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PubMed2001

PS1 N- and C-terminal fragments form a complex that functions in APP processing and Notch signaling.

Presenilin proteins play critical roles in the proteolytic processing of both Notch and amyloid precursor protein (APP). Presenilin itself undergoes endoproteolytic processing to generate an N-terminal and C-terminal fragment. As demonstrated previously, overexpression of presenilin 1 (PS1) holoprotein does not change the levels of the N-terminal and C-terminal fragments (NTF and CTF). When we coexpress the PS1 NTF and CTF, marked increases in the cellular levels of these fragments are seen. By coexpressing the PS1 NTF and CTF, we demonstrate conclusively that a noncovalent complex of the NTF and CTF is the active species of presenilin. However, although the PS1 NTF/CTF complex is necessary for gamma-secretase activity, it is not sufficient. Independent overexpression of the PS1 NTF and CTF was also used to show that the Asp-257 and Asp-385 mutations in PS1 decrease Abeta production by a direct effect on gamma-secretase activity and not by the inhibition of PS1 endoproteolysis.

Amyloid Precursor Protein Secretases↗
PubMed2001

Presenilin-mediated transmembrane cleavage is required for Notch signal transduction in Drosophila.

The cleavage model for signal transduction by receptors of the LIN-12/Notch family posits that ligand binding leads to cleavage within the transmembrane domain, so that the intracellular domain is released to translocate to the nucleus and activate target gene expression. The familial Alzheimer's disease-associated protein Presenilin is required for LIN-12/Notch signaling, and several lines of evidence suggest that Presenilin mediates the transmembrane cleavage event that releases the LIN-12/Notch intracellular domain. However, doubt was cast on this possibility by a report that Presenilin is not required for the transducing activity of N(ECN), a constitutively active transmembrane form of Notch, in Drosophila. Here, we have reassessed this finding and show instead that Presenilin is required for activity of N(ECN) for all cell fate decisions examined. Our results indicate that transmembrane cleavage and signal transduction are strictly correlated, supporting the cleavage model for signal transduction by LIN-12/Notch and a role for Presenilin in mediating the ligand-induced transmembrane cleavage.

Active Transport, Cell Nucleus↗
PubMed1998

Identification of the minimal requirements for binding to the human epidermal growth factor (EGF) receptor using chimeras of human EGF and an EGF repeat of Drosophila Notch.

Many proteins contain so-called epidermal growth factor (EGF)-like domains that share the characteristic spacing of cysteines and glycines with members of the EGF family. They are, however, functionally unrelated, despite the fact that the three-dimensional structure of these EGF-like domains, also, is often very similar to that of the EGF receptor agonists. In the present study, we linked an EGF-like repeat from the Drosophila Notch protein to the N- and C-terminal linear tail sequences of human EGF (hEGF), and we showed that this chimera (E1N6E) is unable to bind or activate the hEGF receptor. This recombinant protein was then used as a basic construct for identifying the minimal requirements for high affinity EGF receptor binding and activation. We selectively reintroduced a limited number of important hEGF-derived residues, and by using this unique approach, we were able to make hEGF/Notch chimeras that, compared with wild type hEGF, showed nearly 100% binding affinity and mitogenic activity on HER-14 cells expressing the hEGF receptor.

Animals↗
PubMed2001

Distinct intramembrane cleavage of the beta-amyloid precursor protein family resembling gamma-secretase-like cleavage of Notch.

The intramembrane cleavage of beta-amyloid precursor protein by gamma-secretase is the final step in the generation of amyloid beta-protein. A 59- or 57-residue C-terminal fragment called CTFgamma is produced concomitantly. Putative CTFgamma generated in rat brain membrane preparations was purified and sequenced. Instead of CTFgamma, shorter 50- and 49-residue fragments were identified. In addition, we found similar C-terminal fragments of beta-amyloid precursor-like proteins 1 and 2; these were also cleaved at corresponding sites. This newly identified cleavage occurs at a site two to five residues inside the cytoplasmic membrane boundary, which is very similar to gamma-secretase-like cleavage of Notch 1.

Amino Acid Sequence↗
PubMed2004

The structure and dynamics of tandem WW domains in a negative regulator of notch signaling, Suppressor of deltex.

WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded beta-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hinge-like motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms.

Amino Acid Sequence↗
PubMed2001

Wasp, the Drosophila Wiskott-Aldrich syndrome gene homologue, is required for cell fate decisions mediated by Notch signaling.

Wiskott-Aldrich syndrome proteins, encoded by the Wiskott-Aldrich syndrome gene family, bridge signal transduction pathways and the microfilament-based cytoskeleton. Mutations in the Drosophila homologue, Wasp (Wsp), reveal an essential requirement for this gene in implementation of cell fate decisions during adult and embryonic sensory organ development. Phenotypic analysis of Wsp mutant animals demonstrates a bias towards neuronal differentiation, at the expense of other cell types, resulting from improper execution of the program of asymmetric cell divisions which underlie sensory organ development. Generation of two similar daughter cells after division of the sensory organ precursor cell constitutes a prominent defect in the Wsp sensory organ lineage. The asymmetric segregation of key elements such as Numb is unaffected during this division, despite the misassignment of cell fates. The requirement for Wsp extends to additional cell fate decisions in lineages of the embryonic central nervous system and mesoderm. The nature of the Wsp mutant phenotypes, coupled with genetic interaction studies, identifies an essential role for Wsp in lineage decisions mediated by the Notch signaling pathway.

Alleles↗
PubMed1996

T cell leukemia-associated human Notch/translocation-associated Notch homologue has I kappa B-like activity and physically interacts with nuclear factor-kappa B proteins in T cells.

Translocation-associated Notch homologue (TAN-1), a gene originally cloned from the translocation breakpoint of a human T cell leukemia carrying a 9:7(q34.3) translocation, encodes a protein belonging to the Notch/Lin-12/Glp-1 receptor family. These receptors mediate the specification of numerous cell fates during development in invertebrates and vertebrates. The intracellular portion of Notch/TAN-1 contains six ankyrin repeats that are similar to those found in cytoplasmic I kappa B proteins. I kappa B proteins are specific inhibitors of nuclear factor (NF)-kappa B/Rel transcription factors. Here we show that TAN-1 has functional properties of an I kappa B-like regulator with specificity for the NF-kappa B p50 subunit. A recombinant polypeptide corresponding to the cytoplasmic portion of TAN-1 (TAN-1C) specifically inhibited the DNA binding of p50-containing NF-kappa B complexes. When overexpressed in an appropriate cell line, TAN-1C prevented kappa B-dependent transactivation in transient reporter gene assays in a fashion similar to the structurally related protein, Bcl-3. TAN-1C could activate kappa B-dependent gene expression by attenuating the inhibitory effect of an excess of p50 homodimers. Immunoprecipitation experiments showed that the TAN-1 from a T cell line is associated with NF-kappa B containing p50 and p65 subunits. These observations indicate that TAN-1C may directly engage NF-kappa B transcription factors and modulate nuclear gene expression.

Animals↗
PubMed2006

Phenotypic plasticity of T cell progenitors upon exposure to Notch ligands.

Despite many efforts, the nature of thymic immigrants that give rise to T cells has remained obscure, especially since it became known that extrathymic lineage-negative, Sca-1-positive, c-kit high progenitor cells differ from intrathymic early T cell progenitors (ETPs) by functional potential and dependence on Notch signaling. After our observation that intrathymic T cell precursors expressing a human CD25 reporter under control of pre-TCRalpha regulatory elements almost exclusively have the ETP phenotype, we have analyzed the phenotypic changes of reporter-expressing common lymphoid progenitor (CLP) cells in the bone marrow when cultured on Delta-like 1-expressing stromal cells. We note that these quickly adopt the phenotype of double negative (DN)2 thymocytes with little display of the ETP phenotype. Our data suggest that common lymphoid progenitor (CLP) cells could be responsible for the rapid reconstitution of thymus function after bone marrow transplantation since CLP cells in the blood have the capacity to rapidly enter the thymus and become DN2 thymocytes.

Animals↗
PubMed2000

Serrate1-induced notch signalling regulates the decision between immunity and tolerance made by peripheral CD4(+) T cells.

Signals derived from antigen-presenting cells (APC) influence the functional differentiation of CD4(+) T cells. We report here that Serrate1 (Jagged1), a ligand for the Notch1 receptor, may contribute to the differentiation of peripheral CD4(+) T cells into either helper or regulatory cells. Our findings demonstrate that antigen presented by murine APC overexpressing human Serrate1 induces naive peripheral CD4(+) T cells to become regulatory cells. These cells can inhibit primary and secondary immune responses, and transfer antigen-specific tolerance to recipient mice. Our results show that Notch signalling may help explain 'linked' suppression in peripheral tolerance, whereby tolerance induced to one epitope encompasses all epitopes on that antigen during the course of an immune response.

Animals↗
PubMed2004

RING1 inhibits transactivation of RBP-J by Notch through interaction with LIM protein KyoT2.

The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1.

Animals↗
PubMed2002

Gamma-secretase/presenilin inhibitors for Alzheimer's disease phenocopy Notch mutations in Drosophila.

Signaling from the Notch (N) receptor is essential for proper cell-fate determinations and tissue patterning in all metazoans. N signaling requires a presenilin (PS)-dependent transmembrane-cleaving activity that is closely related or identical to the gamma-secretase proteolysis of the amyloid-beta precursor protein (APP) involved in Alzheimer's disease pathogenesis. Here, we show that N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester, a potent gamma-secretase inhibitor reported to reduce amyloid-beta levels in transgenic mice, prevents N processing, translocation, and signaling in cell culture. This compound also induces developmental defects in Drosophila remarkably similar to those caused by genetic reduction of N. The appearance of this phenocopy depends on the timing and dose of compound exposure, and effects on N-dependent signaling molecules established its biochemical mechanism of action in vivo. Other gamma-secretase inhibitors caused similar effects. Thus, the three-dimensional structure of the drug-binding site(s) in Drosophila gamma-secretase is remarkably conserved vis-à-vis the same site(s) in the mammalian enzyme. These results show that genetics and developmental biology can help elucidate the in vivo site of action of pharmacological agents and suggest that organisms such as Drosophila may be used as simple models for in vivo prescreening of drug candidates.

Active Transport, Cell Nucleus↗
PubMed1998

Proneural gene self-stimulation in neural precursors: an essential mechanism for sense organ development that is regulated by Notch signaling.

To learn about the acquisition of neural fate by ectodermal cells, we have analyzed a very early sign of neural commitment in Drosophila, namely the specific accumulation of achaete-scute complex (AS-C) proneural proteins in the cell that becomes a sensory organ mother cell (SMC). We have characterized an AS-C enhancer that directs expression specifically in SMCs. This enhancer promotes Scute protein accumulation in these cells, an event essential for sensory organ development in the absence of other AS-C genes. Interspecific sequence comparisons and site-directed mutagenesis show the presence of several conserved motifs necessary for enhancer action, some of them binding sites for proneural proteins. These and other data indicate that the enhancer mediates scute self-stimulation, although only in the presence of additional activating factors, which most likely interact with conserved motifs reminiscent of NF-kappaB-binding sites. Cells neighboring the SMC do not acquire the neural fate because the Notch signaling pathway effectors, the Enhancer of split bHLH proteins, block this proneural gene self-stimulatory loop, possibly by antagonizing the action on the enhancer of the NF-kappaB-like factors or the proneural proteins. These data suggest a mechanism for SMC committment.

Animals↗
PubMed1998

Delta and Serrate are redundant Notch ligands required for asymmetric cell divisions within the Drosophila sensory organ lineage.

Asymmetric divisions allow a precursor to produce four distinct cells of a Drosophila sensory organ lineage (SOL). Whereas this process requires cell-cell communication via Notch (N) receptor, mitotic recombination that removes the N ligand Delta (Dl) or Serrate (Ser) in the SOL had mild or no effect. Removal of both Dl and Ser, however, led to cell fate transformations similar to the N phenotype. Cell fate transformation occurred even when a single SOL cell lost both Dl and Ser. Thus, Dl and Ser are redundant in mediating signaling between daughter cells to specify their distinct cell fates.

Animals↗
PubMed1995

The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by Notch signaling.

The Notch protein (N) acts as a transmembrane receptor for intercellular signals controlling cell fate choices in vertebrates and invertebrates. The signal of N activation may be transduced directly from the cell surface into the nucleus by an evolutionarily conserved transcription factor, Suppressor of Hairless [Su(H)], by its regulated nuclear import. Su(H) is shown here to play a direct role in the immediate response of the genome to N signaling in Drosophila. First, Su(H) mutant embryos derived from mutant germ-line clones exhibited a "neurogenic" phenotype of neural hypertrophy similar to the N phenotype. Second, the lack of N lateral signaling in these Su(H) mutant embryos was associated with a failure to express the m5 and m8 genes from the Enhancer of split Complex [E(spl)-C]. Finally, the Su(H) protein bound to the regulatory sequences of the E(spl)-C m5 and m8 genes, and these binding sites were required for the activation of the m5 and m8 promoters in the ventral neuroectoderm. The expression of the E(spl)-C m8 gene was found to be similarly regulated by Su(H) during wing imaginal disc development. Thus, the transcriptional activation of these E(spl)-C genes by Su(H) appears to be a direct and relatively general response to the activation of N. However, we also present evidence indicating that N signals in an Su(H)-independent manner during mesectoderm formation.

Animals↗
PubMed1995

Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity.

We have investigated the functional relationships among three loci that are required for multiple alternative cell fate decisions during adult peripheral neurogenesis in Drosophila: Notch (N), which encodes a transmembrane receptor protein, Suppressor of Hairless [Su(H)], which encodes a DNA-binding transcription factor, and the Enhancer of split gene complex [E(spl)-C], which includes seven transcription units that encode basic helix-loop-helix (bHLH) repressor proteins. We describe several lines of evidence establishing that Su(H) directly activates transcription of E(spl)-C genes in response to N receptor activity. Expression of an activated form of the N receptor leads to elevated and ectopic E(spl)-C transcript accumulation and promoter activity in imaginal discs. We show that the proximal upstream regions of three E(spl)-C genes contain multiple specific binding sites for Su(H). The integrity of these sites, as well as Su(H) gene activity, are required not only for normal levels of expression of E(spl)-C genes in imaginal disc proneural clusters, but also for their transcriptional response to hyperactivity of the N receptor. Our results establish Su(H) as a direct regulatory link between N receptor activity and the expression of E(spl)-C genes, extending the known linear structure of the N cell-cell signaling pathway.

Animals↗
PubMed2002

Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of Hairless.

The DNA-binding transcription factor Suppressor of Hairless [Su(H)] functions as an activator during Notch (N) pathway signaling, but can act as a repressor in the absence of signaling. Hairless (H), a novel Drosophila protein, binds to Su(H) and has been proposed to antagonize N signaling by inhibiting DNA binding by Su(H). Here we show that, in vitro, H directly binds two corepressor proteins, Groucho (Gro) and dCtBP. Reduction of gro or dCtBP function enhances H mutant phenotypes and suppresses N phenotypes in the adult mechanosensory bristle. This activity of gro is surprising, because it is directed oppositely to its traditionally defined role as a neurogenic gene. We find that Su(H)-H complexes can bind to DNA with high efficiency in vitro. Furthermore, a H-VP16 fusion protein causes dominant-negative phenotypes in vivo, a result consistent with the proposal that H functions in transcriptional repression. Taken together, our findings indicate that "default repression" of N pathway target genes by an unusual adaptor/corepressor complex is essential for proper cell fate specification during Drosophila peripheral nervous system development.

Alcohol Oxidoreductases↗
PubMed2007

Polarized exocytosis and transcytosis of Notch during its apical localization in Drosophila epithelial cells.

Notch (N) and its ligands, Delta (Dl) and Serrate (Ser), are transmembrane proteins that mediate the cell-cell interactions necessary for many cell-fate decisions. In Drosophila, N is predominantly localized to the apical portion of epithelial cells, but the mechanisms and functions of this localization are unknown. Here, we found N, Dl, and Ser were mostly located in the region from the subapical complex (SAC) to the apical portion of the adherens junctions (AJs) in wing disc epithelium. N was delivered to the SAC/AJs in two phases. First, polarized exocytosis specifically delivered nascent N to the apical plasma membrane and AJs in an O-fut1-independent manner. Second, N at the plasma membrane was relocated to the SAC/AJs by Dynamin- and Rab5-dependent transcytosis; this step required the O-fut1 function. Disruption of the apical polarity by Drosophila E-cadherin (DEcad) knock down caused N and Dl localization to the SAC/AJs to fail. N, but not Dl, formed a specific complex with DEcad in vivo. Finally, our results suggest that juxtacrine signaling in epithelia generally depends on the apicobasally polarized structure of epithelial cells.

Adherens Junctions↗
PubMed2006

Cartilage, SOX9 and Notch signals in chondrogenesis.

Cartilage repair is an ongoing medical challenge. Tissue engineered solutions to this problem rely on the availability of appropriately differentiated cells in sufficient numbers. This review discusses the potential of primary human articular chondrocytes and mesenchymal stem cells to fulfil this role. Chondrocytes have been transduced with a retrovirus containing the transcription factor SOX9, which permits a greatly improved response of the cells to three-dimensional culture systems, growth factor stimulation and hypoxic culture conditions. Human mesenchymal stem cells have been differentiated into chondrocytes using well-established methods, and the Notch signalling pathway has been studied in detail to establish its role during this process. Both approaches offer insights into these in vitro systems that are invaluable to understanding and designing future cartilage regeneration strategies.

Cartilage, Articular↗