Transmembrane Protein 147 (TMEM147) Is a Novel Component of the Nicalin-NOMO Protein Complex

Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely γ-secretase and the Nicalin-NOMO (Nodal modulator) complex. The γ-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the β-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200–220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A ∼22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to γ-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with γ-secretase.     

Nicalin and its binding partner Nomo are novel Nodal signaling antagonists

Nodals are signaling factors of the transforming growth factor-beta (TGFbeta) superfamily with a key role in vertebrate development. They control a variety of cell fate decisions required for the establishment of the embryonic body plan. We have identified two highly conserved transmembrane proteins, Nicalin and Nomo (Nodal modulator, previously known as pM5), as novel antagonists of Nodal signaling. Nicalin is distantly related to Nicastrin, a component of the Alzheimer's disease-associated gamma-secretase, and forms a complex with Nomo. Ectopic expression of both proteins in zebrafish embryos causes cyclopia, a phenotype that can arise from a defect in mesendoderm patterning mediated by the Nodal signaling pathway. Accordingly, downregulation of Nomo resulted in an increase in anterior axial mesendoderm and the development of an enlarged hatching gland. Inhibition of Nodal signaling by ectopic expression of Lefty was rescued by reducing Nomo levels. Furthermore, Nodal- as well as Activin-induced signaling was inhibited by Nicalin and Nomo in a cell-based reporter assay. Our data demonstrate that the Nicalin/Nomo complex antagonizes Nodal signaling during mesendodermal patterning in zebrafish.     

Gamma-secretase activity is associated with a conformational change of nicastrin

Gamma-secretase is a high molecular weight multicomponent protein complex with an unusual intramembrane-cleaving aspartyl protease activity. Gamma-secretase is intimately associated with Alzheimer disease because it catalyzes the proteolytic cleavage, which leads to the liberation of amyloid beta-peptide. At least presenilin (PS), Nicastrin (Nct), APH-1, and PEN-2 are constituents of the gamma-secretase complex, with PS apparently providing the active site of gamma-secretase. Expression of gamma-secretase complex components is tightly regulated, however little is known about the assembly of the complex. Here we demonstrate that Nct undergoes a major conformational change during the assembly of the gamma-secretase complex. The conformational change is directly associated with gamma-secretase function and involves the entire Nct ectodomain. Loss of function mutations generated by deletions failed to undergo the conformational change. Furthermore, the conformational alteration did not occur in the absence of PS. Our data thus suggest that gamma-secretase function critically depends on the structural "activation" of Nct.     

Rer1p competes with APH-1 for binding to nicastrin and regulates gamma-secretase complex assembly in the early secretory pathway

The gamma-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this "proteasome of the membrane" are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of gamma-secretase subcomplexes and, concomitantly, total cellular gamma-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates gamma-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular gamma-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value.     

Nicastrin interacts with gamma-secretase complex components via the N-terminal part of its transmembrane domain

Two secretases are involved in the generation of amyloid beta-peptide, the principal component of amyloid plaques in the brains of Alzheimer's disease patients. While beta-secretase is a classical aspartyl protease, gamma-secretase activity is associated with a high molecular weight complex. One of the complex components, which is critically required for gamma-secretase activity is nicastrin (NCT). Here we investigate the assembly of NCT into the gamma-secretase complex. NCT mutants either lacking the entire cytoplasmic tail, the cytoplasmic tail, and the transmembrane domain (TMD), or containing a set of heterologous TMDs were expressed in cells with strongly reduced levels of endogenous NCT. Maturation of exogenous NCT, gamma-secretase complex formation and proteolytic function was then investigated. This revealed that the cytoplasmic tail of NCT is dispensable for gamma-secretase complex assembly and function. In contrast, the authentic TMD of NCT is critically required for the interaction with gamma-secretase complex components and for formation of an active gamma-secretase complex. Neither soluble NCT lacking any membrane anchor nor NCT containing a heterologous TMD were inserted into the gamma-secretase complex. We identified the N-terminal region of the NCT TMD as a functionally important entity of NCT. These data thus demonstrate that NCT interacts with other gamma-secretase complex components via its TMD.     

The presenilin C-terminus is required for ER-retention, nicastrin-binding and gamma-secretase activity

gamma-Secretase is an intramembrane cleaving protease involved in Alzheimer's disease. gamma-Secretase occurs as a high molecular weight complex composed of presenilin (PS1/2), nicastrin (NCT), anterior pharynx-defective phenotype 1 and PS enhancer 2. Little is known about the cellular mechanisms of gamma-secretase assembly. Here we demonstrate that the cytoplasmic tail of PS1 fulfills several functions required for complex formation, retention of unincorporated PS1 and gamma-secretase activity. The very C-terminus interacts with the transmembrane domain of NCT and may penetrate into the membrane. Deletion of the last amino acid is sufficient to completely block gamma-secretase assembly and release of PS1 from the endoplasmic reticulum (ER). This suggests that unincorporated PS1 is actively retained within the ER. We identified a hydrophobic stretch of amino acids within the cytoplasmic tail of PS1 distinct from the NCT-binding site, which is required to retain unincorporated PS1 within the ER. Deletion of the retention signal results in the release of PS1 from the ER and the assembly of a nonfunctional gamma-secretase complex, suggesting that at least a part of the retention motif may also be required for the function of PS1.     

Functional domains in presenilin 1: the Tyr-288 residue controls gamma-secretase activity and endoproteolysis

Processing of the Alzheimer amyloid precursor protein (APP) into the amyloid beta-protein and the APP intracellular domain is a proteolysis event mediated by the gamma-secretase complex where presenilin (PS) proteins are key constituents. PS is subjected to an endoproteolytic cleavage, generating a stable heterodimer composed of an N-terminal and a C-terminal fragment. Here we aimed at further understanding the role of PS in endoproteolysis, in proteolytic processing of APP and Notch, and in assembly of the gamma-secretase complex. By using a truncation protocol and alanine scanning, we identified Tyr-288 in the PS1 N-terminal fragment as critical for PS-dependent intramembrane proteolysis. Further mutagenesis of the 288 site identified mutants differentially affecting endoproteolysis and gamma-secretase activity. The Y288F mutant was endoproteolyzed to the same extent as wild type PS but increased the amyloid beta-protein 42/40 ratio by approximately 75%. In contrast, the Y288N mutant was also endoproteolytically processed but was inactive in reconstituting gamma-secretase in PS null cells. The Y288D mutant was deficient in both endoproteolysis and gamma-secretase activity. All three mutant PS1 molecules were incorporated into gamma-secretase complexes and stabilized Pen-2 in PS null cells. Thus, mutations at Tyr-288 do not affect gamma-secretase complex assembly but can differentially control endoproteolysis and gamma-secretase activity.     

Generation and characterization of mutant cell lines defective in gamma-secretase processing of Notch and amyloid precursor protein

Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. We have set up a genetic screen based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. We selected clones deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. We further analyzed two of these clones and identified previously undescribed mutations in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired.     

Different cofactor activities in gamma-secretase assembly: evidence for a nicastrin-Aph-1 subcomplex

The gamma-secretase complex is required for intramembrane cleavage of several integral membrane proteins, including the Notch receptor, where it generates an active signaling fragment. Four putative gamma-secretase components have been identified-presenilin (Psn), nicastrin (Nct), Aph-1, and Pen-2. Here, we use a stepwise coexpression approach to investigate the role of each new component in gamma-secretase assembly and activation. Coexpression of all four proteins leads to high level accumulation of mature Psn and increased proteolysis of Notch. Aph-1 and Nct may form a subcomplex that stabilizes the Psn holoprotein at an early step in gamma-secretase assembly. Subcomplex levels of Aph-1 are down-regulated by stepwise addition of Psn, suggesting that Aph-1 might not enter the mature complex. In contrast, Pen-2 accumulates proportionally with Psn, and is associated with Psn endoproteolysis during gamma-secretase assembly. These results demonstrate that Aph-1 and Pen-2 are essential cofactors for Psn, but that they play different roles in gamma-secretase assembly and activation.     

Gamma-secretase complex assembly within the early secretory pathway

gamma-Secretase is an aspartyl protease complex composed of the four core components APH-1, nicastrin (NCT), presenilin (PS), and PEN-2. It catalyzes the final intramembranous cleavage of the beta-secretase-processed beta-amyloid precursor protein to liberate the neurotoxic amyloid beta-peptide. Whereas unassembled complex components appear to be unstable and/or to be retained within the endoplasmic reticulum (ER), the fully assembled complex is known to exert its biological function in late secretory compartments, including the plasma membrane. We thus hypothesized that the gamma-secretase complex undergoes a stepwise assembly within the ER. We demonstrate that gamma-secretase-associated NCT can be actively retained within the ER by the addition of a retention signal. Under these conditions, complex assembly occurred in the absence of maturation of NCT, and ER-retained immature NCT associated with APH-1, PEN-2, and PS fragments. Moreover, a biotinylated transition state gamma-secretase inhibitor allowed the preferential isolation of the fully assembled complex containing immature NCT. Furthermore, we observed a conformational change in immature NCT, which is known to be selectively associated with complete gamma-secretase complex assembly. This was also observed for a small amount of immature endogenous NCT. ER-retained NCT also rescued the biochemical phenotype observed upon RNA interference-mediated NCT knockdown, viz. reduced amyloid beta-peptide production; instability of PS, PEN-2, and APH-1; and accumulation of beta-amyloid precursor protein C-terminal fragments. Finally, we demonstrate that dimeric (NCT/APH-1) and trimeric (NCT/APH-1/PS) intermediates of gamma-secretase complex assembly containing endogenous NCT are retained within the ER and that the incorporation of the fourth and last binding partner (PEN-2) also occurs on immature NCT, suggesting a complete assembly of the gamma-secretase complex within the ER.     

Assembly of the gamma-secretase complex involves early formation of an intermediate subcomplex of Aph-1 and nicastrin

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

Co-expressed presenilin 1 NTF and CTF form functional gamma-secretase complexes in cells devoid of full-length protein

The enzyme gamma-secretase catalyzes the intramembrane proteolytic cleavage that generates the amyloid beta-peptide from the beta-amyloid precursor protein. The presenilin (PS) protein is one of the four integral membrane protein components of the mature gamma-secretase complex. The PS protein is itself subjected to endoproteolytic processing, generating stable N- and C-terminal fragment (NTF and CTF, respectively) heterodimers. Here we demonstrate that coexpression of PS1 NTF and CTF functionally mimics expression of the full-length PS1 protein and restores gamma-secretase activity in PS-deficient mammalian cells. The coexpressed fragments re-associate with each other inside the cell, where they also interact with nicastrin, another gamma-secretase complex component. Analysis of gamma-secretase activity following the expression of mutant forms of NTF and CTF, under conditions bypassing endoproteolysis, indicated that the putatively catalytic Asp257 and Asp385 residues have a direct effect on gamma-secretase activity. Moreover, we demonstrate that expression of the wild-type CTF rescues endoproteolytic cleavage of C-terminally truncated PS1 molecules that are otherwise uncleaved and inactive. Recovery of cleavage is critically dependent on the integrity of Asp385. Taken together, our findings indicate that ectopically expressed NTF and CTF restore functional gamma-secretase complexes and that the presence of full-length PS1 is not a requirement for proper complex assembly.     

Presenilin-1, nicastrin,amyloid precursor protein,and gamma-secretase activity are co-localized in the lysosomal membrane

Alzheimer's disease (AD) is caused by the cerebral deposition of beta-amyloid (Abeta), a 38-43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the gamma-secretase (a complex containing presenilin and nicastrin) to produce Abeta occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the gamma-secretase proteins and a neutral pH optimum of in vitro gamma-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic gamma-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that gamma-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of Abeta and suggest a role for lysosomes in the pathophysiology of AD.     

Functional overexpression of gamma-secretase reveals protease-independent trafficking functions and a critical role of lipids for protease activity

Presenilins appear to form the active center of gamma-secretase but require the presence of the integral membrane proteins nicastrin, anterior pharynx defective 1, and presenilin enhancer 2 for catalytic function. We have simultaneously overexpressed all of these polypeptides, and we demonstrate functional assembly of the enzyme complex, a substantial increase in enzyme activity, and binding of all components to a transition state analogue gamma-secretase inhibitor. Co-localization of all components can be observed in the Golgi compartment, and further trafficking of the individual constituents seems to be dependent on functional assembly. Apart from its catalytic function, gamma-secretase appears to play a role in the trafficking of the beta-amyloid precursor protein, which was changed upon reconstitution of the enzyme but unaffected by pharmacological inhibition. Because the relative molecular mass and stoichiometry of the active enzyme complex remain elusive, we performed size exclusion chromatography of solubilized gamma-secretase, which yielded evidence of a tetrameric form of the complex, yet almost completely abolished enzyme activity. Gamma-secretase activity was reconstituted upon addition of an independent size exclusion chromatography fraction of lower molecular mass and nonproteinaceous nature, which could be replaced by a brain lipid extract. The same treatment was able to restore enzyme activity after immunoaffinity purification of the gamma-secretase complex, demonstrating that lipids play a key role in preserving the catalytic activity of this protease. Furthermore, these data show that it is important to discriminate between intact, inactive gamma-secretase complexes and the active form of the enzyme, indicating the care that must be taken in the study of gamma-secretase.     

Detergent-dependent dissociation of active gamma-secretase reveals an interaction between Pen-2 and PS1-NTF and offers a model for subunit organization within the complex

Gamma-secretase is a member of a new class of proteases with an intramembrane catalytic site and cleaves numerous type I membrane proteins, including the amyloid beta-protein precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: a heterodimeric form of presenilin (PS), composed of its N- and C-terminal fragments (PS-NTF and PS-CTF, respectively), a highly glycosylated, mature form of nicastrin (NCT), Aph-1, and Pen-2. However, it is unclear how these components interact physically with each other and assemble into functional complexes. We and others recently found that Aph-1 interacts with a less glycosylated, immature form of nicastrin as an intermediate toward full assembly of gamma-secretase. Here we show that (1) the detergent dodecyl beta-d-maltoside (DDM) mediates the dissociation and inactivation of active gamma-secretase in a concentration-dependent manner, (2) DDM-dependent dissociation of the active gamma-secretase complex generates two major inactive complexes (Pen-2-PS1-NTF and mNCT-Aph-1) and two minor inactive complexes (mNCT-Aph1-PS1-CTF and PS1-NTF-PS1-CTF), and (3) Pen-2 can also associate with the PS holoprotein in complexes devoid of NCT and Aph-1. Taken together, our results demonstrate that Pen-2 interacts with PS-NTF within active gamma-secretase and offer a model for how the components of active gamma-secretase interact physically with each other.     

Regulation of gamma-secretase activity in Alzheimer's disease

The gamma-secretase complex is an intramembrane aspartyl protease that cleaves its substrates along their transmembrane regions. Sequential proteolytic processing of amyloid precursor protein by beta- and gamma-secretase produces amyloid beta-peptides, which are the major components of amyloid plaques in the brains of Alzheimer's disease patients. The gamma-secretase complex is therefore believed to be critical in the pathogenesis of Alzheimer's disease. Here we review the range of factors found to affect the nature and degree of gamma-secretase complex activity; these include gamma-secretase complex assembly and activation, the integral regulatory subunit CD147, transient or weak binding partners, the levels of cholesterol and sphingolipids in cell membranes, and inflammatory cytokines. Integrated knowledge of the molecular mechanisms supporting the actions of these factors is expected to lead to a comprehensive understanding of the functional regulation of the gamma-secretase complex, and this, in turn, should facilitate the development of novel therapeutic strategies for the treatment of Alzheimer's disease.     

Two domains within the first putative transmembrane domain of presenilin 1 differentially influence presenilinase and gamma-secretase activity

Presenilins (PS) are thought to contain the active site for presenilinase endoproteolysis of PS and gamma-secretase cleavage of substrates. The structural requirements for PS incorporation into the gamma-secretase enzyme complex, complex stability and maturation, and appropriate presenilinase and gamma-secretase activity are poorly understood. We used rescue assays to identify sequences in transmembrane domain one (TM1) of PS1 required to support presenilinase and gamma-secretase activities. Swap mutations identified an N-terminal TM1 domain that is important for gamma-secretase activity only and a C-terminal TM1 domain that is essential for both presenilinase and gamma-secretase activities. Exchange of residues 95-98 of PS1 (sw95-98) completely abolishes both activities while the familial Alzheimer's disease mutation V96F significantly inhibits both activities. Reversion of residue 96 back to valine in the sw95-98 mutant rescues PS function, identifying V96 as the critical residue in this region. The TM1 mutants do not bind to an aspartyl protease transition state analog gamma-secretase inhibitor, indicating a conformational change induced by the mutations that abrogates catalytic activity. TM1 mutant PS1 molecules retain the ability to interact with gamma-secretase substrates and gamma-secretase complex members, although Nicastrin stability is decreased by the presence of these mutants. gamma-Secretase complexes that contain V96F mutant PS1 molecules display a partial loss of function for gamma-secretase that alters the ratio of amyloid-beta peptide species produced, leading to the amyloid-beta peptide aggregation that causes familial Alzheimer's disease.     

Complex N-linked glycosylated nicastrin associates with active gamma-secretase and undergoes tight cellular regulation

The intramembranous proteolysis of Notch and the amyloid precursor protein by gamma-secretase exemplifies an unusual and newly recognized mechanism of signal transduction in multicellular organisms. Here, we show that only a form of nicastrin (NCT) containing N-linked complex oligosaccharides is present in active gamma-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 gamma-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.     

Reconstitution of gamma-secretase activity

gamma-Secretase is a membrane protein complex with an unusual aspartyl protease activity that catalyses the regulated intramembranous cleavage of the beta-amyloid precursor protein (APP) to release the Alzheimer's disease (AD)-associated amyloid beta-peptide (Abeta) and the APP intracellular domain (AICD). Here we show the reconstitution of gamma-secretase activity in the yeast Saccharomyces cerevisiae, which lacks endogenous gamma-secretase activity. Reconstituted gamma-secretase activity depends on the presence of four complex components including presenilin (PS), nicastrin (Nct), APH-1 (refs 3-6) and PEN-2 (refs 4, 7), is associated with endoproteolysis of PS, and produces Abeta and AICD in vitro. Thus, the biological activity of gamma-secretase is reconstituted by the co-expression of human PS, Nct, APH-1 and PEN-2 in yeast.