Enzyme-substrate hybrid ß-sheet controls geometry and water access to the γ-secretase active site.

γ-Secretase is an aspartyl intramembrane protease that cleaves the amyloid precursor protein (APP) involved in Alzheimer's disease pathology and other transmembrane proteins. Substrate-bound structures reveal a stable hybrid ß-sheet immediately following the substrate scissile bond consisting of ß1 and ß2 from the enzyme and ß3 from the substrate. Molecular dynamics simulations and enhanced sampling simulations demonstrate that the hybrid ß-sheet stability is strongly correlated with the formation of a stable cleavage-compatible active geometry and it also controls water access to the active site. The hybrid ß-sheet is only stable for substrates with 3 or more C-terminal residues beyond the scissile bond. The simulation model allowed us to predict the effect of Pro and Phe mutations that weaken the formation of the hybrid ß-sheet which were confirmed by experimental testing. Our study provides a direct explanation why γ-secretase preferentially cleaves APP in steps of 3 residues and how the hybrid ß-sheet facilitates γ-secretase proteolysis.

Different transmembrane domains determine the specificity and efficiency of the cleavage activity of the γ-secretase subunit presenilin.

The γ-secretase complex catalyzes the intramembrane cleavage of C99, a carboxy-terminal fragment of the amyloid precursor protein. Two paralogs of its catalytic subunit presenilin (PS1 and PS2) are expressed which are autocatalytically cleaved into an N-terminal and a C-terminal fragment during maturation of γ-secretase. In this study, we compared the efficiency and specificity of C99 cleavage by PS1- and PS2-containing γ-secretases. Mass spectrometric analysis of cleavage products obtained in cell-free and cell-based assays revealed that the previously described lower amyloid-ß (Aß)38 generation by PS2 is accompanied by a reciprocal increase in Aß37 production. We further found PS1 and PS2 to show different preferences in the choice of the initial cleavage site of C99. However, the differences in Aß38 and Aß37 generation appear to mainly result from altered subsequent stepwise cleavage of Aß peptides. Apart from these differences in cleavage specificity, we confirmed a lower efficiency of initial C99 cleavage by PS2 using a detergent-solubilized γ-secretase system. By investigating chimeric PS1/2 molecules, we show that the membrane-embedded, nonconserved residues of the N-terminal fragment mainly account for the differential cleavage efficiency and specificity of both presenilins. At the level of individual transmembrane domains (TMDs), TMD3 was identified as a major modulator of initial cleavage site specificity. The efficiency of endoproteolysis strongly depends on nonconserved TMD6 residues at the interface to TMD2, i.e., at a putative gate of substrate entry. Taken together, our results highlight the role of individual presenilin TMDs in the cleavage of C99 and the generation of Aß peptides.

Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis.

Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here, we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheimer's disease-associated presenilin/γ-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition-state analog inhibitor, the bilayer promoted the formation of the enzyme's catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.

Cutting proteins into pieces is a crucial process in the cell, allowing several important processes to take place, including cell differentiation (which allows cells to develop into specific types), cell death, protein quality control, or even where in the cell a protein will end up. However, the specialized proteins that carry out this task, known as proteases, can also be involved in the development of disease. For example, in the brain, a protease called γ-secretase cuts up the amyloid-ß protein precursor, producing toxic forms of amyloid-ß peptides that are widely believed to cause Alzheimer's disease. Proteases like γ-secretase carry out their role in the membrane, the layer of fats (also known as lipids) that forms the outer boundary of the cell. The environment in this area of the cell can influence the activity of proteases, but it is poorly understood how this happens. One way to address this question would be to compare the activity of γ-secretase in the lipid environment of the membrane to its activity when it is entirely surrounded by different molecules, such as detergent molecules. Unfortunately, γ-secretase is not active when it is removed from its lipid environment by a detergent, making it difficult to perform this comparison. To overcome this issue, Feilen et al. chose to study PSH, a protease similar to γ-secretase that produces the same amyloid-ß peptides but remains active in detergent. When Feilen et al. mixed PSH with lipid molecules like those found in the membrane and amyloid-ß precursor protein, PSH produced amyloid-ß peptides including those that are thought to cause Alzheimer's. However, when a detergent was substituted for the lipid molecules this led to longer amyloid-ß peptides than usual, indicating that PSH was not able to cut proteins as effectively. The change in environment appeared to reduce PSH's ability to progressively trim small segments from the peptides. Computer modelling of the protease's structure in lipids versus detergent supported the experimental findings: the model predicted that the areas of PSH important for recognizing and cutting other proteins would be more stable in the membrane compared to the detergent. These results indicate that the cell membrane plays a vital role in the stability of the active regions of proteases that are cleaving in this environment. In the future, this could help to better understand how changes to the lipid molecules in the membrane may contribute to the activity of γ-secretase and its role in Alzheimer's disease.

Substrate recruitment by γ-secretase.

γ-Secretase is a membrane-embedded protease complex that is crucial for many physiological processes throughout life. Due to its pivotal role in the etiology of Alzheimer's disease (AD), in particular the familial forms of the disease, the enzyme is one of the most studied intramembrane proteases and an important drug target. By cleaving a C-terminal fragment of the ß-amyloid precursor protein (APP), γ-secretase generates several amyloid ß-peptide (Aß) species including longer, neurotoxic forms such as Aß42 that are a widely believed to trigger AD. Besides APP, γ-secretase cleaves numerous other substrates including most prominently Notch1, whose cleavage by γ-secretase is essential for cell differentiation and affected in certain types of cancer. In this review, we will describe the exciting progress made in our understanding of how the γ-secretase complex recognizes and recruits its substrates to its catalytic subunit presenilin for their intramembrane proteolytic cleavage. This complicated process is not well understood and only recently insights from biochemical studies and structural biology are beginning to reveal this secret of γ-secretase.

Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction.

Physiological function and pathology of the Alzheimer's disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.