共查询到20条相似文献,搜索用时 31 毫秒
1.
Macromolecular complexes containing presenilins (PS1 and PS2), nicastrin, anterior pharynx defective phenotype 1 (APH-1), and PS enhancer 2 (PEN-2) mediate the intramembranous, gamma-secretase cleavage of beta-amyloid precursor protein (APP), Notch, and a variety of type 1 membrane proteins. We previously demonstrated that PEN-2 is critical for promoting endoproteolysis of PS1 and that the proximal two-thirds of transmembrane domain (TMD) 1 of PEN-2 is required for binding with PS1. In this study, we sought to identify the structural domains of PS1 that are necessary for binding with PEN-2. To address this issue, we generated a series of constructs encoding PS1 mutants harboring deletions or replacements of specific TMDs of PS1-NTF, and examined the effects of encoded molecules on interactions with PEN-2, stabilization and endoproteolysis of PS1, and gamma-secretase activity. We now show that PS1 TMDs 1 and 2 and the intervening hydrophilic loop are dispensable for binding to PEN-2. Furthermore, analysis of chimeric PS1 molecules that harbor replacements of each TMD with corresponding transmembrane segments from the sterol regulatory element-binding protein cleavage activating protein (SCAP) revealed that the PS1-SCAP TMD4 mutant failed to coimmunoprecipitate endogenous PEN-2, strongly suggesting that the fourth TMD of PS1 is required for interaction with PEN-2. Further mutational analyses revealed that the "NF" sequence within the TMD4 of PS1 is the minimal motif that is required for binding with PEN-2, promoting PS1 endoproteolysis and gamma-secretase activity. 相似文献
2.
Prokop S Shirotani K Edbauer D Haass C Steiner H 《The Journal of biological chemistry》2004,279(22):23255-23261
Gamma-secretase is a protease complex composed of presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, which catalyzes intramembrane cleavage of several type I transmembrane proteins including the Alzheimer's disease-associated beta-amyloid precursor protein. We generated stable RNA interference-mediated PEN-2 knockdown cells to probe mutant PEN-2 variants for functional activity. Knockdown of PEN-2 was associated with impaired NCT maturation and deficient PS1 endoproteolysis, which was efficiently rescued by wild type or N-terminally tagged PEN-2 but not by C-terminally tagged PEN-2 or by the C-terminally truncated PEN-2-DeltaC mutant. Although the latter mutants rescued the PS1 holoprotein accumulation associated with the PEN-2 knockdown, they failed to restore normal levels of the PS1 N- and C-terminal fragments and to maturate NCT. PEN-2-DeltaC was highly unstable and rapidly turned over by proteasomal degradation consistent with its failure to become stably incorporated into the gamma-secretase complex. In addition, expression of PEN-2-DeltaC caused a selective instability of the PS1 N-/C-terminal fragment heterodimer that underwent proteasomal degradation, whereas NCT and APH-1 were stable. Interestingly, when we knocked down PEN-2 in the background of the endoproteolysis-deficient PS1 Deltaexon9 mutant, immature NCT still accumulated, demonstrating that PEN-2 is also required for gamma-secretase complex maturation when PS endoproteolysis cannot occur. Taken together, our data suggest that PEN-2 is required for the stabilization of the PS fragment heterodimer within the gamma-secretase complex following PS endoproteolysis. This function critically depends on the PEN-2 C terminus. Moreover, our data show that PEN-2 is generally required for gamma-secretase complex maturation independent of its activity in PS1 endoproteolysis. 相似文献
3.
Luo WJ Wang H Li H Kim BS Shah S Lee HJ Thinakaran G Kim TW Yu G Xu H 《The Journal of biological chemistry》2003,278(10):7850-7854
Presenilin (PS, PS1/PS2) complexes are known to be responsible for the intramembranous gamma-secretase cleavage of the beta-amyloid precursor protein and signaling receptor Notch. PS holoprotein undergoes endoproteolysis by an unknown enzymatic activity to generate NH(2)- and COOH-terminal fragments, a process that is required for the formation of the active and stable PS/-gamma-secretase complex. Biochemical and genetic studies have recently identified nicastrin, APH-1, and PEN-2 as essential cofactors that physically interact with PS1 and are necessary for the gamma-secretase activity. However, their precise function in regulating the PS complex and gamma-secretase activity remains unknown. Here, we demonstrate that endogenous PEN-2 preferentially interacts with PS1 holoprotein. Down-regulation of PEN-2 expression by small interfering RNA (siRNA) abolishes the endoproteolysis of PS1, whereas overexpression of PEN-2 promotes the production of PS1 fragments, indicating a critical role for PEN-2 in PS1 endoproteolysis. Interestingly, accumulation of full-length PS1 resulting from down-regulation of PEN-2 is alleviated by additional siRNA down-regulation of APH-1. Furthermore, overexpression of APH-1 facilitates PEN-2-mediated PS1 proteolysis, resulting in a significant increase in PS1 fragments. Our data reveal a direct role of PEN-2 in proteolytic cleavage of PS1 and a regulatory function of APH-1, in coordination with PEN-2, in the biogenesis of the PS1 complex. 相似文献
4.
Gamma-secretase is a high molecular mass aspartyl protease complex composed of presenilin (PS1 or PS2), nicastrin (Nct), anterior pharynx-defective-1 (APH-1) and presenilin enhancer-2 (PEN-2). The complex mediates the intramembraneous proteolysis of beta-secretase cleaved beta-amyloid precursor protein (APP) leading to the secretion of the Alzheimer's disease-associated amyloid beta-peptide (Abeta). In order to dissect functionally important domains of Nct required for gamma-secretase complex assembly, maturation, and activity we mutated evolutionary conserved amino acids. The mutant Nct variants were expressed in a cellular background with significantly reduced endogenous Nct. Mutant Nct was functionally investigated by its ability to restore PS, APH-1 and PEN-2 expression as well as by monitoring the accumulation of the APP C-terminal fragments, the immediate substrates of gamma-secretase. We identified three independent mutations within the ectodomain of Nct, which rescued expression of APH-1 but not of PEN-2 or PS and thus failed to restore gamma-secretase activity. Interestingly, these immature Nct variants selectively bound to APH-1, suggesting a stable Nct/APH-1 interaction independent of PS and PEN-2. Consistent with this finding, expression of APH-1 remained largely unaffected in the PS double knock-out and immature Nct co-immunoprecipitated with APH-1 in the absence of PS and PEN-2. Taken together, our findings suggest that immature Nct can stably interact with APH-1 to form a potential scaffold for binding of PS and PEN-2. Moreover, binding of the latter two complex partners critically depends on the integrity of the Nct ectodomain. 相似文献
5.
Gamma-secretase is an aspartyl protease complex that catalyzes the intramembrane cleavage of a subset of type I transmembrane proteins including the beta-amyloid precursor protein (APP) implicated in Alzheimer's disease. Presenilin (PS), nicastrin (NCT), anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2) constitute the active gamma-secretase complex. PEN-2, the smallest subunit, is required for triggering PS endoproteolysis. Stabilization of the resultant N- and C-terminal fragments, which carry the catalytically active site aspartates, but not endoproteolysis itself, requires the C-terminal domain of PEN-2. To functionally dissect the C-terminal domain we created C-terminal deletion mutants and mutagenized several evolutionary highly conserved residues. The PEN-2 mutants were then probed for functional complementation of a PEN-2 knockdown, which displays deficient PS1 endoproteolysis and impaired NCT maturation. Progressive truncation of the C-terminus caused increasing loss of function. This was also observed for an internal deletion mutant as well as for C-terminally tagged PEN-2 with a twofold elongated C-terminal domain. Interestingly, only simultaneous, but not individual substitution of the highly conserved D90, F94, P97 and G99 residues with alanine interfered with PEN-2 function. All loss of function mutants identified allowed PS1 endoproteolysis, but failed to stably associate with the resultant PS1 fragments, which like the PEN-2 loss of function mutants underwent proteasomal degradation. However, complex formation of the PEN-2 mutants with PS1 fragments could be recovered when proteasomal degradation was blocked. Taken together, our data suggest that the PS-subunit stabilizing function of PEN-2 depends on length and overall sequence of its C-terminal domain. 相似文献
6.
Shiraishi H Sai X Wang HQ Maeda Y Kurono Y Nishimura M Yanagisawa K Komano H 《Journal of neurochemistry》2004,90(6):1402-1413
The presenilin (PS) complex, including PS, nicastrin, APH-1 and PEN-2, is essential for gamma-secretase activity, which is required for amyloid beta-protein (Abeta) generation. However, the precise individual roles of the three cofactors in the PS complex in Abeta generation remain to be clarified. Here, to distinguish the roles of PS cofactors in gamma-secretase activity from those in PS endoproteolysis, we investigated their roles in the gamma-secretase activity reconstituted by the coexpression of PS N- and C-terminal fragments (NTF and CTF) in PS-null cells. We demonstrate that the coexpression of PS1 NTF and CTF forms the heterodimer and restores Abeta generation in PS-null cells. The generation of Abeta was saturable at a certain expression level of PS1 NTF/CTF, while the overexpression of PEN-2 alone resulted in a further increase in Abeta generation. Although PEN-2 did not enhance PS1 NTF/CTF heterodimer formation, PEN-2 expression reduced the IC50 of a specific gamma-secretase inhibitor, a transition state analogue, for Abeta generation, suggesting that PEN-2 expression enhances the affinity or the accessibility of the substrate to the catalytic site. Thus, our results strongly suggest that PEN-2 is not only an essential component of the gamma-secretase complex but also an enhancer of gamma-cleavage after PS heterodimer formation. 相似文献
7.
Nicastrin, presenilin, APH-1, and PEN-2 form active gamma-secretase complexes in mitochondria 总被引:10,自引:0,他引:10
Hansson CA Frykman S Farmery MR Tjernberg LO Nilsberth C Pursglove SE Ito A Winblad B Cowburn RF Thyberg J Ankarcrona M 《The Journal of biological chemistry》2004,279(49):51654-51660
Mitochondria are central in the regulation of cell death. Apart from providing the cell with ATP, mitochondria also harbor several death factors that are released upon apoptotic stimuli. Alterations in mitochondrial functions, increased oxidative stress, and neurons dying by apoptosis have been detected in Alzheimer's disease patients. These findings suggest that mitochondria may trigger the abnormal onset of neuronal cell death in Alzheimer's disease. We previously reported that presenilin 1 (PS1), which is often mutated in familial forms of Alzheimer's disease, is located in mitochondria and hypothesized that presenilin mutations may sensitize cells to apoptotic stimuli at the mitochondrial level. Presenilin forms an active gamma-secretase complex together with Nicastrin (NCT), APH-1, and PEN-2, which among other substrates cleaves the beta-amyloid precursor protein (beta-APP) generating the amyloid beta-peptide and the beta-APP intracellular domain. Here we have identified dual targeting sequences (for endoplasmic reticulum and mitochondria) in NCT and showed expression of NCT in mitochondria by immunoelectron microscopy. We also showed that NCT together with APH-1, PEN-2, and PS1 form a high molecular weight complex located in mitochondria. gamma-secretase activity in isolated mitochondria was demonstrated using C83 (alpha-secretase-cleaved C-terminal 83-residue beta-APP fragment from BD8 cells lacking presenilin and thus gamma-secretase activity) or recombinant C100-Flag (C-terminal 100-residue beta-APP fragment) as substrates. Both systems generated an APP intracellular domain, and the activity was inhibited by the gamma-secretase inhibitors l-685,458 or Compound E. This novel localization of NCT, PS1, APH-1, and PEN-2 expands the role and importance of gamma-secretase activity to mitochondria. 相似文献
8.
Gamma-secretase is an intramembrane cleaving aspartyl protease complex intimately implicated in Alzheimer disease pathogenesis. The protease is composed of the catalytic subunit presenilin (PS1 or PS2), the substrate receptor nicastrin (NCT), and two additional subunits, APH-1 (APH-1a, as long and short splice forms (APH-1aL, APH-1aS), or APH-1b) and PEN-2. Apart from the Alzheimer disease-associated beta-amyloid precursor protein, gamma-secretase has been shown to cleave a large number of other type I membrane proteins. Despite the progress in elucidating gamma-secretase function, basic questions concerning the precise organization of its subunits, their molecular interactions, and their exact stoichiometry in the complex are largely unresolved. Here we isolated endogenous human gamma-secretase from human embryonic kidney 293 cells and investigated the subunit architecture of the gamma-secretase complex formed by PS1, NCT, APH-1aL, and PEN-2 by chemical cross-linking. Using this approach, we provide evidence for the close neighborhood of the PS1 N- and C-terminal fragments (NTF and CTF, respectively), the PS1 NTF and PEN-2, the PS1 CTF and APH-1aL, and NCT and APH-1aL. We thus identify a previously unrecognized PS1 CTF/APH-1aL interaction, verify subunit interactions deduced previously from indirect approaches, and provide a model of the gamma-secretase complex subunit architecture. Finally, we further show that, like the PS1 CTF, the PS2 CTF also interacts with APH-1aL, and we provide evidence that these interactions also occur with the other APH-1 variants, suggesting similar subunit architectures of all gamma-secretase complexes. 相似文献
9.
Morais VA Crystal AS Pijak DS Carlin D Costa J Lee VM Doms RW 《The Journal of biological chemistry》2003,278(44):43284-43291
Nicastrin (NCT) is a type I integral membrane protein that is one of the four essential components of the gamma-secretase complex, a protein assembly that catalyzes the intramembranous cleavage of the amyloid precursor protein and Notch. Other gamma-secretase components include presenilin-1 (PS1), APH-1, and PEN-2, all of which span the membrane multiple times. The mechanism by which NCT associates with the gamma-secretase complex and regulates its activity is unclear. To avoid the misfolding phenotype often associated with introducing deletions or mutations into heavily glycosylated and disulfide-bonded proteins such as NCT, we produced chimeras between human (hNCT) and Caenorhabditis elegans NCT (ceNCT). Although ceNCT did not associate with human gamma-secretase components, all of the ceNCT/hNCT chimeras interacted with gamma-secretase components from human, C. elegans, or both, indicating that they folded correctly. A region at the C-terminal end of hNCT, encompassing the last 50 residues of its ectodomain, the transmembrane domain, and the cytoplasmic domain was important for mediating interactions with human PS1, APH-1, and PEN-2. This finding is consistent with the fact that the bulk of the gamma-secretase complex proteins resides within the membrane, with relatively small extramembranous domains. Finally, hNCT associated with hAPH-1 in the absence of PS, consistent with NCT and APH-1 forming a subcomplex prior to association with PS1 and PEN-2 and indicating that the interactions between NCT with PS1 may be indirect or stabilized by the presence of APH-1. 相似文献
10.
Capell A Beher D Prokop S Steiner H Kaether C Shearman MS Haass C 《The Journal of biological chemistry》2005,280(8):6471-6478
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. 相似文献
11.
gamma-Secretase is an intramembrane-cleaving aspartyl protease complex that mediates the final cleavage of beta-amyloid precursor protein to liberate the neurotoxic amyloid-beta peptide implicated in Alzheimer's disease. The four proteins presenilin (PS), nicastrin (NCT), APH-1, and PEN-2 are sufficient to reconstitute gamma-secretase activity in yeast. Although PS seems to contribute the catalytic core of the gamma-secretase complex, no distinct function could be attributed to the other components so far. In Caenorhabditis elegans, mutation of a glycine to an aspartic acid within a conserved GXXXG motif in the fourth transmembrane domain of APH-1 causes a loss of function phenotype. Surprisingly, we now found that the human homologue APH-1a carrying the equivalent mutation G122D is fully active in yeast co-expressing PS1, NCT, and PEN-2. To address this discrepancy, we expressed APH-1a G122D in HEK293 cells. As reported previously, overexpressed APH-1a G122D was not incorporated into the gamma-secretase complex. Separate overexpression of PS1, NCT, or PEN-2 together with APH-1a G122D allowed the formation of heterodimers lacking the other endogenous components. Only the combined overexpression of PS1 and NCT together with APH-1a G122D facilitated the formation of a fully active gamma-secretase complex. Under these conditions, APH-1a G122D supported the production of normal amounts of Abeta. We conclude that cooperative effects may stabilize a trim-eric complex of APH-1a G122D together with PS1 and NCT. Upon successful complex assembly, the GXXXG motif becomes dispensable for gamma-secretase activity. 相似文献
12.
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. 相似文献
13.
Intramembranous "gamma-secretase" processing of beta-amyloid precursor protein (APP) and other transmembrane proteins, including Notch, is mediated by a macromolecular complex consisting of presenilins (PSs), nicastrin (NCT), APH-1, and PEN-2. We now demonstrate that in cells coexpressing PS1, APH-1, and NCT, full-length PS1 accumulates to high levels and is fairly stable. Upon expression of PEN-2, the levels of PS1 holoprotein are significantly reduced, commensurate with an elevation in levels of PS1 fragments. These findings suggest that APH-1 and NCT are necessary for stabilization of full-length PS1 and that PEN-2 is critical for the proteolysis of stabilized PS1. In N2a and 293 cell lines that stably overexpress PS1, APH-1, NCT, and PEN-2, PS1 fragment levels are elevated by up to 10-fold over endogenous levels. In these cells, we find a marked accumulation of the APP-CTF gamma (AICD) fragment and a concomitant reduction in levels of both APP-CTF beta and CTF alpha. Moreover, the production of the gamma-secretase-generated Notch S3/NICD derivative is modestly elevated. However, we failed to observe a corresponding increase in levels of secreted A beta peptides in the medium of these cells. These results lead us to conclude that, although the PS1, APH-1, NCT, and PEN-2 are essential for gamma-secretase activity, the proteolysis of APP-CTF and Notch S2/NEXT are differentially regulated and require the activity of additional cofactors that promote production of AICD, NICD, and A beta. 相似文献
14.
The gamma-secretase complex, consisting of presenilins (PS), nicastrin (NCT), APH-1, and PEN-2, catalyzes the intramembranous proteolysis of truncated beta-amyloid precursor protein (APP) and Notch derivatives to generate the APP intracellular domain (AICD) and Notch intracellular domain (NICD), respectively. To examine the intracellular sites in which active gamma-secretase resides, we expressed NCT variants harboring either an endoplasmic reticulum (ER) retention signal (NCT-ER) or a trans-Golgi network (TGN) targeting motif (NCT-TGN) along with PS1, APH-1, and PEN-2 and examined gamma-secretase activity in these settings. In cells expressing NCT-ER and the other components, PS1 fragments hyperaccumulated, but AICD levels were not elevated. On the other hand, upon coexpression of an ER-retained APP variant or a constitutionally active Notch mutant, NDeltaE, we observed enhanced production of AICD or NICD, respectively, in cells expressing NCT-ER. Moreover, we show that membranes from cells expressing NCT-ER, NCT-TGN, or NCT-WT contain identical levels of PS1 derivatives that can be photoaffinity cross-linked to a biotinylated, benzophenone-derivatized gamma-secretase inhibitor. Finally, our cell-free gamma-secretase assays revealed nearly equivalent gamma-secretase activities in cells expressing PS1, APH-1, PEN-2, and either NCT-WT or NCT-ER. Taken together, we interpret these findings as suggesting that active gamma-secretase complex is generated in the early compartments of the secretory pathway but that these complexes are transported to late compartments in which substrates are encountered and subsequently processed within respective transmembrane segments. 相似文献
15.
16.
Watanabe N Tomita T Sato C Kitamura T Morohashi Y Iwatsubo T 《The Journal of biological chemistry》2005,280(51):41967-41975
gamma-Secretase is a multimeric membrane protein complex comprised of presenilin (PS), nicastrin (Nct), Aph-1, and Pen-2. It is a member of an atypical class of aspartic proteases that hydrolyzes peptide bonds within the membrane. During the biosynthetic process of the gamma-secretase complex, Nct and Aph-1 form a heterodimeric intermediate complex and bind to the C-terminal region of PS, serving as a stabilizing scaffold for the complex. Pen-2 is then recruited into this trimeric complex and triggers endoproteolysis of PS, conferring gamma-secretase activity. Although the Pen-2 accumulation depends on PS, the binding partner of Pen-2 within the gamma-secretase complex remains unknown. We reconstituted PS1 in Psen1/Psen2 deficient cells by expressing a series of PS1 mutants in which one of the N-terminal six transmembrane domains (TMDs) was swapped with those of CD4 (a type I transmembrane protein) or CLAC-P (a type II transmembrane protein). We report that the proximal two-thirds of TMD4 of PS1, including the conserved Trp-Asn-Phe sequence, are required for its interaction with Pen-2. Using a chimeric CD4 molecule harboring PS1 TMD4, we further demonstrate that the PS1 TMD4 bears a direct binding motif to Pen-2. Pen-2 may contribute to the activation of the gamma-secretase complex by directly binding to the TMD4 of PS1. 相似文献
17.
He G Qing H Cai F Kwok C Xu H Yu G Bernstein A Song W 《Journal of neurochemistry》2006,99(5):1403-1412
Gamma-secretase catalyzes intramembraneous proteolysis of several type I transmembrane proteins, including beta-amyloid precursor protein (APP), to generate amyloid beta protein (Abeta), a key player in the pathogenesis of Alzheimer's disease (AD). The critical components of the gamma-secretase complex include presenilin (PS), nicastrin (NCT), presenilin enhancer-2 (PEN-2) and anterior pharynx defective-1 (APH-1). Abnormalities of the ubiquitin-proteasome pathway have been implicated in the pathogenesis of AD; while PS and PEN-2 turnover is regulated by this pathway, it is unknown whether the ubiquitin-proteasome pathway is also involved in the degradation of APH-1 protein. In this study, we found that the expression of endogenous and exogenous APH-1 significantly increased in cells treated with proteasome-specific inhibitors. The effect of the proteasome inhibitors on APH-1 was dose- and time-dependent. APH-1 protein was ubiquitinated. Pulse-chase metabolic labeling experiments showed that the degradation of newly synthesized radiolabeled APH-1 proteins was inhibited by lactacystin. Disruption of the PS1 and PS2 genes did not affect the degradation of APH-1 by the ubiquitin-proteasome pathway. Furthermore, over-expression of APH-1 and inhibition of proteasomal APH-1 degradation facilitated gamma-secretase cleavage of APP to generate Abeta. These results demonstrate that the degradation of APH-1 protein is mediated by the ubiquitin-proteasome pathway. 相似文献
18.
Zhang YW Luo WJ Wang H Lin P Vetrivel KS Liao F Li F Wong PC Farquhar MG Thinakaran G Xu H 《The Journal of biological chemistry》2005,280(17):17020-17026
gamma-Secretase, which is responsible for the intramembranous cleavage of Alzheimer beta-amyloid precursor protein and the signaling receptor Notch, is a multiprotein complex consisting of at least four components: presenilin (PS); nicastrin (Nct); APH-1 (anterior pharynx-defective-1); and presenilin enhancer-2 (PEN-2). Presenilin 1 (PS1) is known to be essential for the stability, interaction, and trafficking of the other PS1/gamma-secretase components. However, the precise functions of the other components remain elusive. Here, we investigated the functions of Nct within the PS1/gamma-secretase complex. We demonstrated that the loss of Nct expression in the embryonic fibroblast cells (Nct KO cells) results in dramatically decreased levels of APH-1, PEN-2, and PS1 fragments accompanied by a significant accumulation of full-length PS1. In the absence of Nct, PEN-2 and full-length PS1 are subjected to proteasome-mediated degradation, whereas the degradation of APH-1 is mediated by both proteasomal and lysosomal pathways. Unlike the case of wild type cells in which the gamma-secretase complex mainly locates in the trans-Golgi network, the majority of residual PEN-2, APH-1, and the uncleaved full-length PS1 in Nct KO cells reside in the endoplasmic reticulum, which remain associated with each other in the absence of Nct. Interestingly, significant amounts of full-length PS1 and PEN-2, but not APH-1, are detected on the plasma membrane in Nct KO cells, suggesting the Nct-independent cell surface delivery of the PEN-2.PS1. Finally, the diminished PEN-2 protein level in Nct-deficient cells can be partially restored by overexpression of exogenous PS1, APH-1, or PEN-2 individually or collectively, indicating a dispensable role for Nct in controlling PEN-2 level. Taken together, our study demonstrates a critical role of Nct in the stability and proper intracellular trafficking of other components of the PS1/ gamma-secretase complex but not in maintaining the association of PEN-2, APH-1, and full-length PS1. 相似文献
19.
Steiner H Winkler E Edbauer D Prokop S Basset G Yamasaki A Kostka M Haass C 《The Journal of biological chemistry》2002,277(42):39062-39065
The Alzheimer disease-associated presenilin (PS) proteins apparently provide the active site of gamma-secretase, an unusual intramembrane-cleaving aspartyl protease. PSs principally occur as high molecular weight protein complexes that contain nicastrin (Nct) and additional so far unidentified components. Recently, PEN-2 has been implicated in gamma-secretase function. Here we identify PEN-2 as a critical component of PS1/gamma-secretase and PS2/gamma-secretase complexes. Strikingly, in the absence of PS1 and PS1/PS2, PEN-2 levels are strongly reduced. Similarly, PEN-2 levels are reduced upon RNA interference-mediated down-regulation of Nct. On the other side, down-regulation of PEN-2 by RNA interference is associated with reduced PS levels, impaired Nct maturation, and deficient gamma-secretase complex formation. We conclude that PEN-2 is an integral gamma-secretase complex component and that gamma-secretase complex components are expressed in a coordinated manner. 相似文献
20.
Shirotani K Edbauer D Prokop S Haass C Steiner H 《The Journal of biological chemistry》2004,279(40):41340-41345
The gamma-secretase complex catalyzes the final intramembraneous cleavage of the beta-amyloid precursor protein, liberating the neurotoxic amyloid beta-peptide implicated in Alzheimer's disease. Apart from the catalytic subunit presenilin (PS), three additional subunits, nicastrin, APH-1, and PEN-2, have been identified. In mammals, two PS homologues, PS1 and PS2, which are part of distinct gamma-secretase complexes, exist. Likewise, two APH-1 homologues, APH-1a and APH-1b, have been identified. Furthermore, two APH-1a splice forms, APH-1aS and APH-1aL, have been reported. Here we show that both APH-1a splice forms and APH-1b are expressed in peripheral and neuronal cells. APH-1aS, APH-1aL, and APH-1b form separate, proteolytically active gamma-secretase complexes containing either one of the two PSs. Deficiency of APH-1a caused a decrease in nicastrin, PS, and PEN-2 levels and an increase in the levels of APH-1b, whereas deficiency of APH-1b did not affect the levels of APH-1a or the other complex components. Consistent with this finding, we found that deficiency of APH-1a was associated with reduced gamma-secretase activity, whereas deficiency of APH-1b was not. Thus, APH-1b gamma-secretase complexes may fulfill redundant functions. Taken together, our results suggest that, dependent on the tissue expression of the individual subunits, six distinct gamma-secretase complexes composed of the known subunits can exist in human cells. 相似文献