Prokop S, Haass C, Steiner HLength and overall sequence of the PEN-2 C-terminal domain determines its function in the stabilization of presenilin fragments. J Neurochem 94:57-62

Adolf-Butenandt-Institute, Department of Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease Research, Ludwig-Maximilians-University, Munich, Germany.
Journal of Neurochemistry (Impact Factor: 4.28). 08/2005; 94(1):57-62. DOI: 10.1111/j.1471-4159.2005.03165.x
Source: PubMed


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.

Download full-text


Available from: Stefan Prokop, Dec 08, 2014
  • Source
    • "After endoproteolysis, PS1 NTF and CTF are stabilised by interactions with the PEN2 C-terminus (Kim and Sisodia, 2005b). Interestingly, altering the length or integrity of either the luminal N-or C-terminus of PEN2 affects binding to other components and ␥-secretase activity (Crystal et al., 2003; Hasegawa et al., 2004; Isoo et al., 2007; Prokop et al., 2005). PEN2 thus seems to use its 'hairpin' topology literally, hooking up and stabilizing the final ␥-secretase complex. "
    [Show abstract] [Hide abstract]
    ABSTRACT: gamma-Secretase is a promiscuous aspartyl protease responsible for the final intramembrane cleavage of various type I transmembrane proteins after their large ectodomains are shed. The vast functional diversity of its substrates, which are involved in cell fate decisions, adhesion, neurite outgrowth and synapse formation, highlights the important role gamma-secretase plays in development and neurogenesis. The most renowned substrates are the amyloid precursor protein and Notch, from which gamma-secretase liberates amyloid beta peptides and induces downstream signalling, respectively. gamma-Secretase is a multiprotein complex containing presenilin (which harbours the catalytic site), nicastrin, APH1 and PEN2. Its assembly occurs under tight control of ER-Golgi recycling regulators, which allows defined quantities of complexes to reach post-Golgi compartments, where gamma-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 gamma-secretase.
    Full-text · Article · Mar 2008 · Journal of Cell Science
  • Source
    • "Pen-2 facilitates the cleavage of full-length PS into active NTF and CTF (Francis et al. 2002; Steiner et al. 2002). Both the sequence and length of the C-terminus of Pen-2 are critical for intermolecular interactions and function of presenilin complexes (Hasegawa et al. 2004; Kim and Sisodia 2005; Prokop et al. 2005). In particular, deletion of the last 12 amino acids of Pen-2 has been shown to abrogate the ability of Pen-2 to stabilize PS fragments and does not restore c-secretase activity in the background of Pen-2 knockdown by RNAi (Hasegawa et al. 2004; Kim and Sisodia 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Gamma-secretase cleavage, mediated by a complex of presenilin, presenilin enhancer (Pen-2), nicastrin, and Aph-1, is the final proteolytic step in generating amyloid beta protein found in brains of Alzheimer's disease patients and Notch intracellular domain critical for proper neuronal development. Here, we employ the zebrafish model to study the role of Pen-2 in neuronal survival. We found that (i) knockdown of Pen-2 using antisense morpholino led to a reduction of islet-1 positive neurons, (ii) Notch signaling was reduced in embryos lacking Pen-2 or other gamma-secretase components, (iii) neuronal loss in Pen-2 knockdown embryos is not as a result of a lack of neuronal precursor cells or cell proliferation, (iv) absence of Pen-2 caused massive apoptosis in the whole animal, which could be suppressed by simultaneous knockdown of the tumor suppressor p53, (v) loss of islet-1 or acetylated tubulin positive neurons in Pen-2 knockdown embryos could be partially rescued by knockdown of p53. Our results demonstrate that knockdown of Pen-2 directly induces a p53-dependent apoptotic pathway that contributes to neuronal loss and suggest that Pen-2 plays an important role in promoting neuronal cell survival and protecting from apoptosis in vivo.
    Full-text · Article · Apr 2006 · Journal of Neurochemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The Aβ peptides that are the principal component of the amyloid plaques in the brains of AD patients are released to the extracellular space by the activity of ɣ-secretase. It was the aim of the present study to provide evidence for a hypothetical quality control mechanism for ɣ-secretase assembly. By applying a reporter protein approach, two novel ER retention signals were identified in the transmembrane domain (TMD) 4 of PS1 and the TMD1 of Pen2. Mutagenesis of the two known signals in PS1 (TMD4 and TMD9) did not result in the ER export of full-length PS1. Thus, it is suggested here that further signals exist in PS1. Furthermore, Retention in the ER 1 (Rer1) was identified as a protein involved in the ER retention of Pen2 but not PS1. Pen2 was the first identified mammalian substrate of Rer1 and Rer1 interacts with Pen2 via the binding to the Pen2-TMD1. The signal in the PS1-TMD4 is independent of Rer1, suggesting that other, so far unknown mechanisms exist which contribute to TMD-based ER retention. Data from our lab and by others already demonstrated that the Pen2-TMD1 and the PS1-TMD4 are necessary for the interaction of PS1 and Pen2. Thus, both TMDs are bifunctional, since they contribute to protein-protein interaction and to ER retention. This protein-protein interaction between the TMDs results in the masking and inactivation of the retention signals in both TMDs. In this way both functions of the TMDs are connected and contribute to the ER quality control for the ɣ-secretase assembly. Additionally, it was shown that the stable overexpression of the PS1-TMD4 interferes with the described quality control mechanism and thus with the assembly of the ɣ-secretase. This finding discloses the possibility that pharmacological targeting of ɣ-secretase TMDs could be used to inhibit this important enzyme under pathological conditions. Die Hauptkomponente der amyloiden Plaques im Gehirn von Alzheimer Patienten sind die Aβ Peptide, welche durch die Aktivität der ɣ-Sekretase in den extrazellulären Raum gelangen. Das Ziel der vorliegenden Arbeit war es, Belege für die einen hypothetischen Qualitätskontrollmechanismus beim Zusammenbau der ɣ-Sekretase zu finden. Durch die Anwendung von Reporterproteinen konnten zwei neuartige ER Retentionssignale identifiziert werden, welche in der Transmembrandomäne (TMD) 4 von PS1 und der TMD1 von Pen2 liegen. Die Mutagenese der zwei bekannten Retentionssignale in PS1 (TMD4 und TMD9) führte nicht zum Export von PS1 aus dem ER. Daher ist zu vermuten, dass es weitere Signale in PS1 gibt. Des Weiteren wurde Retention in the ER 1 (Rer1) als ein Protein identifiziert, das zur ER Retention von Pen2, jedoch nicht von PS1, beiträgt. Pen2 ist das erste bekannte Substrat von Rer1 in Säugetierzellen wobei Rer1 mit Pen2 durch die Bindung an die Pen2-TMD1 interagiert. Die Unabhängigkeit des Signals in der PS1-TMD4 von Rer1 impliziert, dass es weitere bisher unbekannte Mechanismen gibt, welche zur TMD-basierten ER Retention beitragen. Ergebnisse von uns und anderen zeigten bereits, dass die Pen2-TMD1 und die PS1-TMD4 notwendig für die Interaktion von PS1 und Pen2 sind. Dementsprechend sind beide TMD bifunktional, da sie sowohl zu einer Protein-Protein Interaktion als auch zur ER Retention beitragen. Diese Protein-Protein Interaktion führt zu einer Maskierung und somit zur Inaktivierung der Retentionssignale in beiden TMD. Auf diese Art und Weise sind beide Funktionen der TMD aneinander gekoppelt und dienen der Qualitätskontrolle beim Zusammenbau der ɣ-Sekretase. Darüber hinaus wurde hier gezeigt, dass die stabile Überexpression der PS1-TMD4 den beschriebenen Qualitätskontrollmechanismus stört und somit den Zusammenbau der ɣ-Sekretase verhindert. Diese Beobachtung eröffnet eine Möglichkeit zur pharmakologischen Beeinflussung der ɣ-Sekretase über ihre TMD.
    Preview · Article ·
Show more