Membrane Topology and Nicastrin-enhanced Endoproteolysis of APH-1, a Component of the γ-Secretase Complex

Fortna, Ryan R.; Crystal, Adam S.; Morais, Vanessa A.; Pijak, Donald S.; Lee, Virginia M.‐Y.; Doms, Robert W.

doi:10.1074/jbc.m310505200

Cited by 69 publications

(60 citation statements)

References 45 publications

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“…The structure of Osenkowski et al (18) revealed a somewhat porous structure that was interpreted to have several domains on the extracellular side and solvent-accessible low density cavities in the transmembrane region of the complex. In contrast and despite having comparable dimensions, our structure appears to be more compact, with the extracellular region consisting of a single domain that is consistent with the dimensions of the ectodomain of NCT (65)(66)(67). In addition, instead of several low density cavities within the postulated transmembrane region (18), we observed a single central chamber that interfaces with a concave surface on the extracellular region.…”

Section: Discussionsupporting

confidence: 52%

Structure of γ-Secretase and Its Trimeric Pre-activation Intermediate by Single-particle Electron Microscopy

Renzi

Zhang

Rice

et al. 2011

Journal of Biological Chemistry

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The ␥-secretase membrane protein complex is responsible for proteolytic maturation of signaling precursors and catalyzes the final step in the production of the amyloid ␤-peptides implicated in the pathogenesis of Alzheimer disease. The incorporation of PEN-2 (presenilin enhancer 2) into a pre-activation intermediate, composed of the catalytic subunit presenilin and the accessory proteins APH-1 (anterior pharynx-defective 1) and nicastrin, triggers the endoproteolysis of presenilin and results in an active tetrameric ␥-secretase. We have determined the three-dimensional reconstruction of a mature and catalytically active ␥-secretase using single-particle cryo-electron microscopy. ␥-Secretase has a cup-like shape with a lateral belt of ϳ40 -50 Å in height that encloses a water-accessible internal chamber. Active site labeling with a gold-coupled transition state analog inhibitor suggested that the ␥-secretase active site faces this chamber. Comparison with the structure of a trimeric pre-activation intermediate suggested that the incorporation of PEN-2 might contribute to the maturation of the active site architecture.Alzheimer disease is a prevalent cause of dementia in adults (1). The neuropathological hallmarks of Alzheimer disease include the presence of senile plaques and neurofibrillary tangles throughout the cerebral cortex and hippocampus and the loss of neurons in these specific brain areas (2). Senile plaques are composed of dystrophic neurites surrounding extracellular aggregates of amyloid ␤-peptides (A␤) 4 that are derived from the sequential proteolytic degradation of amyloid precursor proteins (APPs) to produce A␤ peptides that range from 34 to 42 amino acids in length (3, 4). APP is first cleaved in its ectodomain segment by BACE1, and the resulting C-terminal membrane-associated derivatives are subsequently hydrolyzed within their transmembrane domain (TMD) by ␥-secretase. This intramembrane proteolytic activity of ␥-secretase is critical in other cellular processes, such as the activation of Notchand ErbB4-dependent signaling (4, 5), and in the processing of a growing list of additional type I integral membrane proteins, including APP-like proteins, E-cadherin, CD44, and lipoprotein receptor-related protein (reviewed in Ref. 6).Reconstitution experiments in Saccharomyces cerevisiae have demonstrated that the integral membrane proteins presenilin (PS; 52.7 kDa), nicastrin (NCT; 78.4 kDa if non-glycosylated), APH-1 (anterior pharynx-defective 1; 29 kDa), and PEN-2 (presenilin enhancer 2; 12 kDa) are essential and sufficient for ␥-secretase activity (7). It is now widely accepted that PS, a polypeptide with nine predicted TMDs (8) and two absolutely conserved catalytic aspartates in the hydrophobic region of adjacent TMD6 and TMD7 (9), is the catalytic subunit of ␥-secretase. In fact, PS is now considered to be the founding member of diaspartyl intramembrane proteases, which are also found in other eukaryotes (10, 11) and in archaea (12). In vivo, PS undergoes endoproteolysis in the intracellular...

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Section: Discussionsupporting

confidence: 52%

Structure of γ-Secretase and Its Trimeric Pre-activation Intermediate by Single-particle Electron Microscopy

Renzi

Zhang

Rice

et al. 2011

Journal of Biological Chemistry

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“…For instance, the APH1 TMD(s) interacting with NCT has not been determined but may reside in the second half of APH1 (Fortna et al, 2004) (Fig. 3).…”

Section: Journal Of Cell Science 416mentioning

confidence: 99%

Building γ-secretase – the bits and pieces

Spasić

Annaert

2008

Journal of Cell Science

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occurs under tight control of ER-Golgi recycling regulators, which allows defined quantities of complexes to reach postGolgi compartments, where ␥-secretase activity is regulated by multiple other factors. 3D-EM rendering reveals a complex with a translucent inner space, suggesting the presence of a water-filled cavity required for intramembrane proteolysis. Despite huge efforts, we are now only beginning to unravel the assembly, stoichiometry, activation and subcellular location of ␥-secretase. Presenilin at the heart of ␥-secretase Mammalian PSs come in two flavours, PS1 and PS2, which are highly homologous. Both are polytopic membrane proteins and have ten hydrophobic domains, of which nine are proposed to span the membrane (Laudon et al., 2005;Oh and Turner, 2005;Spasic et al., 2006) (Fig. 1). PSs are initially translocated in the ER as fulllength proteins but get converted by endoproteolysis within hydrophobic domain 7 to stable N-and C-terminal fragments (NTFs and CTFs, respectively) that associate to form a heterodimer. Although both fragments are part of the catalytic ␥-secretase, endoproteolysis is not a requirement for activity (Li et al., 2000;Steiner et al., 1999) (reviewed in Dillen and Annaert, 2006).Two highly conserved aspartate residues (Asp257 and Asp385 in human PS1) within transmembrane domain 6 (TMD6) and TMD7 constitute the core of the catalytic site. Mutation of either abolishes ␥-secretase activity Wolfe et al., 1999). Together with surrounding residues, they mark a highly conserved YD/GxGD consensus motif (Steiner et al., 2000). Tyr389 may also contribute to the catalytic site . The conformation of the active site also depends on more remote sequences within PS1, such as the C-terminal PAL motif and Cys residues in TMD1 and TMD8 (Kornilova et al., 2006). Binding of an active-site-directed inhibitor prevents the disulphide cross-linking between TMD1 and TMD8, implicating their close proximity to or allosteric interaction with the catalytic site. Finally, catalytic activity must be sensitive to subtle changes in the overall TMD conformation, given the effects of the scattered FAD-linked mutations.These interactions between remote parts of the molecule fit with the idea that PS1 adopts a ring-like topology (Annaert et al., 2001). This model is based on the identification of APP-binding regions in PS1 -TMD1 (extending to part of the first luminal loop domain) and the C-terminus -and led to the idea that the substrate must first bind to a remote docking site at the heterodimer interface prior to entering the active site. Indeed, subsequent imaging and biochemical studies have demonstrated that transition-state ␥-secretase inhibitors that bind to PS1 do not affect the interaction between PS1 and the APP C-terminal fragment (Berezovska et al., 2003; Esler et al., 2002). Similarly, inhibitors based on the substrate TMD do not prevent labelling of the complex by active-sitedirected inhibitors (Kornilova et al., 2003). Interestingly, these peptide inhibitors label only PS heterodimers and not th...

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“…One essential member of ␥-secretase complex is APH-1, a sevenpass membrane protein (Francis et al, 2002;Fortna et al, 2004) that forms a stable subcomplex with nicastrin (Gu et al, 2003;Hu and Fortini, 2003;LaVoie et al, 2003;Takasugi et al, 2003). Unlike Caenorhabditis elegans and Drosophila in which a single Aph-1 gene exits, three Aph-1 genes are found in mice; Aph-1a is localized to chromosome 3, whereas Aph-1b and Aph-1c are arranged in tandem (ϳ20 kb apart) on chromosome 9.…”

Section: Introductionmentioning

confidence: 99%

APH-1a Is the Principal Mammalian APH-1 Isoform Present in γ-Secretase Complexes during Embryonic Development

Ma¹,

Li²,

Price³

et al. 2005

J. Neurosci.

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APH-1 (anterior pharynx defective) along with nicastrin and PEN-2 (presenilin enhancer) are essential components of the presenilin (PS)-dependent ␥-secretase complex. There exist three murine Aph-1 alleles termed Aph-1a, Aph-1b, and Aph-1c that encode four distinct APH-1 isoforms: APH-1aL and APH-1aS derived from differential splicing of Aph-1a, APH-1b, and APH-1c. To determine the contributions of mammalian APH-1 homologs in formation of functional ␥-secretase complexes, we generated Aph-1a Ϫ/Ϫ mice and derived immortalized fibroblasts from these embryos. Compared with littermate controls, the development of Aph-1a Ϫ/Ϫ embryos was dramatically retarded by embryonic day 9.5 and exhibited patterning defects that resemble, but are not identical to, those of Notch1, nicastrin, or PS null embryos. Moreover, in immortalized Aph-1a Ϫ/Ϫ fibroblasts, the levels of nicastrin, PS fragments, and PEN-2 were dramatically decreased. Consequently, deletion of Aph-1a resulted in significant reduction in levels of high-molecular-weight ␥-secretase complex and secretion of ␤-amyloid (A␤). Importantly, complementation analysis revealed that all mammalian APH-1 isoforms were capable of restoring the levels of nicastrin, PS, and PEN-2, as well as A␤ secretion in Aph-1a Ϫ/Ϫ cells. Together, our findings establish that APH-1a is the major mammalian APH-1 homolog present in PS-dependent ␥-secretase complexes during embryogenesis and support the view that mammalian APH-1 isoforms define a set of distinct functional ␥-secretase complexes.

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Membrane Topology and Nicastrin-enhanced Endoproteolysis of APH-1, a Component of the γ-Secretase Complex

Cited by 69 publications

References 45 publications

Structure of γ-Secretase and Its Trimeric Pre-activation Intermediate by Single-particle Electron Microscopy

Structure of γ-Secretase and Its Trimeric Pre-activation Intermediate by Single-particle Electron Microscopy

Building γ-secretase – the bits and pieces

APH-1a Is the Principal Mammalian APH-1 Isoform Present in γ-Secretase Complexes during Embryonic Development

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