16 Sep 2016

Ever since researchers discovered that the γ-secretase complex cleaves amyloid precursor protein (APP), they have puzzled over the precise mechanism. In particular, they cannot explain why the enzyme usually, though not always, lops off three amino acids at a time. In the 30 August eLife online, scientists led by David Bolduc, Michael Wolfe, and Dennis Selkoe at Brigham and Women’s Hospital, Boston, present evidence that the enzyme contains three substrate-binding pockets that accommodate three sequential amino acids in a peptide chain. These pockets stabilize a transition state leading to proteolytic cleavage upstream. However, because one of these pockets is small, it holds on poorly to bulky amino acids, and this causes the secretase to skip to the next amino acid troika, claim the researchers. Supported by cleavage patterns of artificial forms of APP, their hypothesis explains the tripeptide cleavage activity of the secretase complex and why some familial APP mutations increase the Aβ42/40 ratio in the brain.

“The work of Bolduc et al. represents a major advance in our understanding of catalysis by the γ-secretase enzyme,” noted Charles Sanders, Vanderbilt University, Nashville, in an accompanying editorial. Other γ-secretase experts were unavailable for comment before this article posted.

γ-Secretase plays a central role in regulated intramembrane proteolysis, cleaving the transmembrane domains of hundreds of proteins, including Notch and APP. BACE (β-secretase) processing of the latter exposes a 99-amino-acid C-terminal fragment to γ-secretase. This C-99 retains the transmembrane domain, which γ-secretase first cuts internally between amino acids 48 and 49, or 49 and 50 (so-called epsilon cleavage). This releases into the cytosol either of two APP intracellular domains, AICD49-99 or AICD50-99. Then, γ-secretase proceeds in carboxypeptidase fashion to chop off C-terminal amino acids from the remaining transmembrane domain until Aβ peptides are finally released into the lumen.

Seminal work by Yasuo Ihara at Doshisha University, Kizugawa, Japan, revealed that the carboxypeptidase-like cleavage of γ-secretase occurs in three- and sometimes four-amino acid increments (see Takami et al., 2009). This helped explain why both Aβ42 and Aβ40 are predominantly formed from the same protein: Epsilon cleavage at position 48 yields Aβ45, then Aβ42, while initial cleavage at amino acid 49 yields Aβ46, Aβ43, and finally Aβ40.

Why does γ-secretase cut every third amino acid when many carboxypeptidases tend to cut one amino acid at a time? A clue came from inhibitor studies. Previously, researchers led by Wolfe found that chemical inhibitors worked well if they mimicked tripeptides, but less well if they were dipeptide or four-amino-acid analogs (see Esler et al., 2004). Bolduc realized that there must be only three substrate-binding pockets in the protease. Typically, four are found in transmembrane proteases, including the widely studied rhomboid, which cleaves in single-amino-acid increments. This hinted that the three pockets stabilize a transition state necessary for catalytic cleavage in three-amino-acid increments.

If this were true, then destabilizing this tripeptide transition state should disrupt the cleavage pattern. To test this, Bolduc capitalized on another observation Wolfe had made, namely that tripeptide-like inhibitors of γ-secretase worked poorly if the central amino acid was bulky. Bolduc predicted that the S2 substrate pocket was smaller that the S1 and S3 pockets, and therefore, sticking large aromatic amino acids in the second position would disrupt catalysis. Subsequent experiments suggested that he was correct. 

Bolduc found that putting a large amino acid—phenylalanine—in the second peptide position impeded catalysis sufficiently to shift cleavage by one or more amino acids. For example, placing phenylalanine at Aβ position 44 weakened proteolysis between alanine 42 and tyrosine 43, suppressing production of Aβ42 and favoring the Aβ40 pathway (see figure above). Phenylalanine three amino acids downstream at position 47 had the same effect. Similarly, if placed at positions 48 or 45, this aromatic amino acid suppressed production of Aβ43 and Aβ40, increasing the Aβ42/40 ratio. 

According to the authors, the findings explain why the I45F Iberian mutation, at position 716 of APP, causes early onset familial AD. This mutation was previously found to increase the Aβ42/40 ratio, but at that time it was not clear why (see Lichtenthaler et al., 1999). 

What about other FAD mutations? None of the other known mutations substitute an aromatic amino acid at the S2 position. So how else do they cause AD? Researchers have theorized that these mutations somehow alter the initial epsilon cleavage or tweak carboxypeptidase cleavage in such a way as to favor the Aβ42 pathway. Bolduc and colleagues were able to rule out the latter by studying FAD mutations in combination with phenylalanine substitutions at the epsilon cleavage site. Placing phenylalanine at positon 50 suppressed cleavage at amino acid 48, locking in at the 49 position, and in turn reducing the Aβ42/40 ratio. Most of the FAD mutations were unable to prevent this reduction, indicating that they cannot affect subsequent carboxypeptidase cleavages in favor of Aβ42.

Could this new insight lead to better γ-secretase modulators? “Not directly, but we can’t modulate catalysis if we don’t understand the mechanism,” said Bolduc. “The next step the field needs to take is to rationally design modulators to shift the Aβ42/40 ratio,” he suggested.—Tom Fagan

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References

Alzpedia Citations

1. APP

Mutations Citations

1. APP I716F (Iberian)

Paper Citations

1. Takami M, Nagashima Y, Sano Y, Ishihara S, Morishima-Kawashima M, Funamoto S, Ihara Y. gamma-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment. J Neurosci. 2009 Oct 14;29(41):13042-52. PubMed.
2. Esler WP, Das C, Wolfe MS. Probing pockets S2-S4' of the gamma-secretase active site with (hydroxyethyl)urea peptidomimetics. Bioorg Med Chem Lett. 2004 Apr 19;14(8):1935-8. PubMed.
3. Lichtenthaler SF, Wang R, Grimm H, Uljon SN, Masters CL, Beyreuther K. Mechanism of the cleavage specificity of Alzheimer's disease gamma-secretase identified by phenylalanine-scanning mutagenesis of the transmembrane domain of the amyloid precursor protein. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):3053-8. PubMed.

Further Reading

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