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WO2008065624A1 - Partenaire d'interaction de la protéine neuronatine et ses utilisations - Google Patents

Partenaire d'interaction de la protéine neuronatine et ses utilisations Download PDF

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Publication number
WO2008065624A1
WO2008065624A1 PCT/IB2007/054827 IB2007054827W WO2008065624A1 WO 2008065624 A1 WO2008065624 A1 WO 2008065624A1 IB 2007054827 W IB2007054827 W IB 2007054827W WO 2008065624 A1 WO2008065624 A1 WO 2008065624A1
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Prior art keywords
seq
neuronatin
protein
fragment
variant
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Inventor
Leif Kongskov Larsen
Niels Vrang
Philip Just Larsen
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Rheoscience AS
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Rheoscience AS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • the present disclosure relates to identification of neuronatin protein interaction partner particularly amyloid precursor protein binding protein-2 (APPB P2).
  • APPB P2 amyloid precursor protein binding protein-2
  • Diabetes is characterized by a decreased ability to regulate blood glucose concentrations. Diabetes can be grouped in two major classes: diabetes type I, wherein the insulin- producing cells are partly or fully destroyed by an auto-immune reaction and diabetes type II, wherein the insulin-producing cells are intact or essentially intact and wherein the decreased ability to regulate blood glucose is thought to be caused by decreased insulin- sensitivity. Diabetes type II is often accompanying obesity. In the present disclosure, the term diabetes is thus equivalent to diabetes type II. A number of pathological conditions furthermore accompany obesity and/or diabetes such as hypertension, and cardiovascular diseases.
  • Amyloid beta precursor protein-binding protein-2 interacts with microtubules and is functionally associated with beta-amyloid precursor protein transport and/or processing.
  • the beta-amyloid precursor protein (APP) is a cell surface protein with signal-transducing properties, and it is thought to play a role in the pathogenesis of Alzheimer's disease.
  • the gene encoding APPBP2 has been found to be highly expressed in breast cancer. Multiple polyadenylation sites have been found for this gene. Amplification or other mutation of the gene encoding APPBP2 is frequently linked to cancer.
  • WO0155450A2 discloses a strong linkage between genomic amplification of the PATI /APPBP2, and various other genes and cancer. This disclosure further describes the use of an antibody as a probe for the detection of neoplasms, including breast cancers wherein the probes bind selectively with target polynucleotide sequences such as PAT1/APPBP2. This disclosure does not teach or disclose the present invention.
  • the present disclosure relates to identification of one or more interaction partner of neuronatin protein.
  • the disclosure also provides a method of detection of the neuronatin protein interaction partner.
  • the disclosure discloses amyloid precursor protein binding protein-2 (APPBP2) as a neuronatin protein interaction partner.
  • the present disclosure also provides amyloid precursor protein binding protein-2 (APPBP2) as a target for the treatment of obesity and diabetes.
  • the present disclosure further relates to the interaction between APPBP2 and neuronatin protein and compounds that modulates such interaction.
  • An aspect of the disclosure is to provide a method of modulating the interaction between first protein or a homologue or a derivative or a fragment thereof and a second protein or a homologue or a derivative or a fragment thereof, wherein the first protein is ⁇ - neuronatin protein having amino acid sequence as shown in SEQ ID NO: 2 or ⁇ - neuronatin protein having amino acid sequence as shown in SEQ ID NO: 4 and second protein is neuronatin interaction partner having amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10 or a fragment thereof, said method comprising:
  • Another aspect of the disclosure is to provide a method of preventing or treating diabetes and/or obesity in a subject, wherein said method comprising administering an effective amount of at least one compound selected from a group consisting of: • a polynucleotide encoding the neuronatin interaction partner, said polynucleotide comprising the nucleotide sequence selected from a group consisting of SEQ ID NO: 5, SEQ ID NO: 7 , SEQ ID NO: 9, a fragment and a variant thereof;
  • a biologically active neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8 and
  • a biologically active variant of the polypeptide sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and a variant of one of said sequence having at least 50% identity therewith;
  • said biologically active polypeptide comprises at least 50 contiguous amino acids
  • a polynucleotide sequence that is antisense to the transcript encoding neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, a fragment and a variant thereof;
  • a siRNA molecule that is specific to the transcript encoding a neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, a fragment and a variant thereof;
  • Still another aspect of the disclosure relates to a method of identifying a compound that modulates the interaction between first protein which is ⁇ -neuronatin protein having amino acid sequence as shown in SEQ ID NO: 2 or ⁇ -neuronatin protein having amino acid sequence as shown in SEQ ID NO: 4 or a homologue or a derivative or a fragment thereof and a second protein that is neuronatin interaction partner or a homologue or derivative or fragment thereof, said method comprises contacting said first protein with said second protein in the presence of the test compound and determining the level of the complex formed between the first protein and the second protein.
  • vector construct comprising a polynucleotide sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or a variant of one of said sequences having at least 50% identity therewith, or a fragment of SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or a variant of one of said sequences having at least 50% identity therewith, or a fragment of SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or a variant of one of said sequences having at least 50% identity therewith, or a fragment of SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or a variant of one of said sequences having at least 50% identity therewith, or a fragment of SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or a variant of one of said sequences having at least 50% identity therewith, or a fragment of SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or
  • SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 comprising at least 150 nucleotides, or a variant of one of said fragments having at least 60% identity therewith, or a polynucleotide sequence that hybridises with SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9; and a regulatory sequence linked to the polynucleotide sequence, said polynucleotide sequence is in the sense or antisense orientation.
  • the disclosure further provides a packaging cell line capable of producing an infective virion comprising the vector construct to produce APPBP2 protein.
  • the disclosure also provides a host cell comprising a polynucleotide as shown in SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or a variant of one of the sequences having at least 50% identity therewith, or a fragment of SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 comprising at least 150 nucleotides, or a variant of one of the fragments having at least 60% identity therewith, or a sequence that hybridises with SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or a fragment or a variant thereof or the vector construct.
  • compositions for preventing or treating diabetes and/or obesity in a subject, wherein the composition comprising at least one compound selected from a group consisting of:
  • a polynucleotide encoding the neuronatin interaction partner comprising the nucleotide sequence selected from a group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, a fragment and a variant thereof;
  • a polypeptide sequence of neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8,
  • SEQ ID NO: 10 a fragment and a variant thereof
  • a polynucleotide sequence that is antisense to the transcript encoding neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, a fragment and a variant thereof;
  • a siRNA molecule that is specific to the transcript encoding a neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, a fragment and a variant thereof;
  • Another aspect of the disclosure relates to a compound that modulates the interaction between ⁇ -neuronatin protein encoded by the polynucleotide sequence as shown in SEQ ID NO: 1 or ⁇ -neuronatin protein encoded by the polynucleotide sequence as shown in SEQ ID NO: 3 and the neuronatin protein interaction partner encoded by the polynucleotide sequence selected from a group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, a polynucleotide sequence having at least 70% similarity to SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9; a fragment and a variant thereof.
  • Yet another aspect of the disclosure is to provide a compound that modulates the biological activity of the protein complex between first protein which is ⁇ -neuronatin protein or ⁇ -neuronatin protein or a homologue or a derivative or a fragment thereof and a second protein or a homologue or a derivative or a fragment thereof, wherein second protein is neuronatin protein interaction partner having amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, a fragment and a variant thereof.
  • Still yet another aspect of the disclosure provides a compound that modulates the biological activity of a polypeptide as shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment or a variant thereof.
  • the disclosure further provides a compound that modulates the biological activity of a polypeptide comprising amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10; a fragment and a variant thereof.
  • the disclosure also provides neuronatin protein interaction partner, wherein the interaction partner is encoded by the polynucleotide sequence selected from a group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, a fragment and a variant thereof.
  • Figure: 1 shows yeast two-hybrid screening. Reporter expression test of the activation domain-hybrid preys. The candidate (APPBP2) of the human pancreas library screening was positive for all three reporters.
  • Figure: 2 shows expression of Neuronatin (A-E) and APPBP2 (F-J) mRNA at different levels of the hypothalamus and brainstem in male Sprague-Dawley rats. High levels of mRNA expression are observed in the hypothalamus (A,-C, and F-H) and in specific brainstem nuclei (E, J). All control experiments utilizing the sense probes for neuronatin and APPB P2 mRNA were negative (D, I).
  • Figure: 3 show the relative expression of Neuronatin (A) and APPBP2 (B) in a rat cDNA library using RT-PCR. Whereas Nnat expression was found to be more or less restricted to the brain and epididymal white adipose tissue (epiWAT) APPBP2 demonstrated a more widespread expression profile with the highest level of expression in the adrenal gland, testis, epiWAT and brain.
  • epiWAT epididymal white adipose tissue
  • A'Variant of a polypeptide sequence means any naturally occurring or synthetic mutant of the sequence including allelic variants, degenerative variants, isoforms, sequences comprising amino acid substitutions, insertions, deletions and truncations, and derivatives of the sequence, including derivatives containing chemical modifications in the amino acid side chains.
  • a "variant" of a polynucleotide sequence means any natural occurring or synthetic mutant of the sequence including allelic variants, degenerative variants, isoforms, sequences comprising nucleotide substitutions, insertions, deletions and truncations, and derivatives of the sequence, including derivatives containing chemical modifications.
  • oligonucleotide is a strand of nucleotide residues which has a sufficient number of bases to be used as a primer or probe in a polymerase chain reaction (PCR).
  • Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal, or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides.
  • Chimeric molecules may be constructed by introducing all or part of the nucleotide sequence of this disclosure into a vector containing additional nucleic acid sequence which might be expected to change any one or several of the following APPBP2 characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.
  • Animal as used herein may be defined to include human, domestic (e. g. cats, dogs, etc.), agricultural (e. g., cows, horses, sheep, etc.) or test species (e. g., mouse, rat, rabbit, etc.).
  • domestic e. g. cats, dogs, etc.
  • agricultural e. g., cows, horses, sheep, etc.
  • test species e. g., mouse, rat, rabbit, etc.
  • Protein or “polypeptide” as used herein is a biological molecule expressed by a gene and comprised of amino acids.
  • the present disclosure relates to identification of an interaction partner of neuronatin protein.
  • the disclosure discloses amyloid precursor protein binding protein-2 (APPB P2) as neuronatin protein interaction partner.
  • the neuronatin protein interaction partner is referred to as amyloid precursor protein binding protein-2 (APPBP2).
  • the corresponding genetic sequence encoding the amyloid precursor protein binding protein-2 (APPBP2) is referred to herein as APPBP2.
  • the present disclosure also provides amyloid precursor protein binding protein-2 (APPBP2) as a target for the treatment of obesity and diabetes.
  • the present disclosure further relates to the interaction between APPBP2 protein and neuronatin protein and compounds that modulates such interaction.
  • a preferred embodiment of the present invention is directed to APPBP2 protein capable of interacting with neuronatin protein, the product of the gene neuronatin.
  • the ⁇ - neuronatin protein and ⁇ -neuronatin protein comprises at least 7 contiguous amino acids of the polypeptide sequence as shown in SEQ K) NO: 2 and SEQ ID NO: 4 respectively.
  • the ⁇ -neuronatin protein and ⁇ -neuronatin protein is encoded by the polynucleotide sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 3 respectively.
  • APPBP2 polypeptides include those having the amino acid sequences set forth herein e.g. SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 including the APPBP2 polypeptide modified with conservative amino acid substitutions, as well as biologically active fragments, variants, and derivatives thereof.
  • the APPBP2 polypeptides are encoded by the nucleotide sequence substantially as set forth in SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9.
  • the amino acid sequence corresponding to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 is as set forth in SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 respectively.
  • biologically active is used herein to refer to a specific effect of the polypeptide, including but not limited to specific binding, e.g. to a receptor, antibody, or other recognition molecule; activation of signal transduction pathways on a molecular level; and/or induction (or inhibition by antagonists) of physiological effects mediated by the native APPBP2 polypeptide in vivo.
  • APPBP2 polypeptides including fragments, variants, and derivatives, can be prepared synthetically, e.g. using the well known techniques of solid phase or solution phase peptide synthesis. Preferably, solid phase synthetic techniques are employed.
  • APPBP2 polypeptides of the present disclosure can be prepared using well known genetic engineering techniques.
  • the present invention relates to fragments of SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.
  • the said fragment comprises at least 7 amino acids and may contain varying fragments up to the full length.
  • the present disclosure contemplates that naturally occurring fragments of APPBP2 exist and may be important.
  • the protein sequence of APPBP2 includes a number of sites that are frequently the target for proteolytic cleavage. It is possible that the full-length polypeptide may be cleaved at one or more such sites to form biologically active fragments. Such biologically active fragments may either agonize or antagonize the functional activity of the APPB P2 polypeptide to affect body weight.
  • the present disclosure contemplates providing APPBP2 fragments having the minimum amino acid sequence necessary for a biological activity. This can be readily determined, e.g. by testing the activity of fragments of APPB P2 polypeptide for the ability to agonize or antagonize or inhibit the activity of the native neuronatin polypeptide.
  • the variant of SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10 has at least 60 %, more preferably 70 %, more preferably 80 %, more preferably 90 %, and most preferably 95 % identity therewith.
  • the variants of the fragment of SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10 according to the present disclosure preferably has at least 85 %, more preferably 90 % and most preferably 95 % identity with the fragments.
  • the variants of the APPB P2 polypeptide of the present invention includes all naturally occurring APPBP2 variants from different animal species, including e.g. variants originating from mouse, rats, dogs, cats, monkeys and ruminants, such as cow and ox.
  • the variant of SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 has at least 40 %, more preferably 50 %, more preferably 60 %, more preferably 70 %, more preferably 80 %, and most preferably 90 % identity therewith.
  • the variant of the fragment of SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 preferably has at least 60 %, more preferably 70 %, more preferably 80 % and most preferably 90 %, identity with said fragment.
  • the structure of the APPBP2 polypeptide can be analysed by various methods known in the art.
  • the protein sequence can be characterized by a hydrophilicity analysis.
  • a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the APPB P2 polypeptide, which may indicate regions buried in the interior of the folded polypeptide, and regions accessible on the exterior of the polypeptide.
  • secondary structural analysis can also be done, to identify regions of APPB P2 polypeptide that assume specific secondary structures.
  • Manipulation of the predicted or determined structure, including secondary structure prediction can be accomplished using computer software programs available in the art.
  • the APPBP2 peptide sequence may analysed by programs which predict cleavage of signal peptide to release mature peptide.
  • an analog of APPBP2 polypeptide can be tested to determine whether it cross-reacts with an antibody specific for native neuronatin polypeptide, or specific fragments thereof.
  • the degree of cross -reactivity provides information about structural homology or similarity of proteins, or about the accessibility of regions corresponding to portions of the polypeptide that were used to generate fragment- specific antibodies.
  • the present disclosure may be derivatized by the attachment of one or more chemical moieties to the protein moiety.
  • the chemically modified derivatives may be further formulated for intraarterial, intraperitoneal, intramuscular, subcutaneous, intravenous, oral, nasal, rectal, buccal, sublingual, pulmonary, topical, transdermal, or other routes of administration.
  • Chemical modification of biologically active proteins has been found to provide additional advantages under certain circumstances, such as increasing the stability and circulation time of the therapeutic protein and decreasing immunogenicity.
  • the chemical moieties suitable for derivatization may be selected from among water- soluble polymers.
  • the polymer selected should be water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer will be pharmaceutically acceptable.
  • One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/protein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations. For the present proteins and peptides, these may be ascertained using the assays provided herein.
  • the water soluble polymer may be selected from the group consisting of for example, polyethylene glycol, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly- 1,3- dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohol.
  • Polyethylene glycol propionaldehyde may provide advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or un-branched.
  • the preferred molecular weight is between about 2 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g. the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
  • the number of polymer molecules so attached may vary, and one skilled in the art will be able to ascertain the effect on function.
  • One may mono-derivatize, or may provide for a di-, tri-, tetra- or some combination of derivatization, with the same or different chemical moieties (e.g. polymers, such as different weights of polyethylene glycols).
  • the proportion of polymer molecules to protein (or peptide) molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is no excess un-reacted protein or polymer) will be determined by factors such as the desired degree of derivatization (e.g. mono, di-, tri-, etc.), the molecular weight of the polymer selected, whether the polymer is branched or un-branched, and the reaction conditions.
  • polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
  • attachment methods available to those skilled in the art, e.g. EP 401384 incorporated herein by reference (coupling PEG to G-CSF). See for instance Malik et al. [Malik F, Delgado C, Knusli C, Irvine AE, Fisher D, and Francis GE: Polyethylene glycol (PEG)-modified granulocyte-macrophage colony- stimulating factor (GM-CSF) with conserved biological activity; Exp. Hematol.
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues, those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue.
  • Sulfahydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecule(s).
  • Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group. Attachment at residues important for receptor binding should be avoided if receptor binding is desired.
  • polypeptides and nucleic acids of the present disclosure have significant therapeutic potential.
  • a therapeutically effective amount of such an agent is administered in a pharmaceutically acceptable carrier, diluent, or excipient.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similarly untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the federal or a state government or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to reduce by at least about 15%, preferably by at least 50%, more preferably by at least 90%, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host.
  • the APPBP2 polypeptide, or functionally active fragment thereof, or an antagonist thereof can be administered orally or parenterally, preferably parenterally. Because metabolic homeostasis is a continuous process, controlled release administration of APPBP2 polypeptide is preferred.
  • the polypeptide may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, intranasally, by lung inhalation, or other modes of administration.
  • a controlled release system can be placed in proximity of the therapeutic target, i.e. the brain, thus requiring only a fraction of the systemic dose.
  • the therapeutic compound can be delivered in a vesicle, in particular a liposome.
  • recombinant cells that have been transformed with the APPBP2 gene and that express high levels of the polypeptide can be transplanted in a subject in need of APPBP2 polypeptide.
  • autologous cells transformed with APPBP2 are transplanted to avoid rejection; alternatively, technology is available to shield non- autologous cells that produce soluble factors within a polymer matrix that prevents immune recognition and rejection.
  • the APPBP2 polypeptide can be delivered by intravenous, intraarterial, intraperitoneal, intramuscular, or subcutaneous routes of administration.
  • the APPBP2 polypeptide, properly formulated, can be administered by nasal or oral administration.
  • a constant supply of neuronatin can be ensured by providing a therapeutically effective dose (i.e. a dose effective to induce metabolic changes in a subject) at the necessary intervals, e.g. daily, every 12 hours, etc.
  • a therapeutically effective dose i.e. a dose effective to induce metabolic changes in a subject
  • these parameters will depend on the severity of the disease condition being treated, other actions, such as diet modification, that are implemented, the weight, age, and sex of the subject, and other criteria, which can be readily determined according to standard good medical practice by those of skill in the art.
  • One embodiment of the disclosure relates to a method of modulating the interaction between first protein that is ⁇ -neuronatin protein or ⁇ -neuronatin protein or a homologue or a derivative or a fragment thereof and a second protein or a homologue or a derivative or a fragment thereof, said second protein is neuronatin interaction partner having an amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10 or a fragment thereof, said method comprising providing a modulator for modulating the interaction between the neuronatin protein and neuronatin interaction partner, and determining the level of the interaction between the neuronatin protein and neuronatin protein interaction partner
  • the disclosure provides a method of modulating the interaction between ⁇ -neuronatin protein or ⁇ neuronatin protein and neuronatin protein interaction partner, wherein the ⁇ -neuronatm protein and ⁇ -neuronatm protein comprises at least 7 contiguous ammo acids of the polypeptide sequence as shown in SEQ ID NO 2 and SEQ ID NO 4 respectively
  • the ⁇ -neuronatm protein and ⁇ -neuronatin protein is encoded by the polynucleotide sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 3 respectively
  • the disclosure provides a method of preventing or treating diabetes and/or obesity in a subject, said method comprising administering an effective amount of at least one compound selected from a group consisting of.
  • a polynucleotide sequence encoding the neuronatin protein interaction partner comprises the nucleotide sequence selected from a group consisting of SEQ ID NO 5, SEQ ID NO 7 , SEQ ID NO: 9, a fragment and a va ⁇ ant thereof,
  • a biologically active neuronatin protein interaction partner comprising the ammo acid sequence selected from a group consisting of SEQ ID NO 6, SEQ ID NO: 8 and SEQ ID NO 10;
  • a biologically active variant of the polypeptide sequence selected from a group consisting of SEQ ID NO 6, SEQ ID NO. 8, SEQ ID NO. 10, and a variant of one of the sequence having at least 50% identity therewith,
  • a biologically active variant of said fragment having at least 80% identity therewith, • a polynucleotide sequence that is antisense to the transcript encoding neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ K) NO: 8, SEQ ID NO: 10, a fragment and a variant thereof; and
  • a siRNA molecule that is specific to the transcript encoding a neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, a fragment and a variant thereof;
  • Yet another embodiment of the disclosure provides a method of preventing or treating diabetes and/or obesity in a subject, said method comprising administering an effective amount of at least one compound selected from a group consisting of:
  • a polynucleotide sequence encoding the neuronatin protein interaction partner comprises the nucleotide sequence selected from a group consisting of SEQ ID NO: 5, SEQ ID NO: 7 , SEQ ID NO: 9, a fragment and a variant thereof;
  • a biologically active neuronatin protein interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10;
  • a biologically active variant of the polypeptide sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and a variant of one of the sequence having at least 50% identity therewith;
  • Yet another embodiment of the disclosure provides a method of preventing or treating diabetes and/or obesity in a subject, said method comprising administering an effective amount of at least one compound selected from a group consisting of:
  • a polynucleotide sequence that is antisense to the transcript encoding neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ K) NO: 8, SEQ ID NO: 10, a fragment and a variant thereof;
  • a siRNA molecule that is specific to the transcript encoding a neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, a fragment and a variant thereof;
  • Yet another embodiment of the disclosure provides a method of preventing or treating diabetes and/or obesity in a subject, said method comprising administering an effective amount of biologically active neuronatin interaction partner encoded by the polynucleotide sequence selected from a group consisting of:
  • Another embodiment of the disclosure provides a method of identifying a compound that modulates the interaction between first protein which is ⁇ -neuronatin protein or ⁇ - neuronatin protein; or a homologue or a derivative or a fragment thereof and a second protein which is neuronatin interaction partner or a homologue or a derivative or a fragment thereof, said method comprises contacting said first protein with said second protein in the presence of the test compound, and determining the level of the complex formed between the first protein and the second protein.
  • Yet another embodiment of the disclosure provides a method of screening for drug candidates for preventing or treating diabetes and/ or obesity in a subject, the method comprises contacting a compound with neuronatin interaction partner having an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or a fragment thereof and assaying for the presence of the complex between neuronatin interaction partner having amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or a fragment and the compound; and for the presence of a complex between neuronatin interaction partner having amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or a fragment and neuronatin or a fragment thereof and the compound.
  • the present disclosure provides a vector construct comprising:
  • the vector is selected from a group consisting of a viral vector, an adenoviral vector, an adenovirus-associated viral vector, a lentivirus vector, a retroviral vector and a vacciniaviral vector.
  • the regulatory sequence used in the construction of the vector is selected from a group consisting of CMV, MMTV, SV40, metallothionine promoters, trp, lac, tac, 17, ⁇ -factor, alcohol oxidase, PGH, and rous sarcoma virus enhancer.
  • the present disclosure further provides a host cell comprising:
  • the present disclosure further provides a host cell comprising:
  • the present disclosure further provides a host cell comprising: • a fragment of SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 comprising at least 150 nucleotides,
  • the host cell is selected from a group consisting of a human cell, a dog cell, a monkey cell, a rat cell, a mouse cell, Chinese Hamster ovary, insect cell, a yeast cell and E. coli.
  • the present disclosure provides a pharmaceutical composition for preventing or treating diabetes and /or obesity in a subject, wherein said composition comprising at least one compound selected from a group consisting of:
  • a polynucleotide encoding the neuronatin interaction partner comprising the nucleotide sequence selected from a group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, a fragment and a variant thereof;
  • a biologically active variant of a polynucleotide sequence selected from a group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or a variant of one of the said sequence having at least 50% identity therewith;
  • a polypeptide sequence of neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10; a fragment , and a variant thereof;
  • a polynucleotide sequence that is antisense to the transcript encoding neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ K) NO: 8, SEQ ID NO: 10, a fragment and a variant thereof;
  • a siRNA molecule that is specific to the transcript encoding a neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ K) NO: 8, SEQ ID NO: 10, a fragment and a variant thereof.
  • the present disclosure provides a pharmaceutical composition for preventing or treating diabetes and /or obesity in a subject, wherein said composition comprising at least one compound selected from a group consisting of:
  • a polynucleotide encoding the neuronatin interaction partner comprising the nucleotide sequence selected from a group consisting of SEQ K
  • a biologically active variant of a polynucleotide sequence selected from a group consisting of SEQ ID NO: 5, SEQ K) NO: 7, SEQ ID NO: 9, or a variant of one of the said sequence having at least 50% identity therewith;
  • the present disclosure provides a pharmaceutical composition for preventing or treating diabetes and /or obesity in a subject, wherein said composition comprising at least one compound selected from a group consisting of: • a polypeptide sequence of neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10; a fragment , and a variant thereof;
  • the present disclosure provides a pharmaceutical composition for preventing or treating diabetes and /or obesity in a subject, wherein said composition comprising at least one compound selected from a group consisting of:
  • a polynucleotide sequence that is antisense to the transcript encoding neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, a fragment and a variant thereof;
  • a siRNA molecule that is specific to the transcript encoding a neuronatin interaction partner comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ K) NO: 8, SEQ ID NO: 10, a fragment and a variant thereof.
  • compositions of the above are provided. Such pharmaceutical compositions may be for administration for injection, or for oral, pulmonary, nasal or other forms of administration.
  • pharmaceutical compositions comprising effective amounts of protein or derivative products of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers, are comprehended by the invention.
  • Such compositions include diluents of various buffer content (e.g. Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g. Tween 80, Polysorbate 80), anti-oxidants (e.g.
  • compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives.
  • the compositions may be prepared in liquid form, or may be in dried powder, such as lyophilised form. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • the vector construct of the invention may be a viral vector, an adenoviral vector, an adenovirus-associated viral vector, a lentivirus vector, a retroviral vector or a vacciniaviral vector.
  • the packaging cell line of the invention may be a phage.
  • the recombinant host cell of the invention may be a human cell, a dog cell, a monkey cell, a rat cell or a mouse cell.
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets
  • the pharmaceutical composition of the present disclosure can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits.
  • an animal preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of APPB P2 treated by administering antisense compounds in accordance with this disclosure.
  • compositions of the disclosure may contain an effective amount of an antisense compound along with a suitable pharmaceutically acceptable diluent or carrier.
  • Use of the antisense compounds and methods of the disclosure may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.
  • the antisense compounds of the disclosure are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding APPBP2 polypeptide enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the disclosure with a nucleic acid encoding APPBP2 can be detected by means known in the art.
  • Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of APPB P2 in a sample may also be prepared.
  • compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be via any route known to be effective by the physician of ordinary skill, except that parenteral administration directly into the central nervous system is not a route taught or claimed in this invention. Administration may be oral or parenteral. Peripheral, parenteral administration is preferred. Parenteral administration is commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe or some other mechanical device such as an infusion pump. For the purpose of this invention, peripheral parenteral routes include intravenous, intramuscular, subcutaneous, and intraperitoneal routes of administration.
  • Intravenous, intramuscular, and subcutaneous routes of administration of the compounds used in the present invention are more preferred. Intravenous and subcutaneous routes of administration of the compounds used in the present invention are yet more highly preferred.
  • Controlled release preparations may be achieved by the use of polymers to complex or absorb the active compound used in the present invention.
  • Extended duration may be obtained by selecting appropriate macromolecules, for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylenevinyl acetate, methylcellulose, carboxymethylcellulose, or protamine sulphate, and by selecting the concentration of macromolecules, as well as the methods of incorporation, in order to prolong release.
  • Another possible method to extend the duration of action by controlled release preparations is to incorporate an active compound used in the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerisation, for example, hydroxymethylcellulose or gelatin- microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or in macroemulsions.
  • neuronatin interaction partner is encoded by the polynucleotide sequence selected from a group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9; a polynucleotide sequence having at least 70% similarity to SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9; a fragment and a variant thereof.
  • the present further discloses a compound that modulates the biological activity of the protein complex between first protein or a homologue or a derivative or a fragment thereof and a second protein or a homologue or a derivative or a fragment thereof, wherein the first protein is ⁇ -neuronatin protein or ⁇ -neuronatin protein and the second protein is neuronatin interaction partner having amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, a fragment and a variant thereof.
  • the disclosure provides a compound that modulates the biological activity of the protein complex between first protein or a homologue or a derivative or a fragment thereof and a second protein or a homologue or a derivative or a fragment thereof, wherein the first protein is ⁇ -neuronatin protein or ⁇ -neuronatin protein comprises at least 30 contiguous amino acids.
  • Another embodiment of the disclosure provides a compound that modulates the biological activity of a polypeptide as shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment or a variant thereof.
  • Still another embodiment of the disclosure relates to a compound that modulates the biological activity of a polypeptide comprising amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10; a fragment and a variant thereof.
  • Still yet another embodiment of the disclosure relates to neuronatin protein interaction partner, wherein the interaction partner is encoded by the polynucleotide sequence selected from a group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, a fragment and a variant thereof. Any number of approaches may be employed to identify the interaction partner for neuronatin protein.
  • the disclosure describes the Yeast two-hybrid (Y2H) screening method to identify a candidate protein interacting with neuronatin.
  • Y2H screening a ⁇ -neuronatin protein, with a deletion of the N-terminal 35 amino acids comprising amino acid sequence as shown in SEQ ID NO: 2 encoded by the polynucleotide sequence as shown in SEQ ID NO: 1 was amplified using two primers (SEQ ID NO: 11 and SEQ ID NO: 12) and was cloned into a plasmid bait vector, pBCT by yeast homologous recombination.
  • the recombinant vector also contains the DNA binding domain (BD) of GAL4 fused with the neuronatin gene.
  • BD DNA binding domain
  • the cloned bait was checked for autonomous activation of transcription of the reporter genes (self transcriptional activator function).
  • the bait plasmids were introduced with an empty prey vector into a PBN204 yeast strain which contains three Y2H reporters (ADE2, URA3 and Lac3) with different sensitivities to transcriptional activators
  • Y2H screening was performed against two different activation domain (AD) libraries: a rat hypothalamus library and a human pancreas library.
  • AD activation domain
  • Prey plasmids from 18 Ura3+ prey candidates were isolated and amplified by PCR. In order to reconfirm the activation all preys were reintroduced into yeast with the neuronatin bait or without neuronatin (negative control). Clones containing candidate interacting proteins were re-tested for induction of the three independent Y2H reporter genes. Only one prey candidate showed neuronatin-dependent reporter gene expression on URA3, ADE2 and Lac3 ( Figure 1). Details are provided in Example 1.
  • the vector construct of the invention may be a viral vector, an adenoviral vector, an adenovirus-associated viral vector, a lentivirus vector, a retroviral vector or a vacciniaviral vector.
  • the packaging cell line of the invention may be a phage.
  • the recombinant host cell of the invention may be a human cell, a dog cell, a monkey cell, a rat cell or a mouse cell.
  • the non-human animal is a mouse, a rat, a sheep, a dog, a primate, or a reptile. In specific embodiment, the animal is a mammal.
  • the host cell includes yeast, bacteria, insect cell, animal cell including mammalian cell.
  • Glutathione S-transferase (GST) pull-down assay was carried out to study the interaction between neuronatin and APPBP2. Detailed description is provided in Example 2 and Example 3.
  • This system can also be used for evaluating the effect of various compounds on the interaction between neuronatin and APPBP2 and for mapping the interacting domains of the two polypeptides when only parts of the neuronatin and/or APPBP2 polypeptides are cloned into the vectors
  • Co-immunoprecipitation assay can also be used for mapping the interacting domains by cloning only fragments of the two proteins for mapping the interaction, and can in addition be used for assaying the effect of various compounds on the interaction.
  • rat APPBP2 polypeptide comprising amino acid sequence as shown in SEQ ID NO: 8 encoded by the polynucleotide as shown in SEQ ID NO: 7 was carried out according to the procedure described by Jensen et al. J Biol Chem, 271, 18749-58 (1996). Expression profiling can also be carried out by the methods known in the art.
  • Fresh tissue samples are obtained from Sprague Dawley rats and include the following anatomically defined areas: cortex, prefrontral cortex, brain, striatum, colliculus superior, hippocampus, amygdale, cerebellum, thalamus, hypothalamus, raphe nucleus, nucleus tractus solitarius (NTS), brain stem, medulla spinalis, epididymal white adipose tissue, perirenal white adipose tissue, mesenteric white adipose tissue, subcutaneous white adipose tissue, inguinal white adipose tissue, interscapular brown adipose tissue, antrum of stomach, fundus of stomach, corpus of stomach, duodenum, ileum, jejunum, colon, thymus, adrenal, pancreas, pituitary, tongue, muscle, heart, kidney, liver, spleen, and lung.
  • Total RNA is extracted from the tissue samples by using the method known in the art.
  • For the expression profiling of human APPB P2 polypeptide comprising amino acid sequence as shown in SEQ ID NO: 6 encoded by the polynucleotide sequence as shown in SEQ ID NO: 5.
  • RNA from the following tissues is purchased from Biochain Inc.: cerebral cortex, parietal lobe, total brain, pons, olfactory, medulla oblongata, temporal lobe, cerebellum (left), occipital lobe, frontal lobe, diencephalon, hypothalamus, thalamus, hippocampus, spinal cord, adipose, breast, stomach, pylorus of stomach, fundus of stomach, cardia of stomach, corpus of stomach, duodenum, ileum, jejunum, colon, colon from type 2 diabetic patient, thymus, adrenal, pancreas, prostate, uterus, ovary, skeletal muscle, heart, peripheral blood leukocyte, bladder, placenta, kidney, liver, fetal liver, spleen, lung.
  • Example 4 Detailed procedure of the expression profiling of APPBP2 in rat and human is provided in Example 4.
  • APPBP2 cDNA fragment of 781 base-pair was cloned into a plasmid vector. Detailed procedure of total RNA extraction, RT-PCR and cDNA synthesis of APPBP2 fragment are provided in Example 5. In vitro transcription of APPB P2 was carried out as described in Example 4. Specific activity of the generated transcripts was determined using a beta- counter.
  • rat APPBP2 cDNA sequence 2 ⁇ l of the template cDNA reaction is mixed with primers directed against the rat APPBP2 cDNA sequence (Accession No: BC091285).
  • Forward primer (754-774) as shown in SEQ ID NO: 21 and reverse primer (1535-1515) as shown in SEQ ID NO: 22 were designed from the rat APPBP2 cDNA sequence.
  • a and D are obtained from the level of the paraventricular nucleus of the hypothalamus demonstrating the presence of neuronatin and APPBP2 mRNA expression in this area
  • FIG. 2A, 2F; arrow Further caudally, both Neuronatin and APPBP2 gene transcripts were observed in the ventromedial nucleus (Fig. 2B, 2G; arrow), the arcuate nucleus (Fig. 2B, 2G; double arrow) and the dorsomedial (compact) hypothalamic nuclei (Fig. 2C, 2H; black arrow), as well as in the lateral hypothalamic areas. Hypothalamic sections those incubated with a sense cRNA probe showed no signal (Fig. 2D, 21).
  • Neuronatin and APPBP2 gene transcripts were also observed in the nucleus of the solitary tract and area postrema (Fig. 2E, 2J) of the brainstem, as well as in the amygdale, the habenula and in the hippocampus (Fig. 2C, 2H; white arrow). Detailed procedure of in situ hybridization is described in Example 5.
  • One embodiment of the invention relates to antisense analysis of APPB P2.
  • Knowledge of the correct, complete cDNA sequence coding for APPBP2 enables its use as a tool for antisense or siRNA technology (short interfering RNA) in the investigation of gene function (See Example 6 for details).
  • nucleic acid sequences complementary to an mRNA are hybridized to the mRNA intracellularly, thereby blocking the expression of the protein encoded by the mRNA.
  • the antisense sequences may prevent gene expression through a variety of mechanisms.
  • the antisense sequences may inhibit the ability of ribosomes to translate the mRNA.
  • the antisense sequences may block transport of the mRNA from the nucleus to the cytoplasm, thereby limiting the amount of mRNA available for translation.
  • Another mechanism through which antisense sequences may inhibit gene expression is by interfering with mRNA splicing.
  • the antisense nucleic acid may be incorporated in a ribozyme capable of specifically cleaving the target mRNA.
  • the appropriate level of antisense nucleic acids required to inhibit gene expression may be determined using in vitro expression analysis.
  • the antisense molecule may be introduced into the cells by diffusion, injection, infection or transfection using procedures known in the art.
  • the antisense nucleic acids can be introduced into the body as a bare or naked oligonucleotide, oligonucleotide encapsulated in lipid, oligonucleotide sequence encapsulated by viral protein, or as an oligonucleotide operably linked to a promoter contained in an expression vector.
  • the expression vector may be any of a variety of expression vectors known in the art, including retroviral or viral vectors, vectors capable of extrachromosomal replication, or integrating vectors.
  • the vectors may be DNA or RNA.
  • a siRNA is a nucleic acid that is a short, 15-50 base pairs and preferably 21-25 base pairs, double stranded ribonucleic acid.
  • the siRNA contains sequence that is identical or nearly identical to a portion of a gene.
  • RNA may be polymerized in vitro, recombinant RNA, contain chimeric sequences, or derivatives of these groups.
  • the siRNA may contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that expression of the target gene is inhibited.
  • the RNA is preferably double stranded, but may be single, triple, or quadruple stranded.
  • a delivered siRNA can stay within the cytoplasm or nucleus.
  • the siRNA can be delivered to a cell to inhibit expression an endogenous or exogenous nucleotide sequence or to affect a specific physiological characteristic not naturally associated with the cell.
  • Antisense molecule used herein includes antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either DNA or RNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences.
  • Antisense or sense oligonucleotide of the present disclosure comprises at least six nucleotides and are preferably oligonucleotides ranging from 12 to about 50 oligonucleotides. In specific aspects, the oligonucleotide is at least 12 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof and can be single-stranded or double-stranded.
  • the antisense molecules may be polymers that are nucleic acid mimics, such as PNA, morpholino oligos, and LNA.
  • Other types of antisense molecules include short double-stranded RNAs, known as siRNAs, and short hairpin RNAs, and long dsRNA.
  • Antisense molecules may be introduced in cell by employing various methods known in the art, for example by formation of the conjugate with a ligand binding molecule.
  • APPB P2 protein is expressed in bacterial systems.
  • Bacterial expression systems are well known in the art. Promoters from bacteriophage may also be used and are known in the art. In addition synthetic promoters and hybrid promoters are also useful for the expression of desired protein in host cells.
  • the expression vector contains selectable marker gene to allow the selection of transformed host cells.
  • Selection genes are well known in the art and will vary with the host cell used. Suitable selection gene includes genes that render the bacteria resistant to antibiotics such as ampicillin, kanamycin, erythromycin, neomycin, tetracycline, and chloramphenicol.
  • Expression of APPB P2 polypeptide is accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into expression hosts such as E. coll. hi a particular case, the vector was engineered such that it contains a promoter for ⁇ -galactosidase, upstream of the cloning site, followed by sequence containing the amino-terminal Methionin and the subsequent seven residues of ⁇ -galactosidase. Immediately following these eight residues is an engineered bacteriophage promoter useful for artificial priming and transcription and for providing a number of unique endonuclease restriction sites for cloning. Details are provided in Example 7.
  • the cDNA for APPBP2 was cloned into other vectors known to be useful for expression of proteins in specific hosts. Oligonucleotide primers containing cloning sites as well as a segment of DNA (about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA were synthesized chemically by standard methods. These primers were used to amplify the desired gene segment by PCR. The resulting gene segment was digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternatively, similar gene segments were produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, segments of coding sequence from more than one gene were ligated together and cloned in appropriate vectors. It was possible to optimize expression by construction of such chimeric sequences.
  • Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells. Insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae and bacterial cells such as E. coll can be used.
  • a useful expression vector also includes an origin of replication to allow propagation in bacteria, and a selectable marker such as the ⁇ -lactamase antibiotic resistance gene to allow plasmid selection in bacteria.
  • the vector may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells.
  • Vectors for use in eukaryotic expression hosts require RNA processing elements such as 3' polyadenylation sequences that are not part of the cDNA of interest.
  • the vector contains promoters or enhancers which increase gene expression.
  • promoters are host specific and include MMTV, SV40, and metallothionine promoters for CHO cells; trp, lac, tac and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGH promoters for yeast.
  • Transcription enhancers such as the rous sarcoma virus enhancer, are used in mammalian host cells. A person ordinary skilled in the art will know which host systems are compatible with a particular vector.
  • the vectors can be introduced into cells or tissues by any one of a variety of known methods within the art and will vary with the host cell used. Such methods can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al. Once homogeneous cultures of recombinant cells were obtained through standard culture methods, large quantity of recombinant APPBP2 protein was recovered from the conditioned medium and analyzed by using chromatographic methods well known in the art. The APPBP2 DNA can be cloned into the expression vector pcDNA3, as exemplified herein. This product can be used to transform the host cells including mammalian cells.
  • Host cells such as HEK293 or COS can be transformed by standard methodology known in the art for example, by using Lipofectamine mediated gene transfer.
  • APPBP2 can be used for screening therapeutic compounds by using APPBP2 or binding fragments thereof in any of a variety of drug screening techniques.
  • the polypeptide or fragment employed in such a test is either free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. For details see Example 8.
  • the present disclosure provides methods of screening for drug candidates. These methods, well known in the art, comprise contacting such an agent with APPBP2 polypeptide or a fragment thereof and assaying
  • APPBP2 polypeptide or fragment • for the presence of a complex between APPBP2 polypeptide or fragment and Neuronatin or fragment and the cell.
  • the APPBP2 polypeptide or fragment was typically labeled. After suitable incubation, free APPBP2 polypeptide or fragment was separated from that present in bound form, and the amount of free or uncomplexed label was a measure of the ability of the particular agent to bind to APPBP2 or to interfere with the APPBP2-Neuronatin complex.
  • the present disclosure also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding APPBP2 specifically compete with a test compound for binding to APPBP2 polypeptides or fragments thereof. In this manner, the antibodies were used to detect the presence of any peptide which shares one or more antigenic determinants with APPB P2.
  • animal model systems which elucidate the physiological and behavioral roles of the APPB P2 were produced by creating nonhuman transgenic animals in which the activity of the APPBP2 is either increased or decreased or the amino acid sequence of the expressed APPBP2 is altered, by a variety of techniques.
  • Examples of these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a APPBP2, by microinjection, electroporation, retroviral transfection or other means well known to those skilled in the art, into appropriately fertilized embryos in order to produce a transgenic animal or 2) homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these APPB P2 sequences.
  • the technique of homologous recombination is well known in the art.
  • Microinjection adds genes to the genome, but does not remove them, and the technique is useful for producing an animal which expresses its own and added APPBP2, resulting in over expression of the APPBP2. Additional embodiment is to produce a transgenic mouse. Details of the method of producing a transgenic animal are given in Example 9.
  • APPBP2 it can be extremely useful for the elucidation of the function to make tissue-specific knock-outs, particularly in adipose tissue. This will allow for the elucidation of the particular function of APPB P2 in the adipose tissue without disturbing brain- specific pathways including APPBP2.
  • One methodology for inducing tissue-specific knockouts of APPBP2 is to replace the native APPBP2 gene with an APPBP2 gene flanked by loxP sites in one mouse strain and then cross mice from this strain with mice expressing the ere recombinase under control of cell type- specific promoter.
  • Yeast two-hybrid (Y2H) screening was performed by PanBionet (www. Panbionet. com) to identify a candidate protein interacting with neuronatin. The following steps were employed:
  • a truncated ⁇ -neuronatin protein, with a deletion of the N-terminal 35 amino acids comprising amino acid sequence as shown in SEQ ID NO: 2 encoded by the polynucleotide sequence as shown in SEQ ID NO: 1 was cloned into a plasmid bait vector (pBCT) by yeast homologous recombination.
  • pBCT plasmid bait vector
  • the pBCT plasmid encodes the fusion protein of GAL4 binding domain and the requested protein.
  • the yeast GAL4 protein consists of two separable domains: one responsible for DNA-binding and the other for transcriptional activation. The junction between the plasmid GAL4 DNA binding domain and the neuronatin gene was confirmed by DNA sequencing.
  • the cloned bait was checked for autonomous activation of transcription of the reporter genes (self transcriptional activator function).
  • the bait plasmids were introduced with an empty prey vector into a PBN204 yeast strain which contains three Y2H reporters (ADE2, URA3 and Lac3) with different sensitivities to transcriptional activators.
  • the neuronatin bait did not show transcriptional activator function when replica plated on a minimal medium. Consequently, it was considered suitable for Y2H screening.
  • Y2H screening was performed against two different activation domain (AD) libraries: a rat hypothalamus library and a human pancreas library. Transformants of the neuronatin bait and library preys were spread on a selection media where yeasts expressing the interaction of bait and prey proteins will grow. More than 106 transformants were screened from each library.
  • AD activation domain
  • Prey plasmids from 18 Ura3+ prey candidates were isolated and amplified by PCR. In order to reconfirm the activation all preys were reintroduced into yeast with the neuronatin bait or without neuronatin (negative control). Clones containing candidate interacting proteins were re-tested for induction of the three independent Y2H reporter genes. Only one prey candidate showed neuronatin-dependent reporter gene expression on URA3, ADE2 and Lac3 ( Figure 1). The sequence of prey DNA was analyzed by 5' end sequencing and BLAST search. The single prey candidate was identified as amyloid beta precursor binding protein 2 (APPBP2) which is known as a protein interacting with amyloid beta precursor protein (APP). It is suggested that APPBP2 plays a role in e.g. APP transportation.
  • APPBP2 amyloid beta precursor binding protein 2
  • the Y2H system was used to test whether APPBP2 interacts with the full length neuronatin ⁇ and ⁇ isoforms. Both isoforms showed no reporter gene expression. This is most likely a consequence of the hydrophobic residues in both constructs that may block the nuclear localization of bait proteins.
  • the complete coding sequences of human neuronatin alpha and neuronatin beta are cloned into the pGEX-5X-l vector.
  • the fusion proteins are expressed in 0.1 mM isopropyl-1-thio- ⁇ -D-galactopyranoside (IPTG)-induced E. coli strain BL21 and purified by glutathione-Sepharose 4B beads.
  • IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside
  • the DNA sequence coding for the APPBP2 protein is amplified from the plasmid containing the human APPB P2 clone and cloned into the pGBKT7 vector.
  • the Myc- APPBP2 fusion protein is generated by the TNT T7 Quick Coupled Transcription/Translation System and is tested by Western blotting with anti- Myc antibody.
  • Equivalent amounts of GST-neuronatin alpha, GST-neuronatin beta, and GST alone are immobilized on glutathione-Sepharose 4B beads and are incubated in NETN buffer (0.1 M NaCl, 1 mM EDTA, 20 mM EDTA, 20 mM Tris-HCl, pH7.5, 5 ml/1 Nonidet P40, 1 mM phenylmethylsulfonyl fluoride) for 4 h at 4°C.
  • NETN buffer 0.1 M NaCl, 1 mM EDTA, 20 mM EDTA, 20 mM Tris-HCl, pH7.5, 5 ml/1 Nonidet P40, 1 mM phenylmethylsulfonyl fluoride
  • the beads After washing 4 times with H buffer (20 mM HEPES, pH7.7, 50 mM KCl, 200 ml/1 glycerol, 1 ml/1 Nonidet P- 40 and 0.07 ml/1 beta-mercaptoethanol), the beads are boiled in 20 ⁇ l elution buffer.
  • H buffer (20 mM HEPES, pH7.7, 50 mM KCl, 200 ml/1 glycerol, 1 ml/1 Nonidet P- 40 and 0.07 ml/1 beta-mercaptoethanol
  • the proteins bound to the beads are separated by SDS-PAGE and the separated proteins are transferred to PVDF membrane at 500 mA for 2 hours in a blotter and immunoblotted with mouse anti-Myc primary antibody (1:1000 dilution), followed by horseradish peroxidase (HRP) conjugated rabbit anti-mouse IgG (1:20000 dilution)
  • HRP horseradish peroxidase
  • the Myc-tagged protein is visualized with SuperSignal West Femto Maximum sensitivity substrate for HRP.
  • This system can also be used for evaluating the effect of various compounds on the interaction between neuronatin and APPBP2 and for mapping the interacting domains of the two polypeptides when only parts of the neuronatin and/or APPBP2 polypeptides are cloned into the vectors
  • the complete coding sequences of neuronatin alpha and neuronatin beta in-frame are cloned into the vector plasmid pCMV-HA2.
  • the complete coding sequence of APPBP2 in-frame is cloned into the vector plasmid pCMV-myc.
  • COS-7 cells are cultivated in 6- well plates to 70% confluence and co-transfected with 2 ⁇ g plasmid, pCMV-HA2- neuronatin (alpha and beta in separate assays) and pCMV-myc-appbp2. After 48 h of incubation, the cells are collected, washed twice with lysis buffer at 4°C for 20 min.
  • the lysates are pre-cleaned with protein A/G PLUS agarose beads for 1 h at 4°C followed by immunoprecipitation with 200 ng anti-HA antibody at 4°C overnight.
  • the beads are washed with lysis buffer.
  • the precipitated proteins are eluted from the beads by elution buffer and analyzed by Western blotting as described in the pull-down assay.
  • This assay can also be used for mapping the interacting domains by cloning only fragments of the two proteins for mapping the interaction, and can in addition be used for assaying the effect of various compounds on the interaction.
  • rat APPBP2 polypeptide comprising amino acid sequence as shown in SEQ ID NO: 8 encoded by the polynucleotide as shown in SEQ ID NO: 7 as well as for neuronatin polypeptide are carried by real-time quantitative PCR.
  • fresh tissue samples are obtained from Sprague Dawley rats including the following anatomically defined areas:
  • Testis spleen, colon, ileum, jejenum, duodenum, stomach (antrum), stomach (fundus), stomach (corpus), liver, pancreas, adrenal gland, kidney, tongue, thyroid gland, thymus, lung, heart, muscle, brown adipose tissue (BAT), subcutaneous white adipose tissue (WAT), mesenteric WAT, perirenal WAT, epididymal WAT, Vagus nerve, pituitary, mesencephalon, pons, thalamus, medulla spinalis, cerebellum, temporal lobe, hippocampus, hypothalamus, collicolus superior (CoIS), cortex (CTX), prefrontal cortex (PFC), Striatum (caudate putamen, cPu), whole brain
  • First- strand cDNA is prepared using 1 ⁇ g total RNA, the Superscript II RT kit, and random hexamer primers.
  • the cDNA is prepared in a total volume of 20 ⁇ l and is diluted 1:29 in distilled water.
  • a PCR reaction is set up by mixing 12.5 ⁇ l SYBR green mastermix (Stratagene) with 0.25 ⁇ l forward primer (10 ⁇ M), 0.25 ⁇ l reverse primer and 0.375 ⁇ l reference dye (1.6 ⁇ M) and the volume adjusted to a total of 20 ⁇ l and then 5 ⁇ l of diluted cDNA is added.
  • the reaction is incubated at 90 degrees C for 10 min followed by 40 cycles of 95 degrees for 30 seconds, 55 degrees for 30 seconds, 72 degrees for 15 seconds in a Stratagene Mx3000P thermal cycler with fluorescence detection.
  • the resulting fluorescence curves were used for quantification of the APPB P2 cDNA, and the resulting relative numbers normalized with the expression found for the housekeeping gene ubiquitin-conjugating enzyme E2D 3 (Ube2d3).
  • APPBP2 sense 5'-AATATCTGCCAGTCGGTTGC-S'
  • APPBP2 antisense 5'- CCACCAAACACTGACTGTCG-3'
  • Ube2d3 sense 5'- GCTCCCCTATCAGACCTCATC-3'
  • Ube2d3 antisense 5'- ATGAATGGAGGGAGGGAAAC-3 ' .
  • Total RNA is extracted from frozen (-80 0 C) hypothalami obtained from male Sprague- Dawley rats using the RNeasy RNA purification kit. Integrity and concentration of the extracted RNA is evaluated on a gel. RT-PCR is performed in a total volume of 20 ⁇ l using l ⁇ g total RNA, 20 U of Superscript Reverse Transcriptase, enzyme buffer (Ix), 1OmM DTT, 2 pmole/ ⁇ l oligodT(15) primers, ImM dNTP's and 0.5U/ ⁇ l RNase inhibitor.
  • rat APPBP2 cDNA sequence 2 ⁇ l of the template cDNA reaction is mixed with primers directed against the rat APPBP2 cDNA sequence (Accession No: BC091285).
  • Forward primer (754-774) as shown in SEQ ID NO: 19 and reverse primer (1535- 1515) as shown in SEQ ID NO: 20 were designed from the rat APPBP2 cDNA sequence.
  • the final reaction mix (total volume 50 ⁇ l) contains 2 ⁇ l cDNA reaction, 1.5mM MgC12, 0.75mM of each primer, 1.25U Taq DNA polymerase, Ix buffer, and 0.2mM dNTP's.
  • the size of the generated PCR product is checked on a gel.
  • the PCR product is cloned into PCR4-TOPO.
  • E. CoIi (TOPlO chemically competent cells) are transfected with the plasmid DNA and grown on Ampicillin/X-gal containing culture plates.
  • the insert of a positive clone is analyzed by PCR and the PCR-product sequenced.
  • the obtained raw data sequence is analyzed using the BLAST search engine (http://www.ncbi.nlm.nih.gov/BLAST/) verifying a 100% identity to rat APPBP2 cDNA.
  • E. coli containing recombinant plasmid were grown overnight at 37°C in LB medium and plasmid DNA was isolated and purified using the Midi-Prep kit.
  • plasmid DNA was linearized using restriction enzymes (antisense: Notl, sense: Pme ⁇ ).
  • P 33 labeled antisense (T3 polymerase) and sense (T7 polymeras) cRNA probes were prepared as follows: Ix transcription buffer, 1OmM DTT, RNase inhibitor (0.5U/ ⁇ l), CTP/ATP/GTP mix (ImM ), P 33 -alfa-UTP, linearized DNA (l ⁇ g) and polymerase (T3 or T7, 40U) were mixed and incubated for 2 hours at 37°C. Subsequently, DNA was digested by the addition of l ⁇ l RQl, 2 ⁇ l yeast tRNA and l ⁇ l RNase inhibitor (0.5U). The transcripts were purified using chloroform:ethanol extraction followed by precipitation in 2.0M ammoniumacetate and ethanol.
  • the transcripts were diluted in a 1:1 mixture of 100% de-ionized formamide and Tris (1OmM) EDTA (ImM)- DTT (1OmM) buffer (pH 7.5). Specific activity of the generated transcripts was determined using a beta-counter.
  • Two male Spraque Dawley rats (Charles River, Sweden) were used. The animals were sacrificed by decapitation and brains were removed and frozen on dry ice. Twelve micron thick frontal sections were cut in a cryostat and mounted directly on slides. Dried slides were fixed for 5 min in 4% paraformaldehyde. The slides were then rinsed 2 x 5 min in phosphate buffered saline (PBS; pH 7.4) followed by a brief acetylation. 500 ⁇ l acetic anhydride (100%) was added to 200ml 0.1M triethanolamine and the slides were immediately submerged for 2 min.
  • PBS phosphate buffered saline
  • the hybridization buffer consists of 50% demonized formamide, IX salts (300 mM NaCl, 1OmM Tris, 1OmM NaPO 4 (pH 6.8), 5mM EDTA, 0.02% Ficoll 400, 0.2% polyvinylpyrolidone (PVP 40, 40000 MW), 0.2% BSA Fraction V), 10% dextran sulphate, l ⁇ g/ ⁇ l yeast tRNA and 9mM DTT. Probe was added so that the final activity of the hybridization mix is approximately 30.000 cpm/ ⁇ l. The hybridization mix was applied onto the sections (35 ⁇ l/section) that were cover- slipped. Hybridization was performed overnight at 47°C.
  • Sections were subjected to two stringency washes at 62°C and 67°C. Sections were washed for 1 hour at each temperature (lowest first) in a washing buffer consisting of 50% formamide, Ix salts and 1OmM DTT. The sections were rinsed twice (2 x 2 min) in NTE buffer (0.5 M NaCl, 10 mM Tris Cl (pH 7.2), 1 mM EDTA) containing 1OmM DTT, where after they were treated with RNAase- A (20 ⁇ g/ml) for 30 min.
  • NTE buffer 0.5 M NaCl, 10 mM Tris Cl (pH 7.2), 1 mM EDTA
  • the sections were rinsed twice for 5 min in NTE + 1OmM DTT, 30 min in SSC (15mM NaCl, 1.5mM trisodiumcitrate, 2H 2 O, pH 7.0) containing ImM DTT and finally dehydrated through a series of graded ethanol solutions containing 0.3M ammonium acetate (30%, 60%, 80%, 96% and 99%).
  • SSC 15mM NaCl, 1.5mM trisodiumcitrate, 2H 2 O, pH 7.0
  • FIG. 2 shows autoradiograms of frontal hypothalamic sections and brainstem sections hybridized with a Neuronatin (A-F) or APPBP2 probe (G-J).
  • Sections A and D are obtained from the level of the paraventricular nucleus of the hypothalamus demonstrating the presence of neuronatin and APPBP2 mRNA expression in this area (Fig. 2A, 2F; arrow).
  • both Neuronatin and APPBP2 gene transcripts were observed in the ventromedial nucleus (Fig. 2B, 2G; arrow), the arcuate nucleus (Fig. 2B, 2G; double arrow) and the dorsomedial (compact) hypothalamic nuclei (Fig. 2C, 2H; black arrow), as well as in the lateral hypothalamic areas.
  • Hypothalamic sections those incubated with a sense cRNA probe showed no signal (Fig. 2D, 21).
  • Neuronatin and APPBP2 gene transcripts were also observed in the nucleus of the solitary tract and area postrema (Fig. 2E, 2J) of the brainstem, as well as in the amygdale, the habenula and in the hippocampus (Fig. 2C, 2H; white arrow).
  • cDNA sequence coding for APPBP2 enables its use as a tool for antisense or siRNA technology (short interfering RNA) in the investigation of gene function.
  • Oligonucleotides, cDNA or genomic fragments comprising the antisense strand or a small double stranded portion of a polynucleotide coding for APPBP2 were used either in vitro or in vivo to inhibit translation and/or direct degradation of the mRNA.
  • antisense and siRNA molecules can be designed at various locations along the nucleotide sequences.
  • the gene of interest is effectively turned off.
  • the function of the gene is ascertained by observing behavior at the intracellular, cellular, tissue or organismal level (e. g., lethality, loss of differentiated function, changes in morphology, etc.).
  • modifications of gene expression is obtained by designing antisense sequences to intron regions, promoter/enhancer elements, or even to transacting regulatory genes.
  • Example 7 Expression of APPBP2 Expression of APPB P2 is accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into expression hosts such as E. coll.
  • the vector was engineered such that it contains a promoter for ⁇ - galactosidase, upstream of the cloning site, followed by sequence containing the amino- terminal Methionin and the subsequent seven residues of ⁇ -galactosidase.
  • an engineered bacteriophage promoter useful for artificial priming and transcription and for providing a number of unique endonuclease restriction sites for cloning.
  • IPTG isopropyl-P-D-thio- galactopyranoside
  • clones were obtained by deletion or insertion of the appropriate number of bases using well known methods including in-vitro mutagenesis, digestion with Exonuclease III or mungbean nuclease, or the inclusion of an oligonucleotide linker of appropriate length.
  • the APPB P2 cDNA was shuttled into other vectors known to be useful for expression of proteins in specific hosts.
  • Oligonucleotide primers containing cloning sites as well as a segment of DNA (about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA were synthesized chemically by standard methods. These primers were used to amplify the desired gene segment by PCR. The resulting gene segment was digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternatively, similar gene segments were produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, segments of coding sequence from more than one gene were ligated together and cloned in appropriate vectors. It was possible to optimize expression by construction of such chimeric sequences.
  • Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells. Insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae and bacterial cells such as E. coli can be used.
  • a useful expression vector also includes an origin of replication to allow propagation in bacteria, and a selectable marker such as the ⁇ -lactamase antibiotic resistance gene to allow plasmid selection in bacteria.
  • the vector may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells.
  • Vectors for use in eukaryotic expression hosts require RNA processing elements such as 3' polyadenylation sequences that are not part of the cDNA of interest.
  • the vector contains promoters or enhancers which increase gene expression.
  • promoters are host specific and include MMTV, SV40, and metallothionine promoters for CHO cells; trp, lac, tac and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGH promoters for yeast.
  • Transcription enhancers such as the rous sarcoma virus enhancer, are used in mammalian host cells. Once homogeneous cultures of recombinant cells were obtained through standard culture methods, large quantity of recombinant APPBP2 protein was recovered from the conditioned medium and analyzed by using chromatographic methods well known in the art.
  • APPBP2 can be cloned into the expression vector pcDNA3, as exemplified herein.
  • This product can be used to transform, for example, HEK293 or COS by methodology standard in the art for example, by using Lipofectamine mediated gene transfer.
  • APPBP2 can be used for screening therapeutic compounds by using APPBP2 or binding fragments thereof in any of a variety of drug screening techniques.
  • the polypeptide or fragment employed in such a test is either free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, are used for standard binding assays. Complex formation between APPBP2 and the agent being tested was measured. Alternatively, one examines the diminution in complex formation between APPBP2 and Neuronatin caused by the agent being tested.
  • the present disclosure provides methods of screening for drug candidates. These methods, well known in the art, comprise contacting such an agent with APPBP2 polypeptide or a fragment thereof and assaying
  • the APPBP2 polypeptide or fragment was typically labeled. After suitable incubation, free APPBP2 polypeptide or fragment was separated from that present in bound form, and the amount of free or uncomplexed label was a measure of the ability of the particular agent to bind to APPBP2 or to interfere with the APPBP2-Neuronatin complex.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to APPB P2 polypeptides. Briefly stated, large numbers of different small peptide test compounds were synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds were reacted with APPBP2 polypeptide and washed. Bound APPBP2 polypeptide was then detected by methods well known in the art. Purified APPBP2 were also coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies were used to capture the peptide and immobilize it on the solid support.
  • This disclosure also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding APPBP2 specifically compete with a test compound for binding to APPBP2 polypeptides or fragments thereof. In this manner, the antibodies were used to detect the presence of any peptide which shares one or more antigenic determinants with APPBP2.
  • Animal model systems which elucidate the physiological and behavioral roles of the APPBP2 are produced by creating nonhuman transgenic animals in which the activity of the APPBP2 is either increased or decreased, or the amino acid sequence of the expressed APPBP2 is altered, by a variety of techniques.
  • these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a APPBP2, by microinjection, electroporation, retroviral transfection or other means well known to those skilled in the art, into appropriately fertilized embryos in order to produce a transgenic animal or 2) homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these APPBP2 sequences.
  • homologous recombination is well known in the art. It replaces the native gene with the inserted gene and hence is useful for producing an animal that cannot express native APPBP2'S but does express, for example, an inserted mutant APPBP2, which has replaced the native APPBP2 in the animal's genome by recombination, resulting in underexpression of APPB P2.
  • Microinjection adds genes to the genome, but does not remove them, and the technique is useful for producing an animal which expresses its own and added APPBP2, resulting in over expression of the APPBP2.
  • transgenic animal One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice were mated, and the resulting fertilized eggs were dissected out of their oviducts. The eggs were stored in an appropriate medium such as cesium chloride M2 medium. DNA or cDNA encoding APPBP2 was purified from a vector by methods well known to the one skilled in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the transgene.
  • microinjection needle which may be made from capillary tubing using a piper puller
  • the egg to be injected was placed in a depression slide.
  • the needle was inserted into the pronucleus of the egg, and the DNA solution was injected.
  • the injected egg was then transferred into the oviduct of a pseudo pregnant mouse that was stimulated by the appropriate hormones in order to maintain false pregnancy, where it proceeds to the uterus, implants, and develops to term.
  • microinjection is not the only method for inserting DNA into the egg but is used here only for exemplary purposes.
  • APPBP2 it can be extremely useful for the elucidation of the function to make tissue- specific knock-outs, particularly in adipose tissue. This will allow for the elucidation of the particular function of APPB P2 in the adipose tissue without disturbing brain- specific pathways including APPBP2.
  • One methodology for inducing tissue-specific knockouts of APPBP2 is to replace the native APPBP2 gene with an APPB P2 gene flanked by loxP sites in one mouse strain and then cross mice from this strain with mice expressing the ere recombinase under control of an adipocyte-specific promoter.

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Abstract

La présente invention concerne un partenaire d'interaction de la neuronatine. Ce partenaire est une protéine de type 2 se liant à la protéine précurseur amyloïde (APPBP2) et il peut servir de cible pour le traitement de l'obésité et/ou du diabète. L'invention concerne également l'interaction entre l'APPBP2 et la neuronatine ainsi qu'un composé permettant d'augmenter un telle interaction. La présente invention porte en outre sur la modulation de l'activité du partenaire d'interaction de la neuronatine en tant qu'outil de diagnostic, de traitement ou de prévention d'un diabète et/ou d'une obésité. L'invention concerne encore un procédé de criblage de divers composés, qui permet le ciblage du partenaire d'interaction de la neuronatine et se révèle utile pour traiter, soulager, prévenir ou diagnostiquer un diabète et/ou une obésité. La présente invention concerne également une composition pharmaceutique pouvant servir pour le diagnostic, le traitement ou la prévention d'un diabète et/ou d'une obésité.
PCT/IB2007/054827 2006-11-29 2007-11-28 Partenaire d'interaction de la protéine neuronatine et ses utilisations Ceased WO2008065624A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001055450A2 (fr) * 2000-01-28 2001-08-02 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, The National Institutes Of Health GENES AMPLIFIES EN 17q23
WO2002098441A2 (fr) * 2001-06-07 2002-12-12 Azign Bioscience A/S Procede de traitement de troubles metaboliques au moyen de neuronatine polypeptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001055450A2 (fr) * 2000-01-28 2001-08-02 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, The National Institutes Of Health GENES AMPLIFIES EN 17q23
WO2002098441A2 (fr) * 2001-06-07 2002-12-12 Azign Bioscience A/S Procede de traitement de troubles metaboliques au moyen de neuronatine polypeptides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHENG PEIZHONG ET AL: "PAT1, a microtubule-interacting protein, recognizes the basolateral sorting signal of amyloid precursor protein", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 95, no. 25, December 1998 (1998-12-01), pages 14745 - 14750, XP002246589, ISSN: 0027-8424 *

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