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WO2003014152A2 - Proteines de liaison - Google Patents

Proteines de liaison Download PDF

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Publication number
WO2003014152A2
WO2003014152A2 PCT/IB2002/003645 IB0203645W WO03014152A2 WO 2003014152 A2 WO2003014152 A2 WO 2003014152A2 IB 0203645 W IB0203645 W IB 0203645W WO 03014152 A2 WO03014152 A2 WO 03014152A2
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Prior art keywords
binding protein
cell
fusion protein
protein
protein according
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PCT/IB2002/003645
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English (en)
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WO2003014152A3 (fr
Inventor
Stephen Cohen
Aurelio Teleman
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European Molecular Biology Laboratory
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Priority to AU2002328115A priority Critical patent/AU2002328115A1/en
Publication of WO2003014152A2 publication Critical patent/WO2003014152A2/fr
Publication of WO2003014152A3 publication Critical patent/WO2003014152A3/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to proteins that bind to phosphatidyhnositol 5 -phosphate (PI(5)P).
  • the invention also relates to the use of these proteins to measure and visualize PI(5)P in vivo and in vitro and as tools to investigate the role of PI(5)P in vivo.
  • a number of diseases result from dysfunctional cellular signal transduction.
  • Signal transduction ultimately controls gene expression, and cellular proliferation and differentiation, meaning that any deficiency in signal transduction can have wide-ranging consequences.
  • Pis can be rapidly synthesised and degraded in discrete membrane domains or even subnuclear structures, making these molecules ideal as regulators and integrators of very dynamic mechanisms of cell regulation.
  • Pis appear to be specifically involved in the control of a number of cellular events, such as the spatial and temporal organisation of key signalling pathways, the rearrangement of the actin cytoskeleton (see, for example, Tall et al, 2000, Curr. Biol. 10:743-6; Tolias et al, (2000) Curr. Biol. 10: 153-6; Hartwig et al, (1995), Cell 82: 643- 653) and intracellular vesicle trafficking (see Payrastre et al, (2001), Cell. Signal.; 13(6): 377-87 for review; particularly section 4, entitled "Role of the different Pis").
  • Phosphorylated species characterised to date include phosphatidyhnositol 3-phosphate (PI(3)P), phosphatidyhnositol 4-phosphate (PI(4)P), phosphatidyhnositol 5-phosphate (PI(5)P), phosphatidyhnositol 3,4-phosphate (PI(3,4)P 2 ), phosphatidyhnositol 3, 5- phosphate (PI(3,5)P ) and phosphatidyhnositol 3, 4, 5-phosphate (PI(3,4,5)P 3 ).
  • PI(3,4,5)P 3 arises by phosphorylation of PI(4,5)P 2 in response to insulin signaling and is a key component in transmission of the signal to the cell nucleus.
  • PI(3)P in contrast, plays a pivotal role in vesicular traffic and secretion.
  • Phosphoinositides exert many of their effects by recruiting protein components. These protein components bind phosphoinositides via PH (pleckstrin homology) domains or FYVE (Fab-1, YOTB, Nacl, and EEA1) domains (see Lemmon et al (2000) for a review of PH domain function Biochem J. 350, 1). Some of these domains bind to a variety of Pis with low affinity (see Kavran et al (1998) J. Biol Chem. 273 (46), 30497). Other domains bind with high affinity and specificity to one phosphoinositide species. For instance, the PH domain of GRP1 selectively binds PI(3,4,5)P 3 (see US 6,194,173) while the PH domain of phospholipase C- ⁇ , selectively binds PI(4,5)P 2 .
  • PH pleckstrin homology domains
  • FYVE Fab-1, YOTB, Nacl, and EE
  • PI(5)P One of the least understood and studied phosphoinositide species is PI(5)P.
  • PI(5)P was first characterised in 1987 (Rameh et al (1987) Nature 390, 192). Rameh et al established that PI(4,5,)P 2 could be synthesised by phosphorylation of the hydroxyl group at position D4 of PI(5)P and the presence of this PI was demonstrated in mouse fibroblast cells. Since then, a potential pathway for PI(5)P synthesis has been proposed (Tolias et al (1998), J. Biol. Chem. 273(29), 18040) but the role of PI(5)P in vivo remains unclear.
  • a phosphatidyhnositol 5-phosphate (PI(5)P) binding protein that binds selectively to PI(5)P, said protein comprising a pleckstrin homology (PH) domain from a Melted protein, or a functional equivalent thereof.
  • PI(5)P phosphatidyhnositol 5-phosphate
  • PH domain is meant a protein sequence of between 100 and 120 amino acids having at least 30% identity, preferably at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or greater identity, with a PH domain of a Melted protein from Drosophila, human, mouse or C. elegans, which have the amino acid sequence of Figure 3b, Figure 7b, Figure 8b and Figure 9b respectively.
  • Melted proteins contain additional regions of homology in parts of the sequence other than the PH domains, that are herein termed Melted homology (MH) domains.
  • the Melted protein from which the PH domain of the invention is derived contains regions that display significant homology with one or both of the signature MH1 and MH2 domains of the Melted proteins from Drosophila, human, mouse or C. elegans.
  • binding selectively is meant that the PI(5)P binding proteins of the invention bind PI(5)P with a higher affinity than they bind other Pis.
  • the PI(5)P binding proteins of the invention bind PI(5)P with an affinity that is at least twice the affinity with which they bind to other Pis.
  • the PI(5)P binding protein bind PI(5)P with an affinity that is at least five times the affinity with which they bind to other Pis, more preferably at least ten times the affinity with which they bind to other Pis .
  • PI(5)P binding proteins of the invention bind selectively to PI(5)P. They can therefore be used to purify PI(5)P and are also extremely useful as research tools to elucidate the role of PI(5)P.
  • the PI(5)P binding protein of the invention preferably comprises the sequence of a PH domain from a Melted protein that is derived from a vertebrate, a metazoan or from an insect.
  • the Melted protein is from an insect, it is preferably from Drosophila.
  • the PH domain from a Melted protein from Drosophila comprises the sequence in Figure 3b (dMelt PH domain) or a variant thereof.
  • the PI(5)P binding protein may comprise the sequence of Figure 3 a (complete dMelt sequence) or a variant thereof.
  • the Melted protein is isolated from a vertebrate, it is preferably isolated from a mammal, such as a mouse or a human. If from a human, the PH domain from the Melted protein preferably comprises the sequence of Figure 7b (hMelt PH domain) or is a variant thereof. In one embodiment of the invention, the PI(5)P binding protein may comprise the sequence of Figure 7a (complete hMelt sequence) or a variant thereof. If from a mouse, the PH domain from the Melted protein preferably comprises the sequence of Figure 8b (mMelt PH domain) or a variant thereof. In one embodiment of the invention, the PI(5)P binding protein may comprise the sequence of Figure 8a (partial mMelt sequence) or a variant thereof.
  • the Melted protein is isolated from a metazoan, it is preferably isolated from a nematode, such as Caenorhabditis elegans. If from C. elegans, the PH domain from the Melted protein preferably comprises the sequence of Figure 9b (nMelt PH domain) or is a variant thereof. In one embodiment of the invention, the PI(5)P binding protein may comprise the sequence of Figure 9a (complete nMelt sequence) or a variant thereof.
  • PI(5)P binding proteins of the invention that retain the ability to bind selectively to PI(5)P.
  • Functional equivalents of the PI(5)P binding proteins of the invention thus include natural biological variants (e.g. allelic variants or geographical variants within the species from which the PI(5)P binding proteins are derived).
  • variants of the PI(5)P binding proteins of the invention also include, for example, mutants containing amino acid substitutions, insertions or deletions from the wild type sequence.
  • Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
  • Typical such substitutions are among Ala, Nal, Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, i.e.
  • Variants with improved function from that of the wild type sequence may also be designed through the systematic or directed mutation of specific residues in the protein sequence. Improvements in function that may be desired will include greater specificity for PI(5)P or greater affinity for PI(5)P.
  • the term “functional equivalent” also refers to molecules that are structurally similar to the PI(5)P binding proteins of the present invention or that contain similar or identical tertiary structure.
  • the term “functional equivalents” refers to molecules that are structurally similar to the sequences in Figures 3b, 7b, 8b and 9b and which are responsible for PI(5)P binding.
  • Such functional equivalents may be derived from the natural PI(5)P binding proteins or they may be prepared synthetically or using techniques of genetic engineering.
  • synthetic molecules that are designed to mimic the tertiary structure or active site of the naturally-occurring PI(5)P binding proteins of the invention are considered to be functional equivalents.
  • the term “functional equivalent” also refers to homologues of the PI(5)P binding proteins of the invention.
  • homologue is meant a protein exhibiting a high degree of similarity or identity to the amino acid sequence of the PI(5)P proteins of the invention.
  • similarity is meant that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences.
  • identity is meant that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences.
  • homologues possess greater than 30% identity with the sequence of the naturally-occurring PH domain protein sequences. More preferably, homologues according to the invention show greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity with the sequence of the natural protein, as aligned using, for example, the GCG suite of programs (Wisconsin Package Version, 10.1, Genetics Computer Group (GCG), Madison, Wise.) using the default settings.
  • GCG suite of programs Wiconsin Package Version, 10.1, Genetics Computer Group (GCG), Madison, Wise.
  • Such homologues may include proteins in which one or more of the amino acid residues are substituted with another amino acid residue. Any such substituted amino acid residue may or may not be a naturally occurring amino acid.
  • the PI(5)P binding proteins or functional equivalents of the invention may be prepared in recombinant form by expression in a host cell. Suitable expression methods are well known to those of skill in the art and many are described in detail by Sambrook J. et al Molecular cloning: a laboratory manual New York: Cold Spring Harbour Laboratory Press, 2000) and Fernandez J.M. & Hoeffler J.P. (Gene expression systems. Using nature for the art of expression ed. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto, 1998.) The proteins or functional equivalents of the present invention can also be prepared using conventional techniques of protein chemistry, for example by chemical synthesis.
  • the invention provides antibodies which bind to a PI(5)P binding protein or a functional equivalent thereof as described above.
  • Antisera and monoclonal antibodies can be made by standard protocols using the PI(5)P binding protein or functional equivalent thereof as an immunogen (see, for example, Antibodies: A Laboratory Manual ed. By Harlow and Lane, Cold Spring Harbor Press, 1988).
  • the term "antibody” includes fragments of antibodies which also bind specifically to a PI(5)P binding protein or a functional equivalent thereof.
  • the term “antibody” further includes chimeric and humanised antibody molecules having specificity for the PI(5)P binding proteins of the invention or functional equivalents thereof.
  • Antibodies which bind PI(5)P binding proteins are useful in a variety of methods for elucidating the function of PI(5)P as they can be used to demonstrate the presence of a PI(5)P binding protein bound to PI(5)P.
  • the label is an enzyme, a radiolabel or a fluorescent tag.
  • a fusion protein comprising a PI(5)P binding protein of the invention or functional equivalent thereof that is genetically or chemically fused to one or more peptides or polypeptides.
  • the PI(5)P binding protein or functional equivalent thereof is fused to a marker domain.
  • said marker domain is a fluorescent tag, an epitope tag that allows purification by affinity binding, an enzyme tag that allows histochemical or fluorescent labelling, or a radiochemical tag.
  • said fluorescent tag is a green fluorescent protein (GFP) or a fluorescent derivative thereof such as YFP or CFP (see Prasher et al, (1995), Trends in Genetics, 11(8), 320-329).
  • Fusion proteins will aid researchers in establishing the role of PI(5)P in vivo. As indicated previously, no domain which binds selectively to PI(5)P has been identified to date. Fusion proteins comprising a marker domain fused to a domain that binds selectively to PI(5)P can be used, for example, to determine the subcellular location of PI(5)P, measure levels of PI(5)P in cells and screen for compounds that alter the levels of PI(5)P, as described in more detail later.
  • fusion proteins may be most conveniently generated recombinantly from nucleic acid molecules in which two nucleic acid sequences are fused together in frame. These fusion proteins will be encoded by nucleic acid molecules that contain the relevant coding sequence of the fusion protein in question.
  • the invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding a PI(5)P binding protein or functional equivalent thereof according to the first aspect of the invention or a fusion protein according to the second aspect of the invention.
  • nucleic acid molecules include single- or double-stranded DNA, cDNA and RNA, as well as synthetic nucleic acid species.
  • the nucleic acid molecules are DNA or cDNA molecules.
  • the invention also includes cloning and expression vectors incorporating the nucleic acid molecules.
  • expression vectors may additionally incorporate regulatory sequences such as enhancers, promoters, ribosome binding sites and termination signals in the 5' and 3' untranslated regions of genes, that are required to ensure that the coding sequence is properly transcribed and translated, or to regulate the expression of the protein relative to the growth of the cell in which it is expressed.
  • control sequences may be included that encode signal peptides or leader sequences. These leader or control sequences may be removed by the host during post-translational processing.
  • the vectors may incorporate signal sequences which direct the expressed proteins to specific subcellular locations. For example, it may be desirable to assess the effect of a PI(5)P binding protein in the nucleus in which case a nuclear localisation signal may be included in the vector.
  • Vectors according to the invention include plasmids and viruses (including both bacteriophage and eukaryotic viruses), as well as other linear or circular DNA carriers, such as those employing transposable elements or homologous recombination technology.
  • Many such vectors and expression systems are known and documented in the art (see, for example, Fernandez J.M. & Hoeffler J.P. in Gene expression systems. Using nature for the art of expression ed. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto, 1998).
  • Suitable viral vectors include baculovirus-, adenovirus- and vaccinia virus- based vectors.
  • Suitable hosts for recombinant expression include commonly used prokaryotic species, such as E. coli, or eukaryotic yeasts that can be made to express high levels of recombinant proteins and that can easily be grown in large quantities. Mammalian cell lines grown in vitro are also suitable, particularly when using virus-derived expression systems. Another suitable expression system is the baculovirus expression system that involves the use of insect cells as hosts. An expression system may also constitute host cells that have the appropriate encoding nucleic acid molecules incorporated into their genome. Proteins may also be expressed in vivo, for example in insect larvae or in mammalian tissues. A variety of techniques may be used to introduce the vectors according to the present invention into prokaryotic or eukaryotic host cells.
  • Suitable transformation or transfection techniques are well described in the literature (see, for example, Sambrook et al, Molecular cloning: a laboratory manual New York: Cold Spring Harbour Laboratory Press, 2000; Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience, New York, 1991; Spector, Goldman & Leinwald, Spector et a, I Cells, a laboratory manual; Cold Spring Harbour Laboratory Press, 1998).
  • expression systems may either be transient (e.g. episomal) or permanent (such as by chromosomal integration) according to the needs of the system.
  • the invention also provides antisense nucleic acid molecules which hybridise under high stringency hybridisation conditions to the nucleic acid molecules encoding the PIP(5)P binding proteins or functional equivalents thereof.
  • High stringency hybridisation conditions are defined herein as overnight incubation at 42 °C in a solution comprising 50% formamide, 5XSSC (150mM NaCI, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5xDenhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65 °C.
  • a label capable of being detected is attached to these antisense nucleic acid molecules.
  • the label is selected from the group consisting of radioisotopes, fluorescent compounds and enzymes.
  • these antisense nucleic acid molecules can be used as probes to detect defects in the expression of a PI(5)P binding protein.
  • these probes can be used to measure the level of a RNA encoding a PI(5)P binding protein in a cell to establish whether there is a defect in the transcription of the gene encoding the PI(5)P binding protein.
  • the antisense nucleic acid molecules of the invention are therefore useful in diagnostic assays.
  • the antisense nucleic acid molecules can also be used in antisense therapy, inhibiting the transcription or translation of a gene encoding a PI(5)P binding protein or a functional equivalent thereof by hybridising to mRNA or genomic DNA encoding the PI(5)P binding protein.
  • the invention also includes transformed or transfected prokaryotic or eukaryotic host cells containing a nucleic acid molecule encoding a PI(5)P binding protein or variant thereof as described above or an antisense nucleic acid molecule which hybridises to such a nucleic acid molecule.
  • a further aspect of the invention provides a method for preparing a PI(5)P binding protein or a functional equivalent thereof or a fusion protein as defined above, which comprises culturing a host cell containing a nucleic acid molecule encoding a PI(5)P binding protein or functional equivalent thereof according to the invention under conditions whereby said protein is expressed and recovering said protein thus produced.
  • the invention also provides various methods which employ PI(5)P binding proteins and functional equivalents thereof to investigate the role of PI(5)P.
  • the invention provides a method of purifying PI(5)P- containing membrane fractions using the PI(5)P binding proteins of the invention.
  • This method may comprise passing a cell extract through a column to which a PI(5)P binding protein of the invention is bound.
  • a cell extract may be added to a solid surface to which a PI(5)P binding protein is fixed.
  • PI(5)P-containing membrane fractions bound to the PI(5)P binding proteins can be removed following washing. This method will be a useful tool in establishing the role of PI(5)P as it will enable researchers to identify proteins which are copurified with the PI(5)P-containing membrane fractions.
  • the invention further provides a method of visualizing the subcellular location of PI(5)P in cells using a PI(5)P binding protein according to the first aspect of the invention or a fusion protein according to the second aspect of the invention.
  • a PI(5)P binding protein according to the first aspect of the invention or a fusion protein according to the second aspect of the invention.
  • the subcellular location of PI(5)P binding proteins bound to PI(5)P, and hence the subcellular location of can be visualised using antibodies that bind to the PI(5)P binding proteins of the invention.
  • a method of visualizing the subcellular localisation of PI(5)P may comprise expressing a fusion protein according to the second aspect of the invention in a cell and detecting the presence of the marker domain.
  • the marker domain in the fusion protein comprises a green fluorescent protein (GFP) or a fluorescent derivative thereof such as YFP or CFP (see Prasher et al, (1995), Trends in Genetics, 11(8), 320-329).
  • GFP green fluorescent protein
  • Constructs comprising GFP are preferred since it is inherently fluorescent and easily detectable in the cell, for example by fluorescence microscopy, confocal microscopy, spectroscopy (quantitative) or fluorescence activated cell sorting (FACS). Furthermore, it is small, non-toxic and easy to generate in the cell.
  • This method can be used to visualise the subcellulai- location of PI(5)P in cells both in vivo and in vitro.
  • the cells may be transfected with expression constructs encoding the fusion protein.
  • a transgenic animal expressing the fusion protein under control of a ubiquitously expressed promoter or a regulatable promoter may be created.
  • These methods of visualizing the subcellular localisation or level of PI(5)P can be adapted to provide a method of identifying compounds that alter the subcellulai- localisation of PI(5)P in cells.
  • This method comprises visualizing the subcellular locations of PI(5)P by expressing a fusion protein according to the second aspect of the invention in a cell and detecting the presence of a marker domain, contacting said cell with a candidate compound and assessing the effect of the candidate compound on subcellular localisation of PI(5)P by detecting changes in location of the marker domain.
  • the invention also includes compounds that are identified by such a method.
  • Suitable compounds for testing in the methods of the invention may be any one of a number of different compounds.
  • Particularly suitable compounds are small molecules that are suitable for oral delivery, including natural or modified substrates, hormones, small organic molecules, such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, and inorganic molecules.
  • Other suitable compounds might include polypeptides, such as enzymes, receptors, antibodies, and structural or functional mimetics of these polypeptides, including peptides and peptidomimetics, although these compounds would not be suitable for oral delivery.
  • the method of the invention may be modified to test protein drugs that are not orally deliverable, for example, using other delivery routes.
  • candidate polypeptide and peptide compounds for testing in the method of the invention may either be natural compounds, isolated from natural sources, or may be synthetic or recombinant.
  • the invention further provides a method of altering the subcellular localisation of PI(5)P comprising contacting a cell with a compound, as described above, that alters the subcellular localisation of PI(5)P. This may be done, for example, by contacting a cell with a compound known to alter the subcellular location or level of PI(5)P, such as a compound identified by the method outlined above.
  • the invention further provides a method of altering the subcellular localisation of a PI(5)P binding protein or fusion protein as described above by expressing in a cell a PI(5)P binding protein or fusion protein according to the invention that includes a signal sequence that directs the expressed protein to a particular subcellular location.
  • a nuclear localisation sequence may be incorporated into the PI(5)P binding protein coding sequence so that the PI(5)P binding protein is transported to the nucleus. This method will enable researchers to assess the effect of altering the normal subcellular location of the PI(5)P binding proteins.
  • reference to a "cell” includes both an in vitro cell and a cell in vivo, such as in a transgenic animal.
  • the invention further provides a method of measuring PI(5)P levels in a cell or a cell extract using a PI(5)P binding protein according to a first aspect of the invention or a fusion protein according to the second aspect of the invention.
  • the method of measuring PI(5)P levels in a cell extract may comprise separating the cell extract on a matrix, such as on a gel or membrane, and adding a PI(5)P binding protein to the matrix. Following removal of unbound PI(5)P binding protein by washing, the amount of bound PI(5)P binding protein and hence the level of PI(5)P can be determined, for example, using anti-PI(5)P binding protein antibodies.
  • the PI(5)P binding protein may be in the form of a fusion protein.
  • the level of bound fusion protein and hence the level of PI(5)P can be determined by detecting the amount of marker domain present in the cell extract.
  • measurement of PI(5)P levels is facilitated by evaluating the degree of fluorescence intensity.
  • the method of measuring PI(5)P levels in a cell may comprise introducing a construct encoding a fusion protein according to the second aspect of the invention into a cell and detecting the amount of marker domain present in the cell.
  • a transgenic animal expressing the fusion protein under control of a ubiquitously expressed promoter or a regulatable promoter may be created and the amount of marker domain present in the cells in the transgenic animal may be determined.
  • a fluorescent marker domain measurement of PI(5)P levels is facilitated by evaluating the degree of fluorescence intensity, for example using microscopic spectroscopy.
  • the cells are vertebrate cells, metazoan cells or insect cells or extracts therefrom.
  • the cells are Drosophila cells, mouse cells, fish cells or human cells or extracts therefrom.
  • the ability to measure PI(5)P levels in cells and cell extracts is a useful tool in determining the function of PI(5) because it allows researchers to determine how PI(5)P levels differ in different cells. It also enables compounds to be identified which alter the levels of PI(5)P in cells which in turn enables researchers to study the effect of these changes in PI(5)P levels.
  • the invention further provides an in vitro method of screening for compounds that regulate PI(5)P levels in cells, said method comprising:
  • the invention further provides an in vivo method of screening for a compound that regulates PI(5)P levels in cells, said method comprising the steps of: ai) contacting a non-human transgenic organism with a candidate compound, wherein said transgenic organism expresses a fusion protein according to the second aspect oof the invention; and
  • the term "contacting" encompasses both coexpression of the candidate compound with the fusion protein or the use of the exogenously-applied candidate compound to treat cells, for example, by the administration of drugs.
  • the candidate compound is applied to a transgenic organism exogenously, it may be applied by a variety of routes, for example orally, intravenously, intranasally etc.
  • the candidate compound is administered orally.
  • the disclosed in vivo screening method tests the effect of the compound in a eukaryotic organism whose biological context accurately reflects that of the system in which the compound would be used as a therapeutic agent.
  • organisms which may be used in accordance with the present invention include mammals, such as mice, rats, rabbits, chickens, and pigs; amphibians such as frogs; fish, such as Zebrafish, Medakafish; and insects such as Drosophila.
  • mammals such as mice, rats, rabbits, chickens, and pigs
  • amphibians such as frogs
  • fish such as Zebrafish, Medakafish
  • insects such as Drosophila.
  • Even an organism as apparently divergent from humans as Drosophila displays a remarkable level of similarity with the human species that allows ⁇ nferences made in Drosophila to be paralleled in higher eukaryotes, such as humans.
  • cells from these organisms may be used in the in vitro screening methods of the invention.
  • the organism or the cell expresses a fusion protein comprising a PI(5)P binding protein and a marker domain.
  • a change in the level of the particular PI(5)P is generated in a cellular membrane.
  • the change in the amount of PI(5)P in a cellular membrane causes the fusion protein either to bind to and accumulate on (if a greater amount of PI(5)P is generated) or be released from (if a decrease in the amount of PI(5)P is generated) membranes of cells in the transgenic organism.
  • This migration event may be monitored by following the movement of the marker domain in the fusion protein towards or away from membranes of the cell, advantageously using real time techniques such as fluorescence microscopy. Accordingly, the invention provides elegant in vitro and in vivo systems in which the degree of activation or inhibition of a signalling pathway may be monitored in response to the action of a particular compound.
  • the extent of recruitment of the fusion protein to a cellular membrane is compared to a control organism or a control cell.
  • the control organism or control cell should ideally be identical in all respects to the test organism or cell.
  • the control organism or cell is not treated with the candidate compound and the extent of recruitment to the cellular membrane is compared between the two organisms or the two cells. Differential recruitment of the fusion protein between the two systems is indicative of an effect mediated by said candidate compound on said signalling pathway.
  • an “effect” on the signalling pathway is meant that the compound alters the level of PI(5)P in a membrane of a cell.
  • a potentiating effect will increase the signalling through the pathway, generally through effecting an increase in the amount of PI(5)P in the membrane.
  • a compound may also have an inhibitory effect.
  • the transgenic organism that is used may be any non-human transgenic system that is generally and conveniently used on a laboratory scale. Examples include: mammals, such as mice, rats, rabbits, chickens, and pigs; amphibians such as frogs; fish, such as Zebrafish, Medakafish; insects such as Drosophila; and basic organisms such as the nematode Caenorhabditis elegans. Of particular utility are organisms which have a rapid lifecycle and thus short generation time, such as Drosophila and fish. These organisms are additionally advantageous in that the effect of various concentrations of compounds can very easily be tested by adding the candidate compound to the fly food or the fish water.
  • Transgenic organisms can easily be generated according to methods known in the art. Local changes can be incoiporated by modification of somatic cells, whilst germ line therapy may be used to incorporate heritable modifications. Examples of suitable techniques include using retroviral vector infection (see Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, 2nd Ed.), microinjection of DNA into a fertilized egg, transfection of and homologous recombination in embryonic stem cells and nuclear transfer (see Anderson and Seidel, (1998) Science 280: 1400; Wilmut, (1998), Scientific American 279(6): 58). The exact technique that is used will depend on the particular organism that is chosen. For example, in the case of insects such as Drosophila, P-element mediated transformation will be a preferred technique.
  • Visualisation of the degree of recruitment to the cell membrane may be in situ in the transgenic organism using a non-invasive technique such as confocal microscopy, (see, for example, Matsumoto, B., ed. (2001) "Confocal microscopy", Harcourt Publishers Ltd Professional Publications) and monitoring the fluorescence intensity of single cells, calculated using an appropriate software package (such as the ImageQuant software package; Applied Precision Inc.) or magnetic resonance imaging (see, for example, “Magnetic Resonance Imaging”, Stark & Bradley Jr, 1998, 3rd Ed, Mosby).
  • confocal microscopy see, for example, Matsumoto, B., ed. (2001) "Confocal microscopy", Harcourt Publishers Ltd Professional Publications
  • an appropriate software package such as the ImageQuant software package; Applied Precision Inc.
  • magnetic resonance imaging see, for example, “Magnetic Resonance Imaging", Stark & Bradley Jr, 1998, 3rd Ed, Mosby).
  • the method used may be one that only allows tissues to be analysed after their isolation from the organism, either after humane killing or by bioscopy.
  • One example of such a technique is that of immunoblotting or immunohistochemistry.
  • Other examples include the use of spectrofluorimeters and microplate readers, fluorescence scanners, flow cytometers and magnetic resonance imaging. In many cases, the inconvenience of using such invasive techniques may be offset by the advantages they offer in terms of accuracy and quantification.
  • suitable compounds for testing in the methods of the invention may be any one of a number of different compounds.
  • Particularly suitable compounds are small molecules that are suitable for oral delivery, including natural or modified substrates, hormones, small organic molecules, such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, and inorganic molecules.
  • a compound that regulates levels of PI(5)P in cells identified according to any one of the embodiments of the invention recited above.
  • the compound may have either a potentiating or an inhibiting effect on PI(5)P levels.
  • the invention also provides a method of altering the level of PI(5)P in a cell comprising administering to said cell a compound that regulates levels of PI(5)P in cells.
  • This compound may be identified using one of the screening methods of the invention recited above. The ability to increase and decrease PI(5)P levels and assess the effects of such increases and decreases in cells is a useful tool in the investigation of the role of PI(5)P.
  • the PI(5)P binding proteins of the invention along with fusion proteins comprising these binding proteins and proteins identified as being regulators of PI(5)P levels in cells will have many medical applications.
  • the invention further provided a PI(5)P binding protein, a fusion protein or a compound involved in the regulation of PI(5)P levels, as outlined above, for use in therapy.
  • the invention also includes the use of a PI(5)P binding protein, a fusion protein or a compound involved in the regulation of PI(5)P levels in the manufacture of a medicament for the treatment of a disease or disorder in which abnormal levels of PI(5)P are implicated.
  • a further aspect of the invention includes a pharmaceutical composition
  • a pharmaceutical composition comprising a PI(5)P binding protein, a fusion protein or a compound involved in the regulation of PI(5)P levels according to any one of the embodiments of the invention recited above, in conjunction with a pharmaceutically-acceptable carrier molecule.
  • Carrier molecules may be genes, polypeptides, antibodies, liposomes, polysaccharides, polylactic acids, polyglycolic acids and inactive virus particles or indeed any other agent provided that the carrier does not itself induce toxicity effects or cause the production of antibodies that are harmful to the individual receiving the pharmaceutical composition.
  • Carriers may also include pharmaceutically acceptable salts such as mineral acid salts (for example, hydrochlorides, hydrobromides, phosphates, sulphates) or the salts of organic acids (for example, acetates, propionates, malonates, benzoates).
  • Pharmaceutically acceptable carriers may additionally contain liquids such as water, saline, glycerol, ethanol or auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like. Carriers may enable the pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions to aid intake by the patient.
  • a thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • the amount of the active compound in the composition should also be in a therapeutically- effective amount.
  • therapeutically effective amount used herein refers to the amount of agent needed to treat or ameliorate a targeted disease or condition.
  • An effective initial method to determine a "therapeutically effective amount” may be by carrying out assays in the transgenic organism model, although more accurate tests must be carried out on the target organism if initial tests are successful.
  • the transgenic organism model may also yield relevant information such as the preferred routes of administration that will lead to maximum effectiveness.
  • the exact therapeutically-effective dosage will generally be dependent on the patient's status at the time of administration.
  • Factors that may be taken into consideration when determining dosage include the severity of the disease state in the patient, the general health of the patient, the age, weight, gender, diet, time and frequency of administration, drug combinations, reaction sensitivities and the patient's tolerance or response to the therapy. The precise amount can be determined by routine experimentation but may ultimately lie with the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg (mass of drug compared to mass of patient) to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
  • Uptake of a pharmaceutical composition by a patient may be initiated by a variety of methods including, but not limited to enteral, intra-arterial, intrathecal, intramedullary, intramuscular, intranasal, intraperitoneal, intravaginal, intravenous, intraventricular, oral, rectal (for example, in the form of suppositories), subcutaneous, sublingual, transcutaneous applications (for example, see WO98/20734) or transdermal means.
  • Gene guns or hyposprays may also be used to administer pharmaceutical compositions.
  • the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • Direct delivery of the compositions can generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
  • the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • a method of treating a disorder or disease in which abnormal levels of PI(5)P are implicated in a patient comprising administering to the patient, a PI(5)P binding protein, a fusion protein or a compound involved in the regulation of PI(5)P levels or a composition as described above in a therapeutically-effective amount.
  • Preferred patients are mammals, more preferably humans.
  • a transgenic organism comprising a PI(5)P binding protein or a fusion protein comprising a PI(5)P binding protein and a marker domain as generated in a method as described above.
  • the marker domain is GFP.
  • the organism is a mouse, Zebrafish, Medakafish or insect. More preferably, the organism is an insect, such as Drosophila.
  • Figure 1 Sequence comparison of the EST HL03627 and of the predicted gene CG8624 with a Melted protein from Drosophila (dMelt).
  • Figure 4 Assay for phosphoinositide binding by dMelt. dMelt shows specific binding to PI(5)P.
  • FIG. 5 Drosophila S2 cell transfected with dMeltPH-GFP shows a cortical localization of PI(5)P.
  • Figure 6 The ORF of a human Melted protein (hMelt).
  • Figure 10 Multiple sequence alignment of the sequences of dMelt, hMelt and nMelt. Additional regions of similarity outside the PH domain are shown.
  • the MHl domain is underlined.
  • the MH2 domain is in bold.
  • the PH domain is in italic. Examples
  • dMelt has a PH domain that specifically binds PI(5)P
  • dMelt protein can also bind phosphoinositides and whether it has a binding specificity for any one phosphoinositide in particular.
  • E. coli strain BL21 was then transformed with this construct, and protein expression was induced with 0.2mM IPTG at 22C for 2.5 hours.
  • Cells (200mL culture) were harvested and resuspended in 10 mL of lxPBS/lOmM DTT/protease inhibitors.
  • the cell lysate was the added to the PJP strip (lO ⁇ l of lysate in 4mL TBST+3%BSA) and incubated 1 hour at room temperature. After washing with TBST, presence of dMelt-His protein was detected using anti-His-tag antibody, HRP conjugated secondary antibody and ECL. We found that at this concentration of dMelt protein, selective binding to PI(5)P could be detected ( Figure 4). When a 10-fold higher concentration of dMelt-His was used, binding could be detected to all phosphoinositides. Although each individual concentration is arbitrary, the ratio suggests that the PH domain of dMelt can bind PI(5)P ten times with higher affinity than other phosphoinositides.
  • PI(5)P is located cortically in cells
  • Human Melted was cloned by RT-PCR from HeLa-cell cDNA.
  • the ORF was PCRed in 4 separate fragments, using prediction information from the human genome project. Primers used to PCR each fragment are as follows:
  • Fragment #1 5 ' -ataagaatGCGGCCGCccatgcatcaactgttcagactggttttgg-3 '
  • the oligos used for Fragment #1 introduced a Notl restriction site upstream of the ORF, while the oligos used for Fragment #4 introduced a Kpnl restriction site downstream of the ORF.
  • the limits of the four fragments were designed to take advantage of unique restriction sites inside the ORF.
  • Fragment #1 was then digested with Notl & Xhol, Fragment #2 with Xhol & EcoRI, Fragment #3 with EcoRI & BamHI and Fragment #4 with BamHI & Kpnl and the four fragments were ligated together and inserted into a cloning vector. Sequencing of the plasmid revealed the sequence of hMelt.
  • the C. elegans Melted homologue The sequence for nMelt is a predicted sequence. Most of the sequence identified herein as nMelt ORF was predicted by the C.elegans genome project, with the exception of amino acids 125-173. Those were missed by the C. elegans genome project due to an incorrect splicing prediction.
  • the program Genewise was used to compare C. elegans genomic DNA sequence with the Drosophila Melt protein. The program found sequence coding for the 49 'missing' amino acids in a region of DNA that was predicted to be an intron by the C.elegans genome project.

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Abstract

L'invention concerne des protéines qui se lient au phosphatidylinositol 5-phosphate (PI(5)P). L'invention concerne également l'utilisation de ces protéines pour mesurer et visualiser le PI(5)P in vivo et in vitro et comme outils pour étudier le rôle du PI(5)P in vivo.
PCT/IB2002/003645 2001-08-09 2002-08-08 Proteines de liaison WO2003014152A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006061026A (ja) * 2004-08-24 2006-03-09 Japan Science & Technology Agency イノシトールリン脂質可視化プローブ遺伝子導入モデル動物
WO2007017067A2 (fr) * 2005-07-27 2007-02-15 European Molecular Biology Laboratory Modulateurs de l'activite d'une proteine impliquee dans la regulation de l'homeostasie energetique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ADAMS M ET AL: "Melt protein" SWALL, XP002235337 *
MORRIS J B ET AL: "Thrombin stimulation of platelets causes an increase in phosphatidylinositol 5-phosphate revealed by mass assay" FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 475, no. 1, 9 June 2000 (2000-06-09), pages 57-60, XP004337233 ISSN: 0014-5793 *
SHISHEVA ASSIA: "PIKfyve: The road to PtdIns 5-P and PtdIns 3,5-P2." CELL BIOLOGY INTERNATIONAL, vol. 25, no. 12, 2001, pages 1201-1206, XP002245798 2001 ISSN: 1065-6995 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006061026A (ja) * 2004-08-24 2006-03-09 Japan Science & Technology Agency イノシトールリン脂質可視化プローブ遺伝子導入モデル動物
WO2007017067A2 (fr) * 2005-07-27 2007-02-15 European Molecular Biology Laboratory Modulateurs de l'activite d'une proteine impliquee dans la regulation de l'homeostasie energetique
EP1754487A1 (fr) * 2005-07-27 2007-02-21 The European Molecular Biology Laboratory Modulateurs de la protéine Melted et leur utilisation
WO2007017067A3 (fr) * 2005-07-27 2007-04-26 European Molecular Biology Lab Embl Modulateurs de l'activite d'une proteine impliquee dans la regulation de l'homeostasie energetique

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