Wenjuan Ye

γ-Secretase is a membrane protein complex comprised of presenilin, nicastrin, aph-1, and pen-2

γ-Secretase catalyzes the intramembrane proteolysis of Notch, β-amyloid precursor protein, and other substrates as part of a new signaling paradigm and as a key step in the pathogenesis of Alzheimers disease. This unusual protease has eluded identification, though evidence suggests that the presenilin heterodimer comprises the catalytic site and that a highly glycosylated form of nicastrin associates with it. The formation of presenilin heterodimers from the holoprotein is tightly gated by unknown limiting cellular factors. Here we show that Aph-1 and Pen-2, two recently identified membrane proteins genetically linked to γ-secretase, associate directly with presenilin and nicastrin in the active protease complex. Coexpression of all four proteins leads to marked increases in presenilin heterodimers, full glycosylation of nicastrin, and enhanced γ-secretase activity. These findings suggest that the four membrane proteins comprise the limiting components of γ-secretase and coassemble to form the active enzyme in mammalian cells.

Activity-dependent isolation of the presenilin– γ-secretase complex reveals nicastrin and a γ substrate

Presenilin heterodimers apparently contain the active site of γ-secretase, a polytopic aspartyl protease involved in the transmembrane processing of both the Notch receptor and the amyloid-β precursor protein. Although critical to embryonic development and the pathogenesis of Alzheimers disease, this protease is difficult to characterize, primarily because it is a multicomponent complex of integral membrane proteins. Here the functional γ-secretase complex was isolated by using an immobilized active site-directed inhibitor of the protease. Presenilin heterodimers and nicastrin bound specifically to this inhibitor under conditions tightly correlating with protease activity, whereas several other presenilin-interacting proteins (β-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated γ-secretase activity from detergent-solubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-β precursor protein substrate of γ-secretase, was also quantitatively associated with the complex. These results provide direct biochemical evidence that nicastrin is a member of the active γ-secretase complex, indicate that β-catenin, calsenilin, and presenilin-associated protein are not required for γ activity, and suggest an unprecedented mechanism of substrate–protease interaction.

Assembly of the γ-Secretase Complex Involves Early Formation of an Intermediate Subcomplex of Aph-1 and Nicastrin

The γ-secretase complex is an unusual multimeric protease responsible for the intramembrane cleavage of a variety of type 1 transmembrane proteins, including the β-amyloid precursor protein and Notch. Genetic and biochemical data have revealed that this protease consists of the presenilin heterodimer, a highly glycosylated form of nicastrin, and the recently identified gene products, Aph-1 and Pen-2. Whereas current evidence supports the notion that presenilin comprises the active site of the protease and that the other three components are members of the active complex required for proteolytic activity, the individual roles of the three co-factors remain unclear. Here, we demonstrate that endogenous Aph-1 interacts with an immature species of nicastrin, forming a stable intermediate early in the assembly of the γ-secretase complex, prior to the addition of presenilin and Pen-2. Our data suggest 1) that Aph-1 is involved in the early stages of γ-secretase assembly through the stabilization and perhaps glycosylation of nicastrin and by scaffolding nicastrin to the immature γ-secretase complex, and 2) that presenilin, and later Pen-2, bind to this intermediate during the formation of the mature protease.

Direct and Potent Regulation of γ-Secretase by Its Lipid Microenvironment

γ-Secretase is an unusual and ubiquitous aspartyl protease with an intramembrane catalytic site that cleaves many type-I integral membrane proteins, most notably APP and Notch. Several reports suggest that cleavage of APP to produce the Aβ peptide is regulated in part by lipids. As γ-secretase is a multipass protein complex with 19 transmembrane domains, it is likely that the local lipid composition of the membrane can regulate γ-activity. To determine the direct contribution of the lipid microenvironment to γ-secretase activity, we purified the human protease from overexpressing mammalian cells, reconstituted it in vesicles of varying lipid composition, and examined the effects of individual phospholipids, sphingolipids, cholesterol, and complex lipid mixtures on substrate cleavage. A conventional γ-activity assay was modified to include a detergent-removal step to facilitate proteoliposome formation, and this increased baseline activity over 2-fold. Proteoliposomes containing sphingolipids significantly increased γ-secretase activity over a phosphatidylcholine-only baseline, whereas the addition of phosphatidylinositol significantly decreased activity. Addition of soluble cholesterol in the presence of phospholipids and sphingolipids robustly increased the cleavage of APP- and Notch-like substrates in a dose-dependent manner. Reconstitution of γ-secretase in complex lipid mixtures revealed that a lipid raft-like composition supported the highest level of activity compared with other membrane compositions. Taken together, these results demonstrate that membrane lipid composition is a direct and potent modulator of γ-secretase and that cholesterol, in particular, plays a major regulatory role.

Cryoelectron microscopy structure of purified gamma-secretase at 12 A resolution.

Gamma-secretase, an integral membrane protein complex, catalyzes the intramembrane cleavage of the beta-amyloid precursor protein (APP) during the neuronal production of the amyloid beta-peptide. As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimers disease. Existing biochemical studies conflict on the oligomeric assembly state of the protease complex, and its detailed structure is not known. Here, we report that purified active human gamma-secretase in digitonin has a total molecular mass of approximately 230 kDa when measured by scanning transmission electron microscopy. This result supports a complex that is monomeric for each of the four component proteins. We further report the three-dimensional structure of the gamma-secretase complex at 12 A resolution as obtained by cryoelectron microscopy and single-particle image reconstruction. The structure reveals several domains on the extracellular side, three solvent-accessible low-density cavities, and a potential substrate-binding surface groove in the transmembrane region of the complex.

γ-Secretase Substrate Selectivity Can Be Modulated Directly via Interaction with a Nucleotide-binding Site

γ-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid β-protein (Aβ) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of γ-secretase: heterodimeric presenilin composed of its N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified γ-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Aβ, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified γ-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified γ-secretase preparations; the labeling was blocked by ATP itself and APP-selective γ-secretase inhibitors. We concluded that a nucleotide-binding site exists within γ-secretase, and certain compounds that bind to this site can specifically modulate the generation of Aβ while sparing Notch. Drugs targeting the γ-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease.

Complex N-linked Glycosylated Nicastrin Associates with Active γ-Secretase and Undergoes Tight Cellular Regulation

The intramembranous proteolysis of Notch and the amyloid precursor protein by γ-secretase exemplifies an unusual and newly recognized mechanism of signal transduction in multicellular organisms. Here, we show that only a form of nicastrin (NCT) containingN-linked complex oligosaccharides is present in active γ-secretase complexes. Overexpression of NCT does not generate more of this mature protein, a phenomenon analogous to the strictly regulated formation of mature presenilin heterodimers from immature holoprotein. The absence of presenilin severely limits the maturation of NCT, yet combined overexpression of both proteins does not increase respective mature types. Taken together, our findings describe unusual regulatory features of this key signaling protease: the association of NCT with γ-secretase is tightly regulated via glycosylation; at least one other cofactor exists; the least abundant member of the complex becomes limiting; and the cofactor that serves this role may vary by cell type.

Effects of Membrane Lipids on the Activity and Processivity of Purified γ-Secretase

The 19-transmembrane multisubunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimers disease (AD) by intramembrane proteolysis of the β-amyloid precursor protein (APP). Despite substantial advances in elucidating how this protein complex functions, the effect of the local membrane lipid microenvironment on γ-secretase cleavage of substrates is still poorly understood. Using detergent-free proteoliposomes to reconstitute purified human γ-secretase, we examined the effects of fatty acyl (FA) chain length, saturation and double-bond isomerization, and membrane lipid polar headgroups on γ-secretase function. We analyzed γ-secretase activity and processivity [i.e., sequential cleavages in the APP transmembrane domain that convert longer Aβ species (e.g., Aβ(46)) into shorter ones (e.g., Aβ(40))] by quantifying the APP intracellular domain (AICD) and various Aβ peptides, including via a bicine/urea gel system that detects multiple Aβ lengths. These assays revealed several trends. (1) Switching from a cis to a trans isomer of a monounsaturated FA chain in phosphatidylcholine (PC) increased γ-activity, did not affect Aβ(42):Aβ(40) ratios, but decreased the ratio of long (≥42) versus short (≤41) Aβ peptides. (2) Increasing the FA carbon chain length (14, 16, 18, and 20) increased γ-activity, reduced longer Aβ species, and reduced the Aβ(42):Aβ(40) ratio. (3) Shifting the position of the double bond in 18:1(Δ9-cis) PC to the Δ6 position substantially reduced activity. (4) Gangliosides increased γ-activity but decreased processivity, thus elevating the Aβ(42):Aβ(40) ratio. (5) Phosphatidylserine decreased γ-activity but increased processivity. (6) Phosphatidylinositol strongly inhibited γ-activity. Overall, our results show that subtle changes in membrane lipid composition can greatly influence γ-secretase activity and processivity, suggesting that relatively small changes in lipid membrane composition may affect the risk of AD at least as much as presenilin or APP mutations do.

Rapid purification of active γ‐secretase, an intramembrane protease implicated in Alzheimer’s disease

γ‐Secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid‐β precursor protein generates the amyloid‐β protein (Aβ) of Alzheimer’s disease, partially inhibiting γ‐secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of γ‐secretase, but at low resolution (15 Å). The limited amount of purified γ‐secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X‐ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S‐20) that overexpresses strikingly high levels of all four γ‐secretase components (presenilin, nicastrin, Aph‐1 and Pen‐2). We then used these cells to develop a rapid protocol for the high‐grade purification of proteolytically active γ‐secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme.

Discovery of Notch-Sparing γ-Secretase Inhibitors

Overwhelming evidence supports a central role for the amyloid β-peptide (Aβ) in the pathogenesis of Alzheimers disease (AD), and the proteases that produce Aβ from its precursor protein APP are top targets for therapeutic intervention. Considerable effort has focused on targeting γ-secretase, which generates the C-terminus of Aβ; however, γ- secretase inhibitors cause serious toxicities due to interference with the Notch signaling pathway. We have been working toward compounds that directly alter γ-secretase activity to reduce Aβ production without affecting the proteolysis of Notch. Using purified enzyme and substrate, we have shown that γ-secretase can be selectively inhibited in this way by naphthyl-substituted γ-aminoketones and γ-aminoalcohols. These early hits, however, suffered from chemical instability and/or poor potency. Iterative design, synthesis and evaluation have led to the discovery of Notch-sparing γ-secretase inhibitors with substantially increased potencies in biochemical and cellular assays. These compounds are of low molecular weight and are under evaluation for drug-like properties. The discovery and development of these compounds will be discussed.