Nicastrin is required for Presenilin-mediated transmembrane cleavage in Drosophila

Chung, Huimin; Struhl, Gary

doi:10.1038/ncb1201-1129

Cited by 145 publications

(125 citation statements)

References 33 publications

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“…Next, we assessed which step of constitutive Notch signal transduction was affected by TSPAN33 depletion by employing a functional assay previously used to demonstrate that the transmembrane cleavage of Notch depends on presenilin and nicastrin (24)(25)(26)(27). This assay utilizes constitutively active truncated forms of Notch that mimic cleavage products produced during signal transduction (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…I. Aifantis at New York University, New York, NY (29). The pcDNA3 NOTCH1 Jurkat JME17 mutant was generated by cloning a partial NOTCH1 transcript (exons [19][20][21][22][23][24][25][26][27][28][29] amplified by PCR from Jurkat cells, which contains an internal tandem duplication of 51 bases within exon 28 of the NOTCH1 gene, into the unique Bam 1 H and NotI restriction sites of pcDNA3 NOTCH1. The pcDNA3 NOTCH1 P12 ICHIKAWA mutant was generated by cloning a partial NOTCH1 transcript (exons [19][20][21][22][23][24][25][26][27][28][29] amplified by PCR from P12-ICHIKAWA cells, which harbor an internal tandem duplication of 42 bases within exon 27 of the NOTCH1 gene, into the unique Bam 1 H and NotI restriction sites of pcDNA3 NOTCH1.…”

Section: Methodsmentioning

confidence: 99%

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A conserved tetraspanin subfamily promotes Notch signaling in Caenorhabditis elegans and in human cells

Dunn

Sulis

Ferrando

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The cytosolic domain of Notch is a membrane-tethered transcription factor. Ligand binding ultimately leads to γ-secretase cleavage within the transmembrane domain, allowing the intracellular domain to translocate to the nucleus and activate target gene transcription. Constitutive Notch signaling has been associated with human cancers such as T cell acute lymphoblastic leukemia (T-ALL). As tetraspanins have been implicated in many different signaling processes, we assessed their potential contribution to Notch signaling. We used a genetic assay in Caenorhabditis elegans to identify TSP-12 as a positive factor for Notch activity in several cellular contexts. Then, using a cell culture system, we showed that two human TSP-12 orthologs, TSPAN33 and TSPAN5, promote Notch activity and are likely to act at the γ-secretase cleavage step. We also acquired evidence for functional redundancy among tetraspanins in both C. elegans and human cells. Selective inhibition of tetraspanins may constitute an anti-NOTCH therapeutic approach to reduce γ-secretase activity.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A conserved tetraspanin subfamily promotes Notch signaling in Caenorhabditis elegans and in human cells

Dunn

Sulis

Ferrando

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…39 Several reports have provided strong evidence that nicastrin is a component of the PS1 complex. 27,28,43 Recent studies indicate that nicastrin is required for the stabilization of PS1/g-secretase activity 44,45 and is essential for g-secretase activity. However, the addition of nicastrin to the baculovirus system at MOIs of 5 and 10 resulted in suppression of levels of the Ablike species associated with expression of APP-C99 alone or in combination with PS1DE9.…”

Section: Discussionmentioning

confidence: 99%

Baculoviruses expressing the human familial Alzheimer's disease presenilin 1 mutation lacking exon 9 increase levels of an amyloid beta-like protein in Sf9 cells

et al. 2004

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Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-b protein (Ab) that is central to the pathogenesis of Alzheimer's disease. PS1 regulates the intramembranous proteolysis of a 99-amino-acid C-terminal fragment of the amyloid precursor protein (APP-C99), a cleavage event that releases Ab following a reaction catalyzed by an enzyme termed 'c-secretase'. The molecular mechanism of PS1-mediated, c-secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the c-secretase protease or whether instead PS1 mediates c-secretase activity indirectly, perhaps by regulating the trafficking or presentation of substrates to the 'authentic' protease, which may be a molecule distinct from PS1. To address this issue, the baculovirus expression system was used to co-express: (i) APP-C99; (ii) a pathogenic, constitutively active mutant form of PS1 lacking exon 9 (PS1DE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an Ab-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1DE9 mutation. The addition to APP-C99-infected cells of baculoviruses bearing nicastrin, also a transmembrane protein, had a neutral or inhibitory effect on the reaction; tropomyosin viruses had the same effect as nicastrin viruses. These results suggest that PS1DE9 molecules expressed in Sf9 cells retain the ability to modulate Ab levels. Baculoviral-expressed PS1DE9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting c-secretase activity from its individual purified component parts.

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“…NOTCH signaling activation requires the proteolytic processing of this type I integral membrane protein by a two-step cleavage process catalyzed first by a metalloprotease and then by the γ-secretase complex composed of the integral membrane proteins presenilin, nicastrin, Aph1, and Pen-2 (14)(15)(16)(17). Increased activation of the NOTCH signaling has been reported in several tumor types (18).…”

Section: Introductionmentioning

confidence: 99%

z-Leucinyl-Leucinyl-Norleucinal Induces Apoptosis of Human Glioblastoma Tumor–Initiating Cells by Proteasome Inhibition and Mitotic Arrest Response

Monticone

Biollo

Fabiano

et al. 2009

Molecular Cancer Research

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γ-secretase inhibitors have been proposed as drugs able to kill cancer cells by targeting the NOTCH pathway. Here, we investigated two of such inhibitors, the Benzyloxicarbonyl-Leu-Leu-Nle-CHO (LLNle) and the N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), to assess whether they were effective in killing human glioblastoma tumor-initiating cells (GBM TIC) in vitro. We found that only LLNle was able at the micromolar range to induce the death of GBM TICs by apoptosis. To determine the cellular processes that were activated in GBM TICs by treatment with LLNle, we analyzed the amount of the NOTCH intracellular domain and the gene expression profiles following treatment with LLNle, DAPT, and DMSO (vehicle). We found that LLNIe, beside inhibiting the generation of the NOTCH intracellular domain, also induces proteasome inhibition, proteolytic stress, and mitotic arrest in these cells by repressing genes required for DNA synthesis and mitotic progression and by activating genes acting as mitotic inhibitors. DNA content flow cytometry clearly showed that cells treated with LLNle undergo arrest in the G 2 -M phases of the cell cycle. We also found that DAPT and L-685,458, another selective Notch inhibitor, were unable to kill GBM TICs, whereas lactacystin, a pure proteasome inhibitor, was effective although at a much less extent than LLNle. These data show that LLNle kills GBM TIC cells by inhibiting the proteasome activity. We suggest that LLNle, being able to target two relevant pathways for GBM TIC survival, may have a potential therapeutic value that deserves further investigation in animal models.

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Nicastrin is required for Presenilin-mediated transmembrane cleavage in Drosophila

Cited by 145 publications

References 33 publications

A conserved tetraspanin subfamily promotes Notch signaling in Caenorhabditis elegans and in human cells

A conserved tetraspanin subfamily promotes Notch signaling in Caenorhabditis elegans and in human cells

Baculoviruses expressing the human familial Alzheimer's disease presenilin 1 mutation lacking exon 9 increase levels of an amyloid beta-like protein in Sf9 cells

z-Leucinyl-Leucinyl-Norleucinal Induces Apoptosis of Human Glioblastoma Tumor–Initiating Cells by Proteasome Inhibition and Mitotic Arrest Response

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