This very interesting study by Tolia et al. provides biochemical evidence for the existence of a water-filled cavity in the catalytic core of presenilin. The authors convincingly demonstrate by using a cysteine scanning mutagenesis strategy, combined with sulfhydryl-specific chemical cross-linkers, that the two aspartates (D257 and D385), which reside within the catalytic core of γ-secretase, have access to water within the lipid bilayer. While the requirement for water molecules to accomplish peptide bond hydrolysis is expected, the authors provide the first experimental evidence that the catalytic aspartic residues are both accessible to water and face each other with a maximal distance of 5.2A°.

We recently reported the 3D electron microscopic structure of the purified, proteolytically active γ-secretase, which revealed a large aqueous intramembrane chamber of 20-40A° in length, consistent with a proteinaceous proteolytic site that is occluded from the hydrophobic environment of the lipid bilayer (1). The present study by Tolia et al. is in accordance with and verifies...  Read more

This very interesting study by Tolia et al. provides biochemical evidence for the existence of a water-filled cavity in the catalytic core of presenilin. The authors convincingly demonstrate by using a cysteine scanning mutagenesis strategy, combined with sulfhydryl-specific chemical cross-linkers, that the two aspartates (D257 and D385), which reside within the catalytic core of γ-secretase, have access to water within the lipid bilayer. While the requirement for water molecules to accomplish peptide bond hydrolysis is expected, the authors provide the first experimental evidence that the catalytic aspartic residues are both accessible to water and face each other with a maximal distance of 5.2A°.

We recently reported the 3D electron microscopic structure of the purified, proteolytically active γ-secretase, which revealed a large aqueous intramembrane chamber of 20-40A° in length, consistent with a proteinaceous proteolytic site that is occluded from the hydrophobic environment of the lipid bilayer (1). The present study by Tolia et al. is in accordance with and verifies our data, and interestingly proposes that Y389 is involved in the catalytic mechanism through the formation of a hydrogen bond with the side chain of D385.

Interestingly, a new concept is proposed in this paper. The authors found that the membrane-permeable and biotinylated sulfhydryl-specific cross-linker did not label D257C (inaccessible to water?), whereas a homobifunctional sulfhydryl-specific cross-linker did target both D257C and D385C (both accessible to water). A model is proposed to explain this apparent discrepancy, based on functionally distinct complexes. This model suggests that D257 of PS1 remains inaccessible to water in inactive γ-secretase, but does get access to water during hydrolysis in active γ-secretase, possibly upon conformational modulations within TMD6 initiated after binding of the substrates.

In a different vein, I also found it very interesting that the F388C and Y256C substitutions cause a remarkable decrease in the levels of Aβ40 without affecting the Aβ42 levels. First, it is consistent with the observation that several PS1 and PS2 clinical mutations, including PS1δE9, PS1-L166P and PS2-N141I, cause partial loss of function of γ-secretase without affecting the cleavage that generates Aβ42. It also explains the relative increase in the Aβ42/Aβ40 ratio (2). When taken together, the data suggest that the Aβ42 activity is much more “stable” when compared to the Aβ40 activity—could this difference in “stability” be a major and/or common cause of AD including the sporadic forms of the disease? In this regard, F388 has not (yet?) been identified as a clinical mutation, whereas Y256S is a known FAD mutation (3).

In addition, the F388C substitution affects the cleavage of an APP-based substrate without significantly affecting Notch processing and the production of NICD. This apparently supports the idea that γ-secretase can be directly affected in a way that allows selective inhibition of total Aβ production without interfering with Notch processing. Thus, the disturbing specter of Notch-associated toxicities previously reported in relation to in vivo administration of γ-secretase inhibitors may be avoidable. We have recently reported chemical compounds that specifically target PS1-CTF and modulate the generation of Aβ while sparing Notch (4). Investigating whether such compounds target the F388 region within PS1 could provide a structural explanation for how substrates can be differentially handled in the catalytic site of γ-secretase.

References:
1. Lazarov VK, Fraering PC, Ye W, Wolfe MS, Selkoe DJ, Li H. Electron microscopic structure of purified, active gamma-secretase reveals an aqueous intramembrane chamber and two pores. Proc Natl Acad Sci U S A. 2006 May 2;103(18):6889-94. Epub 2006 Apr 24. Abstract

2. Bentahir M, Nyabi O, Verhamme J, Tolia A, Horre K, Wiltfang J, Esselmann H, De Strooper B. Presenilin clinical mutations can affect gamma-secretase activity by different mechanisms. J Neurochem. 2006 Feb;96(3):732-42. Epub 2006 Jan 9. Abstract

3. Miklossy J, Taddei K, Suva D, Verdile G, Fonte J, Fisher C, Gnjec A, Ghika J, Suard F, Mehta PD, McLean CA, Masters CL, Brooks WS, Martins RN. Two novel presenilin-1 mutations (Y256S and Q222H) are associated with early-onset Alzheimer's disease. Neurobiol Aging. 2003 Sep;24(5):655-62. Abstract

4. Fraering PC, Ye W, LaVoie MJ, Ostaszewski BL, Selkoe DJ, Wolfe MS. gamma-Secretase substrate selectivity can be modulated directly via interaction with a nucleotide-binding site. J Biol Chem. 2005 Dec 23;280(51):41987-96. Epub 2005 Oct 19. Abstract

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