Dysfunction of the intramembrane protease γ-secretase is thought to cause Alzheimer's disease (AD), with most AD-derived mutations mapping to the catalytic subunit presenilin 1 (PS1). Here we report an atomic structure of human γ-secretase at 3.4 Å resolution, determined by singleparticle cryo-electron microscopy. AD-derived mutations in PS1 affect residues at two hotspots, each located at the center of a distinct four transmembrane segment (TM) bundle. TM2, and to a lesser extent TM6, exhibit considerable flexibility, yielding a plastic active site and adaptable surrounding elements. The active site of PS1 is accessible from the convex side of the TM horseshoe, suggesting considerable conformational changes in nicastrin extracellular domain (ECD) following substrate recruitment. Aph-1 serves as a scaffold, anchoring the lone TM from nicastrin and supporting the flexible conformation of PS1. Ordered phospholipids stabilize the complex inside the membrane. Our structure serves as a molecular basis for mechanistic understanding of γ-secretase function.A hallmark of Alzheimer's disease (AD) is accumulation of β-amyloid plaque in patient brain 1 . The intramembrane protease γ-secretase is thought to contribute to AD development by generating β-amyloid peptides, particularly those that are prone to aggregation such as Aβ42 2-5 . A mature γ-secretase consists of four components: presenilin, Pen-2, nicastrin, and Aph-1 6 . Among these components, presenilin is responsible for the Aβ-producing proteolytic activity 7,8 .

Cleavage of amyloid precursor protein (APP) by the intramembrane protease γ-secretase is linked to Alzheimer’s disease (AD). We report an atomic structure of human γ-secretase in complex with a transmembrane (TM) APP fragment at 2.6-angstrom resolution. The TM helix of APP closely interacts with five surrounding TMs of PS1 (the catalytic subunit of γ-secretase). A hybrid β sheet, which is formed by a β strand from APP and two β strands from PS1, guides γ-secretase to the scissile peptide bond of APP between its TM and β strand. Residues at the interface between PS1 and APP are heavily targeted by recurring mutations from AD patients. This structure, together with that of γ-secretase bound to Notch, reveal contrasting features of substrate binding, which may be applied toward the design of substrate-specific inhibitors.

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A hallmark of Alzheimer's disease (AD) is the aggregation of β-amyloid peptides (Aβ) into amyloid plaques in patient brain. Cleavage of amyloid precursor protein (APP) by the intramembrane protease γ-secretase produces Aβ of varying lengths, of which longer peptides such as Aβ42 are thought to be more harmful. Increased ratios of longer Aβs over shorter ones, exemplified by the ratio of Aβ42 over Aβ40, may lead to formation of amyloid plaques and consequent development of AD. In this study, we analyzed 138 reported mutations in human presenilin-1 (PS1) by individually reconstituting the mutant PS1 proteins into anterior-pharynx-defective protein 1 (APH-1)aL-containing γ-secretases and examining their abilities to produce Aβ42 and Aβ40 in vitro. About 90% of these mutations lead to reduced production of Aβ42 and Aβ40. Notably, 10% of these mutations result in decreased Aβ42/Aβ40 ratios. There is no statistically significant correlation between the Aβ42/Aβ40 ratio produced by a γ-secretase variant containing a specific PS1 mutation and the mean age at onset of patients from whom the mutation was isolated.Alzheimer's disease | γ-secretase | Aβ peptides | cleavage activity | amyloid hypothesis T he first case of Alzheimer's disease (AD) was reported more than 100 y ago, and the brain of the deceased patient contained characteristic senile plaques (1). These plaques were found to be amyloid in nature by electron microscopy (2), and analysis of amino acid sequence revealed the amyloid to be derived from amyloid precursor protein (APP) (3). APP is first cleaved by β-secretase to produce a 99-residue transmembrane fragment C99, which then undergoes additional cleavages by γ-secretase to generate a series of amyloidogenic β-amyloid peptides (Aβ) with 39-43 amino acids (4, 5). The slightly longer Aβ peptides, particularly Aβ42 and Aβ43, are thought to be more prone to aggregation than the shorter ones such as Aβ40 (6-8). Indeed, the amyloid plaques contain mostly longer Aβ peptides such as Aβ42 (9).Familial cases of AD were found to be genetically linked to missense mutations in APP (10, 11). These observations, together with prior knowledge on amyloid plaques, prompted proposition of the amyloid hypothesis, which regards the formation of β-amyloid plaques as the cause of AD through apoptosis induction of neuronal cells (12). Consistent with this hypothesis, Aβ42 was found to induce cell death (13,14). Human presenilin-1 (PS1) was cloned and found to be targeted by missense mutations in early-onset familial AD (EOFAD) (15). Remarkably, these EOFAD-linked PS1 mutants led to elevated molar ratios of Aβ42 over Aβ40 both in cell lines and in the brains of transgenic animals (16), and the plasma levels of Aβ42 and Aβ43 in FAD patients with PS1 or APP mutations were significantly increased compared with those of healthy individuals (17). These observations led to refinement of the amyloid hypothesis, which identifies increased ratios of longer Aβ peptides over shorter ones as a key early event in AD development (18). The increa...

The γ-secretase complex, comprising presenilin 1 (PS1), Pen-2, Aph-1, and Nicastrin, is a membrane-embedded protease that controls a number of important cellular functions through substrate cleavage. Aberrant cleavage of the amyloid precursor protein results in aggregation of β-amyloid peptide, which accumulates in the brain and consequently causes Alzheimer's disease. Here we report the three-dimensional structure of an intact human γ-secretase complex at 4.5 Å resolution, determined by cryo-EM single-particle analysis. The γ-secretase complex comprises a horseshoe-shaped transmembrane domain, which contains 19 transmembrane segments (TMs), and a large extracellular domain (ECD) from Nicastrin, which sits right above the hollow space formed by the TM horseshoe. Intriguingly, Nicastrin ECD is structurally similar to a large family of peptidases exemplified by the glutamate carboxypeptidase PSMA. This structure serves as an important basis for understanding the functional mechanisms of the γ-secretase complex.γ-Secretase is a membrane-embedded aspartyl protease that cleaves a large number of transmembrane substrate proteins within their membrane-spanning regions, with the cleavage products serving as signaling molecules 1,2 . This process is known as regulated intramembrane proteolysis (RIP) 3 . Two extensively studied substrates of γ-secretase are the amyloid precursor protein (APP) and the Notch receptor 2 . Successive cleavages of APP give

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Despite its potentially adverse effects on lung development and function, supplemental oxygen is often used to treat premature infants in respiratory distress. To understand how neonatal hyperoxia can permanently disrupt lung development, we previously reported increased lung compliance, greater alveolar simplification, and disrupted epithelial development in adult mice exposed to 100% inspired oxygen fraction between postnatal days 1 and 4. Here, we investigate whether oxygen-induced changes in lung function are attributable to defects in surfactant composition and activity, structural changes in alveolar development, or both. Newborn mice were exposed to room air or 40%, 60%, 80%, or 100% oxygen between postnatal days 1 and 4 and allowed to recover in room air until 8 wk of age. Lung compliance and alveolar size increased, and airway resistance, airway elastance, tissue elastance, and tissue damping decreased, in mice exposed to 60 -80% oxygen; changes were even greater in mice exposed to 100% oxygen. These alterations in lung function were not associated with changes in total protein content or surfactant phospholipid composition in bronchoalveolar lavage. Moreover, surface activity and total and hydrophobic protein content were unchanged in large surfactant aggregates centrifuged from bronchoalveolar lavage compared with control. Instead, the number of type II cells progressively declined in 60 -100% oxygen, whereas levels of T1␣, a protein expressed by type I cells, were comparably increased in mice exposed to 40 -100% oxygen. Thickened bundles of elastin fibers were also detected in alveolar walls of mice exposed to Ն60% oxygen. These findings support the hypothesis that changes in lung development, rather than surfactant activity, are the primary causes of oxygen-altered lung function in children who were exposed to oxygen as neonates. Furthermore, the disruptive effects of oxygen on epithelial development and lung mechanics are not equivalently dose dependent. bronchopulmonary dysplasia; epithelium; hyperoxia; type II cells BRONCHOPULMONARY DYSPLASIA (BPD) is a chronic lung disease often seen in premature infants with very low birth weight (21). At autopsy, lungs of infants who die from BPD are less vascularized, with fewer and larger alveoli (7). Although the pathophysiology of BPD is complex and related in part to gestational age, neonatal hyperoxia is recognized as an important contributing factor to this disease in many infants (see Refs. 3,12,17, 37 for review). Premature infants with BPD have low plasma levels of glutathione (59), and hyperoxia in the context of an immature antioxidant defense increases the potential for oxidative stress injury. The use of exogenous surfactant, antenatal steroids, and milder ventilation strategies has markedly increased survival and other improved outcomes for premature infants over the past two decades. However, many patients continue to show decreased lung capacity, even as young adolescents (19,20,55). Moreover, these children are often rehospitalized following r...

Cinnamoyl CoA reductases (CCR) convert hydroxycinnamoyl CoA esters to their corresponding cinnamyl aldehydes in monolignol biosynthesis. We identified two CCR genes in the model legume Medicago truncatula. CCR1 exhibits preference for feruloyl CoA, but CCR2 prefers caffeoyl and 4-coumaroyl CoAs, exhibits sigmoidal kinetics with these substrates, and is substrate-inhibited by feruloyl and sinapoyl CoAs. M. truncatula lines harboring transposon insertions in CCR1 exhibit drastically reduced growth and lignin content, whereas CCR2 knockouts grow normally with moderate reduction in lignin levels. CCR1 fully and CCR2 partially complement the irregular xylem gene 4 CCR mutation of Arabidopsis. The expression of caffeoyl CoA 3-O-methyltransferase (CCoAOMT) is up-regulated in CCR2 knockout lines; conversely, knockout of CCoAOMT up-regulates CCR2. These observations suggest that CCR2 is involved in a route to monolignols in Medicago whereby coniferaldehyde is formed via caffeyl aldehyde which then is 3-Omethylated by caffeic acid O-methyltransferase.