␥-Secretase catalyzes the intramembrane proteolysis of Notch, ␤-amyloid precursor protein, and other substrates as part of a new signaling paradigm and as a key step in the pathogenesis of Alzheimer's 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 ␥-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 ␥-secretase activity. These findings suggest that the four membrane proteins comprise the limiting components of ␥-secretase and coassemble to form the active enzyme in mammalian cells.

Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

Presenilin heterodimers apparently contain the active site of ␥-secretase, a polytopic aspartyl protease involved in the transmembrane processing of both the Notch receptor and the amyloid-␤ precursor protein. Although critical to embryonic development and the pathogenesis of Alzheimer's disease, this protease is difficult to characterize, primarily because it is a multicomponent complex of integral membrane proteins. Here the functional ␥-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 (␤-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated ␥-secretase activity from detergentsolubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-␤ precursor protein substrate of ␥-secretase, was also quantitatively associated with the complex. These results provide direct biochemical evidence that nicastrin is a member of the active ␥-secretase complex, indicate that ␤-catenin, calsenilin, and presenilin-associated protein are not required for ␥ activity, and suggest an unprecedented mechanism of substrate-protease interaction.

Gamma-secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a approximately 100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high Abeta40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.

␥-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 A␤ peptide is regulated in part by lipids. As ␥-secretase is a multipass protein complex with 19 transmembrane domains, it is likely that the local lipid composition of the membrane can regulate ␥-activity. To determine the direct contribution of the lipid microenvironment to ␥-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 ␥-activity assay was modified to include a detergentremoval step to facilitate proteoliposome formation, and this increased baseline activity over 2-fold. Proteoliposomes containing sphingolipids significantly increased ␥-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 ␥-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 ␥-secretase and that cholesterol, in particular, plays a major regulatory role. Alzheimer disease (AD)3 is a neurodegenerative disorder marked by progressive memory loss and cognitive failure. A major pathogenic feature of AD is the accumulation of amyloid ␤-protein (A␤) in brain regions serving memory and cognition (1). A␤ is generated by sequential proteolytic cleavages of the ␤-amyloid precursor protein (APP) by ␤-and ␥-secretases. ␥-Secretase is a unique aspartyl protease that comprises a multiprotein complex composed of presenilin, nicastrin, Aph-1, and Pen-2 (2-4). The novel intramembranous catalytic site of ␥-secretase is now known to be required for the processing of a wide range of type-I transmembrane proteins, most notably APP and the Notch receptor, and the list is rapidly expanding (for reviews, see Refs. 5 and 6). Therefore, it is of great interest to gain a better understanding of factors that can regulate ␥-secretase cleavage activity and substrate specificity.Although scientific attention has focused on identifying and characterizing proteins that affect ␥-secretase activity or trafficking, there has been far less study of the lipid requirements and lipid modulation of the activity of ␥-secretase. Because ␤-secretase, ␥-secretase, and APP are all integral membrane proteins implicated in AD, it is highly likely that their functions are sensitive to the surrounding lipid environment. Seve...

Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.

The ␥-secretase complex is an unusual multimeric protease responsible for the intramembrane cleavage of a variety of type 1 transmembrane proteins, including the ␤-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 ␥-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 ␥-secretase assembly through the stabilization and perhaps glycosylation of nicastrin and by scaffolding nicastrin to the immature ␥-secretase complex, and 2) that presenilin, and later Pen-2, bind to this intermediate during the formation of the mature protease.