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WO2000026364A1 - Regulateur de l'activite de signalisation notch - Google Patents

Regulateur de l'activite de signalisation notch Download PDF

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Publication number
WO2000026364A1
WO2000026364A1 PCT/IB1999/001891 IB9901891W WO0026364A1 WO 2000026364 A1 WO2000026364 A1 WO 2000026364A1 IB 9901891 W IB9901891 W IB 9901891W WO 0026364 A1 WO0026364 A1 WO 0026364A1
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Prior art keywords
protein
notch
fragment
notchless
sequence
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PCT/IB1999/001891
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English (en)
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Stephen Cohen
Antonius Bouwmeester
Julien Royet
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European Molecular Biology Laboratory
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Priority to CA002349477A priority Critical patent/CA2349477A1/fr
Priority to EP99971453A priority patent/EP1127127A1/fr
Publication of WO2000026364A1 publication Critical patent/WO2000026364A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a novel regulator protein and to nucleic acid that encodes this protein.
  • the invention also relates to methods of diagnosis and therapy of disease using this protein and nucleic acids encoding therefor.
  • the Notch signalling pathway comprises an intracellular signalling mechanism that is essential for proper embryonic development. This pathway was first identified and studied in Drosophila, although comparative analyses have since demonstrated a remarkable degree of functional conservation between many of these developmentally important genes in Drosophila and their vertebrate counterparts.
  • the Notch locus has been shown to comprise an evolutionarily conserved cell interaction mechanism that plays a fundamental role in controlling the progression of immature cells to a more differentiated state.
  • Notch genes encode transmembrane receptors that help to determine cell fate during development. Notch acts as a receptor in a signalling pathway that when activated, may block or delay the progression of immature cells toward a more committed state. Inappropriate activation or inactivation of Notch can lead cells to adopt an incorrect fate.
  • Notch genes are linked to various diseases including some cancers (Zagouras et al, 1995; Capobianco et al, 1997; Gallahan and Callahan, 1997; Gridley, 1997). Indeed, two members of the mammalian Notch gene family were initially identified as oncogenes and at least three distinct members of the Notch gene family (Notch 1 , Notch 2 and Notch 4) can contribute to neoplasia in mammals. It seems that truncation of human Notch 1 plays a causal role in the formation of T cell neoplasms.
  • Notch function may also be ⁇ linked to neurodegenerative disease by virtue of its connection to Presenilin (Levitan and Greenwald, 1995; de Strooper et al, 1998).
  • Presenilins are linked to processing of amyloid precursors, suggesting a potential role in the genesis of Alzheimer's disease.
  • a protein comprising an amino acid sequence as identified in SEQ. ID. No. 1, or a functional equivalent thereof.
  • Notchless This novel protein, termed Notchless, was identified in a screen for dominant modifiers of a Notch mutant phenotype in the Drosophila wing.
  • the mutant dominantly suppresses the wing notching phenotype of notchoid mutations and the Notchless protein is shown herein to bind to the cytoplasmic domain of Notch.
  • Notchless modifies Notch signalling activity in a variety of Notch-dependent signalling processes in both Drosophila and Xenopus embryos.
  • functional equivalent is meant any compound that possesses the same conformation as a domain of the Notchless protein that is responsible for its physiological function.
  • this term is meant to include any macromolecule or molecular entity that mimics the conformation of the Notchless protein or that possesses an equivalent complementarity of shape to that possessed by the binding sites of the Notchless protein whose sequence is identified in SEQ ID 1.
  • invertebrate and vertebrate homologues of the Drosophila Notchless protein with the proviso that the protein is not the Xenopus protein, the sequence of which is available on the accession number AF069737.
  • a mouse EST (AA396500), a human EST (AA341327) and an S. cerevisiae DNA sequence (1351791).
  • the C. elegans sequence was compiled by the inventors from multiple clones (C48486, D70156, C35601, M89091) and has a gap in the 6th D40 repeat (this sequence was not identified as a gene by the C. elegans genome project). Until now, no function has been ascribed to any of these nucleic acid sequences in regulating Notch.
  • fragments or variants of the Notchless protein or closely related proteins exhibiting significant sequence homology are also intended to include fragments or variants of the Notchless protein or closely related proteins exhibiting significant sequence homology.
  • fragments is meant any portion of the entire protein sequence that retains a physiological function of the wild type Notchless protein, such as for example, an ability to bind specifically to Notch. Accordingly, fragments containing single or multiple amino acid deletions from either terminus of the protein or from internal stretches of the primary amino acid sequence form one aspect of the present invention.
  • “Variants” include mutants containing amino acid substitutions, insertions or deletions from the wild type Notchless sequence.
  • the Notchless protein is thought to function by binding to the cytoplasmic domain of Notch. It is thought (although the applicant does not wish to be limited by this theory) that Notchless also binds to protein factors other than Notch, since mutants that reduce or remove Notchless expression have been shown to increase Notch activity (see Examples).
  • the predicted Notchless protein has a novel highly conserved N-terminal domain followed by 9 D40 repeats ( Figure 6A).
  • the WD40 repeat is found in a wide variety of proteins of diverse function and is thought to be a protein interaction domain (reviewed in Neer et al, 1994). Typically, WD40 proteins contain 7 repeats. Structure analysis of ⁇ -transducin suggests that these form a propeller-like structure and that 7 repeats can pack to make a flat cylinder (Neer and Smith, 1996).
  • Blast searches using the amino-terminal sequence identified closely related sequences identified in yeast, C. elegans, man and mouse. In all cases the N-terminal domain is followed by WD repeats. In the human and mouse, only short EST fragments have been sequenced; until now, no function has been assigned to these sequences.
  • Xenopus Nle cDNA contains 9 WD repeats with strong similarity to the Drosophila and C. elegans proteins.
  • WD40 repeats are more similar between species than they are to other WD40 repeats in the protein of the same species. Together this suggests that these proteins represent true orthologues.
  • Database searches suggest that there may only be one member of this gene family in C. elegans. mouse and the human.
  • a Notchless protein or functional equivalent thereof modified at one or more positions in the sequence of the protein by the substitution, insertion or deletion of one or more amino acids from the wild type sequence shown in SEQ ID No. 1.
  • such proteins may contain single or multiple amino acid deletions from either terminus of the protein or from internal stretches of the primary amino acid sequence.
  • the Notchless protein or functional equivalent thereof may be prepared by any suitable means as will be clear to the man of skill in the art.
  • the protein is generated by recombinant DNA technology by expression of the encoding DNA in an expression vector in a host cell.
  • Such expression methods are well known to those of skill in the art and many are described in detail in DNA cloning: a practical approach, Volume 11:
  • Protein compounds may also be prepared using the known techniques of genetic engineering such as site-directed or random mutagenesis as described, for example, in
  • a protein according to the present invention may be fused to an effector or reporter molecule such as a label, toxin or bioactive molecule.
  • Such molecules may comprise an additional protein or polypeptide fused to the Notchless protein, or functional equivalent, at its amino- or carboxy-terminus or added internally.
  • the purpose of the additional polypeptide may be to aid detection, expression, separation or purification of the protein or may be to lend additional properties to the protein as desired.
  • fusion proteins comprising Notchless, or a functional equivalent thereof may be used as a "platform" to deliver biologically active protein domains to Notch.
  • an enzyme could be fused to Notchless or to a functional equivalent, that could be targeted to Notch in this fashion.
  • a fusion partner would preferably be regulated in some fashion so that it would only be activated when the Notch and Notchless proteins physically interact. This may be through some conformational change, so that only when the Notch binding domain of Notchless interacts with the Notch protein is the fusion partner activated.
  • Another mechanism may be through the inclusion in the fusion protein of a regulatable domain such as a hormone regulation domain, so that the activity of the fusion partner may be limited until the relevant drug is provided.
  • a particular enzyme of choice as a fusion partner might be a protease. Such an enzyme would act to "clip" the activated form of Notch that is expressed in some cancers, so inactivating it.
  • any effector protein or effector domain
  • Post- translational modifications such as phosphorylation (where kinase or phosphatase is the effector enzyme), or ubiquitination would also have the desired effect.
  • reporter molecules such as luciferase, green fluorescent protein, or horse radish peroxidase.
  • Labels of choice may be radiolabels or molecules that are detectable spectroscopically, for example fluorescent or phosphorescent chemical groups.
  • Linker molecules such as streptavidin or biotin may also be used.
  • other peptides or polypeptides may be fused to a Notchless protein.
  • Suitable peptides may be, for example, ⁇ -galactosidase, glutathione-S-transferase, luciferase, polyhistidine tags, secretion signal peptides, the Fc region of an antibody, the FLAG peptide, cellulose binding domains, calmodulin and the maltose binding protein.
  • Antibodies or peptides used to target the Notchless protein more efficiently towards a site of action may also be fused to the Notchless protein.
  • fusion molecules may be fused chemically, using methods such as chemical cross- linking. Suitable methods will be well known to those of skill in the art and may comprise for example, cross-linking of the thiol groups of cysteine residues or cross-linking using formaldehydes. Chemical cross-linking will in most instances be used to fuse non-protein compounds, such as cyclic peptides and labels.
  • the method of choice will often be to fuse the molecules genetically.
  • the genes or gene portions that encode the proteins or protein fragments of interest are engineered so as to form one contiguous gene arranged so that the codons of the two gene sequences are transcribed in frame.
  • a process for the identification of an agent capable of modifying the levels of expression or activity of a Notch protein comprising screening a Notchless mutant in a sensitised Notch genetic background with a drug and selecting for an altered phenotype.
  • the Notchless mutant is an insect larva.
  • compound screening may be performed by feeding larvae of a suitable genetic background on food containing the compound of interest. Modified function is then assessed in the adult fly. This allows the identification of compounds suitable for oral delivery that would either enhance or suppress the severity of the mutant phenotype through either increasing or decreasing the activity of Nle with respect to Notch.
  • One advantage of such a screen is that it makes possible the selection of drugs that modify Notch activity. Furthermore, these drugs will be suitable for oral delivery. According to a further aspect of the present invention there is provided a drug identified by such a screen.
  • nucleic acid sequence comprising:
  • nucleic acid molecule comprises a nucleotide fragment identical to or complementary to any portion of the nucleotide sequence shown in SEQ ID No 2, that encodes a Notchless protein. It will be appreciated that individual or multiple nucleotide insertions, deletions and substitutions may be made without departing from this aspect of the invention.
  • the nucleic acid molecule may comprise DNA, cDNA or RNA.
  • the nucleic acid molecule comprises DNA.
  • a probe capable of screening for Notchless and prepared from the DNA sequence of SEQ ID No 2.
  • the probe preferably comprises at least 15 oligonucleotides, more preferably between 15 and 300 oligonucleotides, most preferably between 15 and 50 oligonucleotides.
  • the invention also includes cloning and expression vectors containing the DNA sequences of the invention.
  • expression vectors will incorporate the appropriate transcriptional and translational control sequences, for example enhancer elements, promoter-operator regions, termination stop sequences, mRNA stability sequences, start and stop codons or ribosomal binding sites, linked in frame with the nucleic acid molecules of the invention.
  • nucleic acid sequences encoding secretion signalling and processing sequences.
  • Vectors according to the invention include plasmids and viruses (including both bacteriophage and eukaryotic viruses). Many such vectors and expression systems are well known and documented in the art. Particularly suitable viral vectors include baculovirus-, adenovirus- and vaccinia virus-based vectors.
  • heterologous polypeptides and polypeptide fragments in prokaryotic cells such as E. coli
  • prokaryotic cells such as E. coli
  • DNA cloning a practical approach, Volume II: Expression systems, edited by D.M. Glover (IRL Press, 1995).
  • Expression in eukaryotic cells in culture is also an option available to those skilled in the art for the production of heterologous proteins; see for example O'Reilly et al, (1994) Baculovirus expression vectors - a laboratory manual (Oxford University Press) or DNA cloning: a practical approach, Volume IV: Mammalian systems, edited by D.M. Glover (IRL Press, 1995).
  • Suitable vectors can be chosen or constructed for expression of Notchless proteins, containing the appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. bacteriophage, or phagemid, as appropriate.
  • Molecular Cloning a Laboratory Manual. Many known techniques and protocols for manipulation of nucleic acid, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al.
  • the vectors of choice are virus-based.
  • a further aspect of the present invention provides a host cell containing a nucleic acid encoding a Notchless protein or functional equivalent.
  • a still further aspect provides a method comprising introducing such nucleic acid into a host cell or organism.
  • Introduction of nucleic acid may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection or transduction using retrovirus or other viruses, such as vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation or transfection using bacteriophage.
  • nucleic acid of the invention may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for allowing expression of the gene.
  • the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
  • Transgenic animals transformed so as to express or overexpress in the germ line one or more Notchless proteins or functional equivalents as described herein form a still further aspect of the invention, along with methods for their production.
  • Many techniques now exist to introduce transgenes into the embryo or germ line of an organism such as for example, illustrated in Watson et al, (1994) Recombinant DNA (2nd edition), Scientific American Books.
  • a method of gene therapy of a pathological condition caused by a gene mutation in a patient comprising administering to a patient a nucleic acid encoding a Notchless protein or functional equivalent, in a therapeutically-effective amount.
  • Suitable diseases include various cancers such as human acute lymphoblastic leukaemia (Capobianco et al., 1997), cervical squamous carcinoma and adenocarcinoma (Zygouros et al., 1995), and mammary tumours (Gallahan & Callahan, 1997; Gridley, 1997) and certain neurodegenerative diseases, for example familial Alzheimer's disease.
  • the nucleic acid may be introduced into a patient by any suitable means, as will be clear to those of skill in the art.
  • Effective methods of introduction include the use of adenovirus, adeno-associated virus, herpes virus, alpha virus, pox virus and other virus vectors that serve as delivery vehicles for expression of the gene. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.
  • Retroviral vectors may also be used (see Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.)
  • Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukaemia, Virus, Murine Leukaemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus.
  • terapéuticaally effective amount refers to an amount of a therapeutic agent to treat, ameliorate, or prevent the disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the precise effective amount for a subject for a given situation can be determined by routine experimentation and is within the judgement of the clinician.
  • An effective dose will typically be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of nucleic acid construct.
  • Non- viral strategies for gene therapy also exist that utilise agents capable of condensing nucleic acid molecules, delivering these molecules to cells and protecting them from degradation inside the cell.
  • Vehicles for delivery of gene therapy constructs may be administered either locally or systemically.
  • Such strategies include, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see Curiel (1992) Hum Gene Ther 3:147-154) and ligand linked DNA (see Wu (1989) J Biol Chem 264:16985-16987). Naked DNA may also be employed, optionally using biodegradable latex beads to increase uptake. Other methods will be known to those of skill in the art.
  • Liposomes can act as gene delivery vehicles encapsulating nucleic acid comprising a gene cloned under the control of a variety of tissue-specific or ubiquitously-active promoters. Mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585 may also be used.
  • Direct delivery of gene therapy compositions will generally be accomplished, in either a single dose or multiple dose regime, by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue.
  • the compositions may also be administered directly into a tumour or lesion.
  • Other modes of administration include oral and pulmonary administration, using suppositories, and transdermal applications, needles, and gene guns or hyposprays.
  • a pharmaceutical composition comprising a Notchless protein or functional equivalent according to the first aspect of the invention, in conjunction with a pharmaceutically-acceptable excipient.
  • Suitable excipients will be well known to those of skill in the art and may, for example, comprise a phosphate-buffered saline (0.01M phosphate salts, 0.138M NaCl, 0.0027M KC1, ⁇ H7.4), a liquid such as water, saline, glycerol and ethanol, optionally also containing mineral acid salts such as hydrochlorides, hydrobromides, phosphates.
  • a carrier may also be used that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.
  • Pharmaceutical compositions may also contain additional preservatives to ensure a long shelf life in storage.
  • the present invention provides a method of treatment of cancer or of a neurodegenerative disease in a patient comprising administering to a patient a Notchless protein or functional equivalent in a therapeutically-effective amount.
  • the present invention provides for the use of a Notchless protein or functional equivalent, a nucleic acid encoding a Notchless protein or functional equivalent or of a pharmaceutical composition containing a Notchless protein or functional equivalent in therapy.
  • a Notchless protein or functional equivalent according to the invention in conjunction with a pharmaceutically-acceptable carrier in the manufacture of a medicament for the treatment or prevention of cancer or of a neurodegenerative disease in a human or an animal.
  • the invention also comprises the use of components according to the invention in a diagnostic kit for the detection of mutations present in the genome of a patient.
  • a diagnostic kit for the detection of mutations present in the genome of a patient.
  • a component will comprise a Notchless protein or functional equivalent, a nucleic acid encoding a Notchless protein or functional equivalent or an antibody that specifically recognises a Notchless protein or functional equivalent.
  • Other desirable components for inclusion in such a kit will be clear to those of skill in the art. All documents mentioned in the text are incorporated herein by reference.
  • Figure 1 Comparison of sequences of Notchless proteins from Drosophila, yeast, C. elegans, mouse, human dxx ⁇ X. laevis.
  • Notchless phenotype (suppressed nd phenotype) produced when one copy of Nle is mutated in a nd fly.
  • Figure 7 Expression of Xenopus Notchless during embryonic development and phenotypic effects of Notchless overexpression on formation of primary neurons.
  • l(2)kl3714 is from the BDGP P-element lethal collection.
  • P-element excisions were generated by providing a chromosomal source of transposase activity. 105 w excision lines were isolated. One of these was not able to suppress the nd 1 phenotype and was therefore reverted to wild type. Others were analysed for imprecise excision of the P- element by Southern blots. Su(H) SF8 and Su(H) AR9 are described in Schweisguth and Posakony, 1992. Ax 28 is described by de Celis and Garcia-Bellido, 1994.
  • deltex 1 and pCaSpeR hs-dx are described by Diederich, et ai, 1994; Matsuno et al., 1995.
  • nd 1 , nd 2 , ndfa and Dp(l;2)51b are described in Lindsley and Zimm (1992) "The genome of
  • Mouse monoclonal anti-Wg is described in Brook and Cohen, 1996.
  • Mouse monoclonal anti-Notch C17.9C6 is described in Fehon et al., 1990.
  • Mouse (12CA5) anti-HA and rabbit (HA-11) anti-HA were obtained form BabCo.
  • a 15Kb Sail genomic fragment of phage Y2-6 was inserted into the Xhol site of the transformation vector pCaSpeR4.
  • UAS-Nle was prepared by cloning the 1.5Kb Nle cDNA as a Notl-Xhol fragment into pUAST (Brand and Perrimon, 1993).
  • An HA- tagged version of Nle was generated by introducing three copies of the HA epitope (YPYDVPDYA) immediately downstream of the first Methionine residue.
  • the BamHl- Asc ⁇ fragment of pKS-Nle was replaced by a corresponding PCR fragment amplified using the following primers:
  • pMT-HA-N/e was generated by cloning HA-N/e as a BamHl-Sall fragment into the inducible expression vector pRmHa-3.
  • pRmHa-3 -Notch is described in Fehon et al, 1990.
  • GST-fusion protein binding assay 35 S-labelled Numb-N (aa 1-224), Numb-C (aa 224-547) and full length Nle were synthesised by in vitro transcription/translation using the TNT system (Promega). Binding reactions were carried out with lO ⁇ l of labeled protein and 5 ⁇ l of GST or GST- NICD coupled beads in 400 ⁇ l of PBS 0.1 % NP-40 for 1 hour at room temperature. The beads were washed 6 times in PBS, proteins eluted in SDS-gel sample buffer, separated on 10% SDS-polyacrylamide gels and visualised by autoradiography.
  • Schneider S2 cells were grown at 25°C in Schneider's medium (Gibco-BRL) with 1% fetal calf serum and 1% Gentamicin. Cells were harvested and transferred into 6- well 30 mm diameter tissue culture plates at 75% confluence. Each well was then rinsed 3 times with Schneider medium without serum and incubated with 10 ⁇ g of DNA in 500 ⁇ l of Schneider medium and 50 ⁇ l of Lipofectin (Gibco-BRL) for 6 hours. Cells were incubated overnight in medium without Lipofectin. Expression was induced by adding CuSO4 to 0.7 mM and incubating for 12 hours.
  • Schneider's medium Gibco-BRL
  • Gentamicin Gentamicin
  • Cells were harvested and lysed by sonication in PBS, 50 mM NaCl, 5 mM EDTA, 5 mM DTT, 1% Triton X-100 containing protease inhibitors (1 mM PMSF, 5 ⁇ g/ml aprotinin and leupeptin). Cells debris was removed by 10,000 xG centrifugation. 500 ⁇ l of extract (corresponding to lxl ⁇ 6 cells) was incubated with 3 ⁇ l of Rabbit anti-HA antibody for 1 hour at 4°C followed by 1 hour at 4°C with 20 ⁇ l of a 50% slurry of Protein A-Sepharose beads (Pharmacia).
  • the beads were washed 4 times with lysis buffer, proteins eluted in SDS- gel sample buffer and run on a 6% SDS-polyacrylamide gel.
  • the gel was electrophoretically transferred to Immobilon-P membrane (Millipore), blocked for 1 hour at room temperature in 5% dry milk in TTBS (10 mM Tris pH 8.0, 150 mM NaCl, 0.2% Tween-20) and incubated overnight at 4°C with mouse-anti Notch (9C6; used at 1 :2000) or mouse anti-HA (1 : 1000).
  • the membrane was washed 3 times 5 min in TTBS and incubated for 1 hour with peroxidase-conjugated goat-anti-mouse IgG (Jackson labs) diluted 1 :5000 in TTBS.
  • the blot was washed 3 times for 5 min in TTBS and developed using ECL reagents (Amersham).
  • notchoid 1 (nd 1 ) is a viable mutant allele of Notch that causes scalloping of the wing
  • nd ⁇ provides a sensitised genetic background in which to screen for modifiers of Notch signalling activity.
  • strains were generated in which the original P-element had been removed by transposase-mediated excision. These chromosomes differ from the original l(2)kl3714 chromosome only by the lack of the P-element and fail to suppress the nd ⁇ phenotype (data not shown).
  • l(2)kl3714 comes from a collection of P-elements that are supposed to be lethal mutations, it was noted that homozygous mutant individuals are recovered in this stock. They are morphologically normal, though males are sterile (not shown).
  • nd ⁇ mutant wings The scalloping of nd ⁇ mutant wings is thought to be caused by reduced Wingless activity because overexpression of Wingless can suppress the phenotype (Couso and Martinez Arias, 1994) and because further reducing wingless activity enhances the nd ⁇ phenotype (Hing et al, 1994).
  • Removing one copy of the Su(H) gene enhances the severity of the nd ⁇ phenotype and causes an obvious reduction of Wingless expression at the DV boundary (relative to the level in wild-type, compare Figure 2B and E; nd ⁇
  • the 1.5Kb transcript was identified as the Notchless gene by two criteria: (i) the 15Kb Sail fragment of phage Y2-6 was able to restore Notchless activity when introduced into a ndl Nle/+ mutant background (data not shown). The transgene contains all of the 1.5Kb transcription unit but only part of the other transcription unit, (ii) Expression of the 1.5Kb cDNA under GAL4 control restores full Me activity, nd 1; Me/+ mutant flies carrying a GAL4 driver show the suppressed nd ⁇ phenotype ( Figure 3B).
  • the P-element insertion that causes the mutation is located 310 bp 5' to the start of the Me open reading frame. It is therefore likely that the P-element mutant reduces the level of Me expression.
  • mutants were generated by mobilisation of the P-element.
  • An excision mutant named Me ⁇ deletes sequences on both sides of the insertion ( Figure 3 A).
  • Abruptex alleles of Notch have been classified as mutations that increase Notch activity. Their phenotypes are enhanced by increasing the level of wild-type Notch gene product and are suppressed by reducing it (de Celis and Garcia-Bellido, 1994; Brennan et ai, 1997). Like other gain-of-function Abruptex alleles A ⁇ 2& flies show reduced numbers of some bristles on the head and thorax, as well as shortening of wing veins ( Figure 4A, B). These phenotypes are made more severe by introducing an extra copy of the wild-type Notch gene (data not shown). They are also enhanced by removing one copy of the Notchless gene (Figure 4C).
  • a ⁇ 2& Nlel+ flies show increased loss of both small bristles in the thorax (note the large bare patch outlined in red, Figure 4C), and of large bristles in the head compared with Ax 28 flies. Blue shading on the head indicates the cluster of orbital bristles.
  • Deltex is thought to function as a positive regulator of Notch activity (Diederich et al, 1994; Matsuno et al, 1995).
  • deltex mutant flies show a phenotype resembling a reduction of Notch activity: nicking of the distal region of the wing blade and thickening of the wing veins ( Figure 5 A).
  • Removing one copy of Notchless restores the deltex mutant wing to normal ( Figure 5B).
  • Notchless activity can be compensated for by simultaneously reducing Notchless activity.
  • removing one copy of Notchless enhances the effects of overexpressing Deltex using a heat shock- e/tex transgene (Matsuno et al, 1995).
  • Under conditions where Deltex overexpression produces no visible abnormality in an otherwise wild-type wing (Figure 5C)
  • it causes loss of veins in a Me/+ background (arrow, Figure 5D). This resembles the effects of increasing Notch activity in Abruptex mutants.
  • XNle was isolated by PCR using the degenerate primers, F 5'-CGC AGA ATT CCI TTY GAY GTI CCI GTI GAY AT-3' and R 5'-GGT GCT CGA GCY TGI GGY TGR TAI ATD ATR TC-3 ', designed against conserved peptides, PFDVPVDI and DIIYQPQ respectively, found in the Nle domain of the vertebrate proteins identified as expressed sequence tags.
  • Phage stock of a stage 30 library (Stratagene) was used as template to amplify a 200 bp fragment that spans the Nle domain. Five independent clones were sequenced and found to be identical. This fragment was used to screen the stage 30 library, which resulted in the isolation of 25 positive clones of which the longest of 2.2Kb was sequenced on both strands.
  • Temporal expression was assayed by RT-PCR analysis as described by Bouwmeester et al, 1996 using the following primer set that amplifies a XNle fragment of 135 bp; F 5'-CAC CAG ATA AAC TGC AGT TAG-3', R 5'-CTG TTT CAA CTG ATT GCT TCT-3' (28 cycles).
  • pCS2- Me was constructed by subcloning of a 2.2Kb EcoRI fragment in the complementary site of pCS2+.
  • Capped RNA was synthesised using pCS2- ⁇ 7V7e, pCS2-Drosophila Nle (kindly provided by J. Wittbrodt; Max-Planck Institut fur Biophysikalischechemie, Gottingen, Germany) and pCS2-NOTCHI-ICD (kindly provided by C. Kintner; Salk Institute, San Diego CA) digested with Not-1 and transcribed with Sp6.
  • Synthetic RNA (2.5 - 5 ng of XNle and DNle RNA, 100-200 pg XN-ICD) was injected into one blastomere of the 2-cell stage embryo. Embryos were harvested at early neurula stage (st. 13-15). ⁇ -galactosidase activity, a lineage marker for injections, was revealed using X-gal as substrate prior to whole mount in situ. Primary neurons were identified by ⁇ -tubulin staining. Antisense ⁇ -tubulin RNA was synthesised from pBS- ⁇ -tubulin digested with Not I and transcribed with T3 polymerase.
  • Figure 7 shows the temporal expression of XNle.
  • Total RNA isolated from the indicated stages of development was analyzed by RT-PCR analysis for expression of XNle and Histone H4 (loading control).
  • E egg; 4C, 4 cell stage; all other lanes are labelled with stage numbers according to Nieuwkoop and Faber (1956) Normal table of Xenopus Laevis A. Systematical and chronologicla survey of the development from the fertilised egg until the end of metamorphosis. North Holland publishing company, Amsterdam.
  • B Spatial expression of XNle. Whole mount in situ hybridization was used to visualize expression of XNle at neural plate stage (st. 17), tailbud stage (st. 25) and tadpole stage (st. 35).
  • Expression patterns are described in the text, symbols: NC, neural crest; pm, paraxial mesoderm; b, brain; e, eye; ba, branchial arches; s, somites; sp, segmental plate; vbi, ventral blood islands.
  • C Phenotypic consequence of overexpression of XNle, DNle and an activated form of Xenopus Notchl (XN-ICD) on primary neurogenesis. LacZ RNA was co-injected to mark the injected side. Control embryo: 1, i, m denote lateral, intermediate and medial rows of ⁇ -tubulin expressing primary neurons.
  • Xenopus Notchless gene (XNle) is maternally transcribed and that expression remains relatively constant during the early stages of embryonic development without obvious signs of localisation. Elevated levels arise at the end of gastrulation and are maintained during neurulation and organogenesis ( Figure 7A). Localised expression is observed in two lateral domains adjacent to the rostral neural plate, which correspond to the premigratory neural crest cells, and in a region at the anterior end of the neural plate, which corresponds to placodal precursors ( Figure 7B). There is also increased expression in the involuting paraxial mesoderm and in two patches lateral to the closing slit blastopore, through which future somitic cells involute.
  • XNle expression resembles that of other components of the Notch pathway, including Delta and Kuzbanian (Chitnis et al, 1995; Pan and Rubin, 1997). These expression domains correspond to regions where Notch signalling has been implicated in cell fate specification events (Chitnis et al, 1995; Coffman et al, 1993; Jen et ⁇ /., 1997).
  • penotvpe mean ⁇ SE (n) k ⁇ est
  • Wild-type flies have on average 260 small bristles per thorax (Brennan et al, 1997). This number is reduced in Apterous-GAL4/+ flies. Expression of Nle further reduces the number of bristles.
  • Notchless protein binds to the intracellular domain of Notch -
  • Figure 8A shows weak non-specific binding of GST control beads to all three proteins, but this is well below the level of specific binding observed with Numb- N and Notchless.
  • Notchless and Notch in Drosophila S2 cells were tested by immunoprecipitation.
  • Expression of full-length Notch and HA-tagged Notchless proteins was monitored by immunoblotting of total cell extracts (see below). Extracts from induced and uninduced cells were immunoprecipitated using antibody to the HA- tag, and a blot of the gel was probed with a monoclonal antibody directed against the intracellular part of Notch and reprobed subsequently with anti-HA to visualise the immunoprecipitated HA-Notchless.
  • Figure 8B shows the results of the immunoprecipitation of Notch and Nle expressed in S2 cells.
  • Upper panel blot probed with mouse monoclonal anti-Notch (9C6).
  • Lower panel same blot probed subsequently with mouse anti-HA.
  • Lanes 1-3 total cell lysates from S2 cells expressing (1) Notch (2) HA-Nle or (3) both proteins. Note that low levels of endogenous Notch are seen in the Nle-expressing cells (lane 2).
  • Lanes 4-7 Immunoprecipitates from cells expressing (4)Notch, (5) HA- Notchless or (6, 7) both proteins. + indicates immunoprecipitated with Rabbit anti-HA and protein A beads.
  • - indicates control precipitation with protein A beads alone.
  • Notch protein was thus found to immunoprecipitate with HA-Notchless from cells expressing both proteins (Figure 8B lane 7). No precipitation was observed in controls lacking HA-Nle or anti-HA (lanes 4, 6). Together these results indicate that Notchless binds directly to the intracellular domain of Notch.
  • Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann's organizer. Nature 382, 595-601.
  • Deltex acts as a positive regulator of Notch signaling through interactions with the Notch ankyrin repeats. Development 121, 2633-2644.
  • Pan, D. and Rubin,G.M. (1997) Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis.

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Abstract

L'invention concerne une nouvelle protéine régulatrice dénommée Notchless et l'acide nucléique codant pour ladite protéine; ainsi que des méthodes permettant de diagnostiquer et de traiter certaines maladies, notamment le cancer et les maladies neurodégénératives, au moyen de ladite protéine et des acides nucléiques qui la codent.
PCT/IB1999/001891 1998-11-03 1999-11-03 Regulateur de l'activite de signalisation notch WO2000026364A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004062686A3 (fr) * 2003-01-09 2004-11-18 Lorantis Ltd Traitement medical
WO2004087195A3 (fr) * 2003-04-01 2005-02-24 Lorantis Ltd Dosages et traitements medicaux

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997011716A1 (fr) * 1995-09-29 1997-04-03 Yale University Manipulation de cellules incompletement differenciees en utilisant la fonction de notch
WO1997018822A1 (fr) * 1995-11-22 1997-05-29 Yale University Proteines, acides nucleiques et anticorps deltex de vertebres, et procedes et compositions relatifs a ceux-ci
WO1998020142A1 (fr) * 1996-11-07 1998-05-14 Lorantis Limited Notch

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997011716A1 (fr) * 1995-09-29 1997-04-03 Yale University Manipulation de cellules incompletement differenciees en utilisant la fonction de notch
WO1997018822A1 (fr) * 1995-11-22 1997-05-29 Yale University Proteines, acides nucleiques et anticorps deltex de vertebres, et procedes et compositions relatifs a ceux-ci
WO1998020142A1 (fr) * 1996-11-07 1998-05-14 Lorantis Limited Notch

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ROYET, J. ET AL.: "Notchless encodes a novel WD40-repeat-containing protein that modulates Notch signalling activity", THE EMBO JOURNAL, vol. 17, no. 24, 15 December 1998 (1998-12-15), pages 7351 - 7360, XP002133042 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004062686A3 (fr) * 2003-01-09 2004-11-18 Lorantis Ltd Traitement medical
WO2004087195A3 (fr) * 2003-04-01 2005-02-24 Lorantis Ltd Dosages et traitements medicaux

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