23 May 2012. 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.
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 14. Abstract