discussed in this study. As the authors point out, it is likely that only crystallization studies will provide a definitive answer to this question, and, in fact, we completely agree with this conclusion.

    2. In our previous study, we proposed that the nicastrin ectodomain was necessary for maturation of γ-secretase, but not for its activity (Chávez-Gutiérrez et al., 2008). In reference to nicastrin as a substrate acceptor, the authors state of our findings:

    “This model was challenged by the demonstration that expression of NCT harboring an E333A mutation in NCT-/- cells leads to inefficient oligosaccharide maturation of the NCT variant and reduced levels of mature γ-secretase complexes, but that the specific activity of the remaining complexes was no different from that of complexes containing WT NCT. However, the methods used to calculate specific activity in the latter report had several significant technical limitations....”

    This statement reiterates a criticism initially raised by Dries and colleagues (Dries et al., 2009), and we would like to take the opportunity to respond to these comments here. This criticism emerges from a misunderstanding of how the specific activities (activity per unit mass of total γ-secretase) of the WT and E332A-mutant γ-secretase complexes were calculated.

    In Chavez-Gutiérrez et al., 2008, we stated that “for specific activities AICD was normalized against Ps1 CTF and PEN-2 present in the reactions.” Actually, AICD product was normalized to the average of Ps1 CTF and PEN-2 levels present in the reactions and not to the sum of Ps1 CTF and PEN-2 levels, as assumed by Dries et al. In fact, Figure 5 (Chávez-Gutiérrez et al., 2008) shows in white bars the levels of WT- and E332A-Nct γ-secretase complexes and, accordingly, the legend indicates “γ-secretase levels as average of Ps1 CTF and Pen-2 levels, assessed by Western blot in the in vitro reactions.” We would like to highlight that Ps1 CTF and PEN-2 levels are decreased to a very similar extent in the Nct mutant complexes, as indicated by the error bars (white bars). Therefore, our work shows that rescue of Nct-deficient cells with the E332A-Nct mutant restores approximately 10 percent of the -secretase complex levels, relative to rescue with WT-Nct. Most importantly, our study demonstrated that the -secretase complexes that contain the E332A-Nct are as active as the WT-Nct -secretase complexes.

    3. Zhang et al. also claim in the discussion that “Dries et al. established that complexes expressed in Sf9 cells that contained the E333A NCT were indeed inactive.”

    According to the model proposed by Shah et al., the E332-NCT is the counterpart of a glutamate residue (anionic binding site) involved in the recognition of the free amino group of substrates and inhibitors in aminopeptidases. Importantly, mutation of this glutamate in aminopeptidases leads to complete enzymatic inactivation (Luciani et al., 1998; Vazeux et al., 1998). Thus, if the Shah model is valid for Nct, a drastic effect on the activity of the mutant E332A-NCT γ-secretase complex is expected. However, in actuality, Dries et al. reported a mild effect on the activity of the E333A-NCT γ-secretase complex. The E332A substitution in NCT lowers AICD production by a maximum of 30 percent relative to the WT-NCT, when γ-secretase assembly is overcome in Sf9 cells. This result contrasts with the critical role assigned to the E333 residue by Shah et al. and, in fact, supports our findings (Chávez-Gutiérrez et al., 2008).

    In conclusion, it is clear that the substrate binding site in γ-secretase will remain a hot topic of research for the time to come, especially since it might be possible that specific modulation of such a site could provide alternative therapies. As said, we will probably need to wait for the atomic structure of the γ-secretase.

    References:

    Chávez-Gutiérrez L, Tolia A, Maes E, Li T, Wong PC, De Strooper B. Glu(332) in the Nicastrin ectodomain is essential for gamma-secretase complex maturation but not for its activity. J Biol Chem. 2008 Jul 18;283(29):20096-105. PubMed.

    Dries DR, Shah S, Han YH, Yu C, Yu S, Shearman MS, Yu G. Glu-333 of nicastrin directly participates in gamma-secretase activity. J Biol Chem. 2009 Oct 23;284(43):29714-24. PubMed.

    Luciani N, Marie-Claire C, Ruffet E, Beaumont A, Roques BP, Fournié-Zaluski MC. Characterization of Glu350 as a critical residue involved in the N-terminal amine binding site of aminopeptidase N (EC 3.4.11.2): insights into its mechanism of action. Biochemistry. 1998 Jan 13;37(2):686-92. PubMed.

    Shah S, Lee SF, Tabuchi K, Hao YH, Yu C, LaPlant Q, Ball H, Dann CE, Südhof T, Yu G. Nicastrin functions as a gamma-secretase-substrate receptor. Cell. 2005 Aug 12;122(3):435-47. PubMed.

    Vazeux G, Iturrioz X, Corvol P, Llorens-Cortes C. A glutamate residue contributes to the exopeptidase specificity in aminopeptidase A. Biochem J. 1998 Sep 1;334 ( Pt 2):407-13. PubMed.

    View all comments by Bart De Strooper

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References

News Citations

  1. Nicastrin—A DAPper Role in γ-Secretase
  2. Think You Know γ-secretase and p25? Think Again
  3. Barcelona: Allosteric γ Modulation Moves Toward Clinic

Paper Citations

  1. Zhao G, Liu Z, Ilagan MX, Kopan R. Gamma-secretase composed of PS1/Pen2/Aph1a can cleave notch and amyloid precursor protein in the absence of nicastrin. J Neurosci. 2010 Feb 3;30(5):1648-56. PubMed.
  2. Chávez-Gutiérrez L, Tolia A, Maes E, Li T, Wong PC, De Strooper B. Glu(332) in the Nicastrin ectodomain is essential for gamma-secretase complex maturation but not for its activity. J Biol Chem. 2008 Jul 18;283(29):20096-105. PubMed.
  3. D'Andrea LD, Regan L. TPR proteins: the versatile helix. Trends Biochem Sci. 2003 Dec;28(12):655-62. PubMed.

Further Reading

Papers

  1. Frykman S, Teranishi Y, Hur JY, Sandebring A, Goto Yamamoto N, Ancarcrona M, Nishimura T, Winblad B, Bogdanovic N, Schedin-Weiss S, Kihara T, Tjernberg LO. Identification of two novel synaptic γ-secretase associated proteins that affect amyloid β-peptide levels without altering Notch processing. Neurochem Int. 2012 Jul;61(1):108-18. PubMed.
  2. Gwon AR, Park JS, Arumugam TV, Kwon YK, Chan SL, Kim SH, Baik SH, Yang S, Yun YK, Choi Y, Kim S, Tang SC, Hyun DH, Cheng A, Dann CE, Bernier M, Lee J, Markesbery WR, Mattson MP, Jo DG. Oxidative lipid modification of nicastrin enhances amyloidogenic γ-secretase activity in Alzheimer's disease. Aging Cell. 2012 Mar 10; PubMed.
  3. Pamrén A, Wanngren J, Tjernberg LO, Winblad B, Bhat R, Näslund J, Karlström H. Mutations in nicastrin protein differentially affect amyloid beta-peptide production and Notch protein processing. J Biol Chem. 2011 Sep 9;286(36):31153-8. PubMed.

Primary Papers

  1. Zhang X, Hoey RJ, Lin G, Koide A, Leung B, Ahn K, Dolios G, Paduch M, Ikeuchi T, Wang R, Li YM, Koide S, Sisodia SS. Identification of a tetratricopeptide repeat-like domain in the nicastrin subunit of γ-secretase using synthetic antibodies. Proc Natl Acad Sci U S A. 2012 May 29;109(22):8534-9. PubMed.

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