[go: up one dir, main page]

WO2003056032A1 - Domaine de liaison de l'oligosaccharide de la sous-unite beta de l'amp kinase - Google Patents

Domaine de liaison de l'oligosaccharide de la sous-unite beta de l'amp kinase Download PDF

Info

Publication number
WO2003056032A1
WO2003056032A1 PCT/AU2002/001769 AU0201769W WO03056032A1 WO 2003056032 A1 WO2003056032 A1 WO 2003056032A1 AU 0201769 W AU0201769 W AU 0201769W WO 03056032 A1 WO03056032 A1 WO 03056032A1
Authority
WO
WIPO (PCT)
Prior art keywords
oligosaccharide
polypeptide
seq
amp kinase
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU2002/001769
Other languages
English (en)
Inventor
David Stapleton
Bruce E. Kemp
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
St Vincents Institute of Medical Research
Original Assignee
St Vincents Institute of Medical Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by St Vincents Institute of Medical Research filed Critical St Vincents Institute of Medical Research
Priority to AU2002357407A priority Critical patent/AU2002357407A1/en
Publication of WO2003056032A1 publication Critical patent/WO2003056032A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • AMPK has been purified from rat liver as an ⁇ heterotrimer comprising an ⁇ catalytic subunit and ⁇ non catalytic subunits (Mitchelhill et al., 1994; Davies, et al., 1994).
  • the catalytic ⁇ subunit turned out to be the mammalian homolog of the yeast Snflp kinase (Mitchelhill et al., 1994; Carling et al., 1994) and the non catalytic ⁇ subunits also have yeast homologs.
  • AMPK/Snflp kinase homologs have now been detected in all species examined and this has led to the understanding that they represent an ancient metabolite sensing protein kinase subfamily.
  • yeast Although yeast has a single SNF1 gene most mammals, insects, nematodes and plants tend to have multiple genes encoding this catalytic subunit. In mammals the two ( ⁇ l and ⁇ 2) subunits share approximately 90% identity in their N-terminal catalytic domains (residues 1-270) but only approximately 60% identity in their C-terminal ⁇ sequence (Stapleton et al., 1996). Structure functions studies on both Snflp and AMPK ⁇ subunits have shown that the C-terminal domain is responsible for binding the non catalytic subunits ⁇ (Crute et al., 1998). Thus, truncating the ⁇ subunit from 1-548 to 1-392 results in loss of ⁇ binding and catalytic activity.
  • the present inventors have unexpectedly found that the ⁇ subunit of AMP kinase binds oligosaccharides and have identified the oligosaccharide binding domain within the ⁇ subunit.
  • the present inventors have also found that the binding of oligosaccharide to the ⁇ subunit modulates the activity of AMP kinase. As the inventors have identified a previously unknown binding event, this allows for known assay systems to be used to screen for agonists or antagonists which modulate the binding of an oligosaccharide to AMP kinase.
  • the oligosaccharide is a homopolymer of glucose. More preferably, the oligosaccharide is selected from the group consisting of glycogen, starch, amylose, amylopectin and dextran. Most preferably, the oligosaccharide is glycogen.
  • the compound when bound to the AMP kinase, does not adversely affect the kinase activity of the enzyme.
  • the oligosaccharide is detectably labeled.
  • the oligosaccharide can be detectably labeled using known labels such as those selected from the group consisting of: radioisotopes, fluorophores and chromophores.
  • the agent can be considered as "a lead compound" which is tested by various means to determine if it is useful for various methods as outlined below. Depending on the nature of the identified compound the testing means will vary significantly but will be well within the skill of those in the art.
  • the compound does not adversely affect the kinase activity of the AMP kinase.
  • the condition is associated with a lack of glucose uptake by cells of the subject. More preferably, the condition is selected from the group consisting of: Type II diabetes, atherosclerosis, obesity, cancer, hypercholesterolemia, hypertriglyceridemia and elevated glucose in pre-diabetes
  • the present invention can also be used to facilitate athletic training or improve the metabolic profile of sedentary people. In each instance, the invention would be useful through promoting catabolic pathways.
  • the present invention provides a method of regulating transcription of a gene under control of AMP kinase, the method comprising administering a compound identified according to the invention. It is known that cancer cells generally are in a stressed state, relying on pathways involving AMP kinase to provide the necessary energy requirements of the cell. Thus, it is expected that reducing the activity of AMP kinase in cancer cells will be detrimental to the proliferation of the cancer cells.
  • the compound is administered directly to the cancer in the patient.
  • the present invention provides a method of reducing levels of free glycogen in the cell of a subject, the method comprising administering to the subject a beta subunit of AMP kinase, or a mutant and/or fragment thereof which binds an oligosaccharide, or a compound identified according to the invention.
  • the present invention provides a method of modulating AMP kinase activity in a patient, the method comprising administering to the patient a polynucleotide encoding a polypeptide selected from the group consisting of: i) a beta subunit of AMP kinase, ii) a mutant and/or fragment of i) which binds an oligosaccharide, or iii) a mutant and/or fragment of i) which does not bind an oligosaccharide but still maintains at least some AMK kinase activity.
  • the polypeptide is attached to a molecule that is identified by the detection means. More preferably, the polypeptide is a fusion protein. Even more preferably, the fusion protein comprises an enzyme that can be detected by the production of an assayable product. Examples of such enzymes include luciferases, fluorescent proteins such as the green fluorescent protein, or chloramphenicol acetyl transferase.
  • the oligosaccharide is a homopolymer of glucose. More preferably, the oligosaccharide is selected from the group consisting of glycogen, starch, amylose, amylopectin and dextran. Most preferably, the oligosaccharide is glycogen.
  • the present invention provides a fusion protein comprising a beta subunit of AMP kinase, or a mutant and/or fragment thereof which binds an oligosaccharide, fused to at least one other polypeptide sequence.
  • a polypeptide that assists in the detection of the oligosaccharide bound to the polypeptide of the invention is selected from the group consisting of: a luciferase, a fluorescent protein such as the green fluorescent protein, or chloramphenicol acetyl transferase.
  • the present invention provides an isolated polynucleotide encoding a polypeptide of the invention, or encoding a fusion polypeptide of the invention.
  • the present invention provides a method of stimulating phosphorylation of AMP kinase, the method comprising exposing the AMP kinase to an agonist which binds the oligosaccharide binding domain of the AMP kinase, and an enzyme capable of phosphorylating AMP kinase.
  • the agonist is an oligosaccharide.
  • the oligosaccharide is a homopolymer of glucose. More preferably, the oligosaccharide is selected from the group consisting of glycogen, starch, amylose, amylopectin and dextran. Most preferably, the oligosaccharide is glycogen.
  • Figure 13 Structural alignment of Snfl family of ⁇ subunits based on the structure of E.coli glycogen branching enzyme.
  • Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
  • Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, micro injection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
  • agonists or antagonists which can be used to regulate oligosaccharide binding to AMP kinase are employed for therapeutic and prophylactic purposes for such diseases or disorders as those detailed hereinbefore, among others.
  • the agonists or antagonists which can be used to regulate oligosaccharide binding to AMP kinase can act indirectly on the oligosaccharide binding domain. More specifically, as outlined above, AMPK is a heterotrimer comprising the ⁇ , ⁇ and ⁇ subunits.
  • the filters can then be placed in a solution containing, for example, a radioactive of a beta subunit of AMP kinase, or a mutant and/or fragment thereof (e.g., a polypeptide having an amino acid sequence comprising SEQ ID NO:l). After a specified incubation period, the filters can be thoroughly washed and developed for autoradiography. Plagues containing the phage that bind to the radioactive polypeptide are then isolated. These phages can be further cloned and then retested for their ability to bind to the of a beta subunit of AMP kinase, or a mutant and/or fragment thereof as before.
  • a radioactive of a beta subunit of AMP kinase or a mutant and/or fragment thereof (e.g., a polypeptide having an amino acid sequence comprising SEQ ID NO:l).
  • polypeptides, agonists and antagonists that are polypeptides may be employed in accordance with the present invention by expression of such polypeptides in treatment modalities often referred to as "gene therapy".
  • mutant beta subunit AMP kinase which cannot bind an oligosaccharide but maintain kinase activity may be employed in gene therapy techniques for the treatment of disease.
  • cells from a patient may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo. The engineered cells can then be provided to a patient to be treated with the polypeptide.
  • cells may be engineered ex vivo, for example, by the use of a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention can be used to transform stem cells or differentiated stem cells.
  • a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention can be used to transform stem cells or differentiated stem cells.
  • Retroviruses from which the retroviral plasmid vectors hereinabove-mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, Spleen Necrosis Virus, Rous Sarcoma Virus, Harvey Sarcoma Virus, Avian Leukosis Virus, Gibbon Ape Leukemia Virus, Human Immunodeficiency Virus, Adenovirus, Myeloproliferative Sarcoma Virus, and Mammary Tumor Virus.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs herein above described); the ⁇ -actin promoter; and human growth hormone promoters.
  • the promoter may also be the native promoter which controls the gene encoding the polypeptide.
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, Y-2, Y-AM, PA12, T19-14X, VT- 19-17-H2, YCRE, YCRJP, GP+E-86, GP+envAml2, and DAN cell lines as described by Miller (1990).
  • the vector may be transduced into the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO 4 precipitation.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • compositions of the present invention comprise an acceptable carrier.
  • the carrier will also be considered as a "pharmaceutically acceptable carrier", meaning that it is suitable to be administered to an animal, preferably a human.
  • Suitable carriers include isotonic saline solutions, for example phosphate-buffered saline.
  • Polynucleotides/vectors encoding polypeptide components may be administered directly as a naked nucleic acid construct, preferably further comprising flanking sequences homologous to the host cell genome.
  • the amount of nucleic acid administered may typically be in the range of from 1 ⁇ g to 10 mg, preferably from 100 ⁇ g to 1 mg. Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents.
  • transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectamTM and transfectamTM).
  • the PCR reaction included an initial 95°C denaturation / 5 minutes followed by 25 cycles of 95°C / 40 seconds, 55°C / 40 seconds and 72°C / 1 minute, and a final extension time of 10 minutes at 72°C.
  • the resulting product was excised, gene cleaned (Qiaex), digested sequentially with EcoRI, ligated into similarly digested pProEX HT vector and transformed into DH5 alpha cells. Transformants were analysed by restriction analysis and positive clones sequenced by the Big Dye method.
  • the AMPK ⁇ l (42-183) fragment was eluted with PBS containing 500 mM added NaCl and 300 mM imidazole, precipitated with 60% (NLLi) 2 SO 4 for 30 min / 4°C and the resulting precipitate collected by centrifugation.
  • the protein pellet was resuspended in 2 ml 50 mM Tris pH 8.5, desalted on a PD-10 gel filtration column, pooled and stored at -20°C until required. Tris-tricine gel electrophoresis (16.5%) was used to analyse the purification of the AMPK ⁇ l (42-183) fragment.
  • ISO ⁇ l purified AMPK ⁇ l (42-183) fragment was digested with lO ⁇ g/ml sequencing grade Endoproteinase Asp-N (Roche) in the presence of 10 mM CaCl 2 at 25°C / 10 minutes.
  • the entire sample was chromatographed on a C18 column on the SMART system at 40 ⁇ l min flow rate with a 0-40% CH 3 CN gradient / 60 min and 40 ⁇ l fractions were collected. Fractions were analysed by Tris-tricine gel electrophoresis (16.5%), electrospray and Maldi-TOF mass spectrometry and N- terminal sequencing.
  • Endoproteinase AspN generated a fragment corresponding to ⁇ l (66-158) resulting from cleavage on the NH -terminal side of Asp66 and Aspl59.
  • Endoproteinase LysC generated a fragment corresponding to ⁇ l (73-172) resulting from cleavage on the COOH side of Lys72 and Lysl72.
  • Cells were harvested following centrifugation and lysed in PBS, 500 mM NaCl in an Avestin cell crusher. Lysates were clarified by centrifugation at 48,000g / 30 min / 4°C and chromatographed on Ni-Agarose. Non-specific proteins were removed by washing the Ni-Agarose with PBS / 500 mM NaCl / 20 mM imidazole.
  • This step bound the His-TEV together with the cleaved His-tag from the N-terminus of the AMPK ⁇ l (68-163) fragment, whilst allowing the cleaved product to be collected in the flow through fraction.
  • This fraction containing the AMPK ⁇ l (68-163) fragment was concentrated in Centricon-lOs (Millipore) to a volume of 4 ml before further purifying on an SI 00 gel filtration column equilibrated in 50 mM Hepes pH7.0. Fractions were analysed by Tris-tricine gel electrophoresis (Figure 3) and appropriate fractions were pooled and stored in aliquots at -70°C until required.
  • the resultant AMPK ⁇ l (68-163) fragment was subjected to N-terminal sequencing and was found to include the amino acids, GAMDPEF (SEQ ID NO: 30), that consisted of part of the ProEx's multi-cloning site.
  • GAMDPEF SEQ ID NO: 30
  • the protein's concentration was determined by measuring the absorbance at 280nm and utilizing the theoretical extinction coefficient of AMPK ⁇ l (68-163), this being approximately 0.55 for a 1 mg/ml solution.
  • starch binding proteins A characteristic of starch binding proteins is that they can bind glycogen and ⁇ cyclodextran can inhibit the binding to starch.
  • AMPK ⁇ l OBD glycogen-binding assay 20 ⁇ g of AMPK ⁇ l OBD, 20 ⁇ g of purified maltose-binding protein or 20 ⁇ g of bovine serum albumin was incubated on its own or in the presence of 0.5% glycogen +/- ⁇ - cyclodextrin in PBS / 0.1% NP-40 for 60 minutes at 4°C. Samples were centrifuged at 200,000g / 60 minutes / 4°C. The supernatant was discarded and the pellet briefly washed with 500 ⁇ l PBS.
  • the AMPK alpha (1-312) was expressed in bacteria with an N-terminal MBP tag and a C-terminal Histidine tag.
  • This fusion protein was purified by Ni-Agarose chromatography, eluted and phosphorylated with purified CAMKIK, resulting in an active AMPK alpha (1-312).
  • This protein was incubated with increasing concentrations (2 - 4 mM) of sulfo- ⁇ -cyclodextrin in a buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl and 0.1 % NP-40, for 60 minutes at 4°C with mixing.
  • the AMPK ⁇ l OBD when aligned to the glucoamylase SBD appears to contain at least binding site 1 since these residues are completely conserved. Binding site2 residues however are not conserved.
  • Figure 8B shows the relationship between and AMPK oligosaccharide-binding domain, PPP1R4 (a regulatory protein that binds to a phosphatase, and is known to bind to glycogen) and GS (glycogen synthase, also known to bind to glycogen). It appears that the conservation is not as high as with the above-mentioned starch-binding domain. This suggests that the AMPK oligosaccharide-binding domain is a new type of oligosaccharide-binding domain family compared those already described.
  • COS-7 cells in 6 well plates were transiently co- transfected, using Fugene (Roche), with 50 ng GP- ⁇ dsRED2-Nl and AMPK ⁇ l- pEGFP cDNA and incubated for 24 hours at 37°C.
  • Cells were fixed with 4 % fomaldehyde for 15 minutes, permeabilized in PBS / 0.5 % Tween 20 and 150 mM glycine for 15 minutes and the nuclei were stained with Hoescht (Molecular Probes).
  • GP and AMPK bl expression was detected by fluorescence using an Olympus inverted microscope and images were captured using a Spot camera.
  • the AMPK ⁇ subunit colocalizes with GP to glycogen bodies.
  • the subcellular localization of the AMPK ⁇ l subunit was investigated by transiently expressing an AMPK ⁇ l-GFP (green fluorescent protein) construct in COS-7 cells together with glycogen phosphorylase (GP) tagged to the red variant of GFP (green fluorescent protein).
  • AMPK ⁇ l-GFP co-labelled glycogen bodies together with GP (data not shown), demonstrating that AMPK localizes to glycogen.
  • the glycogen bodies were similar in size and shape to those observed when laforin was over expressed (data not shown).
  • Laforin is a dual-specificity phosphatase encoded by the EPM2A gene that contains a glycogen-binding domain that is more closely related to the starch-binding domain.
  • MIR Multiple isomorphoas replacement
  • Model building Model building is performed with an Indigo Silicon Graphics (SGI) workstations, using the tools within the software package 'O'.
  • SGI Indigo Silicon Graphics
  • crystals of the ⁇ -OBD has been grown in the presence of 30% PEG 5000 and cyclodextrin in 0.1 M MES buffer ranging from pH 6.5-7.0.
  • the crystals diffracted to 2.2-2.A with a PI space group and cell dimensions of 43.7A, 44.7A, 50.45A (data not shown).
  • a model of ⁇ -OBD structure was made using the N-terminal domains of E. coli branching enzyme (PDB entry 1MX7) residues 117-223, glycosyltrehalose trehalohydrolase from Sulfo lobus solfataricus residues 1-88 (PDB entry 1EH9) and Pseudomonas isoamylase residues 1-163 (PDB entry 1BF2). (data not shown).
  • the pattern of structurally important residues comprising the hydrophobic core of the ⁇ - sandwich structure was noted.
  • the sequence of ⁇ - OBD 72-155 was added (data not shown) and the model was constructed based on that alignment using the program O.
  • the quality of the model ( Figure 13) was checked by calculating the 3D profile through the Verify3D-structure evaluation server (details not shown). The model was considered to of good quality since the 3D-1D score never fell below 0.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne l'identification d'un domaine de liaison de l'oligosaccharide dans la sous-unité bêta de l'AMP kinase. L'invention concerne également des procédés permettant de cribler des composés qui modulent la liaison de l'oligosaccharide à la sous-unité bêta de l'AMP kinase. Enfin, l'invention concerne des procédés d'utilisation de tels composés dans le traitement d'états pathologiques associés à l'activité de l'AMP kinase non couplées.
PCT/AU2002/001769 2001-12-21 2002-12-23 Domaine de liaison de l'oligosaccharide de la sous-unite beta de l'amp kinase Ceased WO2003056032A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002357407A AU2002357407A1 (en) 2001-12-21 2002-12-23 Amp kinase beta-subunit oligosaccharide binding domain

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPR9728A AUPR972801A0 (en) 2001-12-21 2001-12-21 Oligosaccharide binding domains
AUPR9728 2001-12-21

Publications (1)

Publication Number Publication Date
WO2003056032A1 true WO2003056032A1 (fr) 2003-07-10

Family

ID=3833340

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2002/001769 Ceased WO2003056032A1 (fr) 2001-12-21 2002-12-23 Domaine de liaison de l'oligosaccharide de la sous-unite beta de l'amp kinase

Country Status (2)

Country Link
AU (1) AUPR972801A0 (fr)
WO (1) WO2003056032A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997025341A1 (fr) * 1996-01-08 1997-07-17 St. Vincent's Institute Of Medical Research Nouvelles proteines kinases activees par l'amp
US5846720A (en) * 1989-07-18 1998-12-08 Oncogene Science, Inc. Methods of determining chemicals that modulate expression of genes associated with cardiovascular disease
WO2000028076A1 (fr) * 1998-11-06 2000-05-18 St. Vincent's Institute Of Medical Research Regulation de l'activite de la monoxyde d'azote-synthase

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5846720A (en) * 1989-07-18 1998-12-08 Oncogene Science, Inc. Methods of determining chemicals that modulate expression of genes associated with cardiovascular disease
WO1997025341A1 (fr) * 1996-01-08 1997-07-17 St. Vincent's Institute Of Medical Research Nouvelles proteines kinases activees par l'amp
WO2000028076A1 (fr) * 1998-11-06 2000-05-18 St. Vincent's Institute Of Medical Research Regulation de l'activite de la monoxyde d'azote-synthase

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CHEN Z.-P. ET AL.: "Expression of the AMP-activated protein kinase beta1 and beta2 subunits in skeletal muscle", FEBS LETTERS, vol. 460, 1999, pages 343 - 348, XP004498706, DOI: doi:10.1016/S0014-5793(99)01371-X *
DATABASE EMBL [online] Database accession no. (AF182717) *
GAO G. ET AL.: "Non-catalytic beta- and gamma-subunit isoforms of the 5'-AMP-activated protein kinase", J. BIOL. CHEM., vol. 271, no. 15, 1996, pages 8675 - 8681, XP002181599, DOI: doi:10.1074/jbc.271.15.8675 *
MITCHELHILL K.I. ET AL.: "Posttranslational modifications of the 5'-AMP-activated protein kinase beta1 subunit", J. BIOL. CHEM., vol. 272, no. 39, 1997, pages 24475 - 24479 *
STAPLETON D. ET AL.: "AMP-activated protein kinase isoenzyme family: subunit structure and chromosomal location", FEBS LETTERS, vol. 409, 1997, pages 452 - 456, XP002181600, DOI: doi:10.1016/S0014-5793(97)00569-3 *
THORNTON C. ET AL.: "Identification of a novel AMP-activated protein kinase beta subunit isoform that is highly expressed in skeletal muscle", J. BIOL. CHEM., vol. 273, no. 20, 1998, pages 12443 - 12450 *
WOODS A. ET AL.: "Characterization of AMP-activated protein kinase beta and gamma subunits: assembly of the heterotrimeric complex in vitro", J. BIOL. CHEM., vol. 271, no. 17, 1996, pages 10282 - 10290, XP002907303, DOI: doi:10.1074/jbc.271.48.30517 *

Also Published As

Publication number Publication date
AUPR972801A0 (en) 2002-01-24

Similar Documents

Publication Publication Date Title
Wedegaertner et al. Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha.
Dong et al. Molecular characterization of a cDNA that encodes six isoforms of a novel murine A kinase anchor protein
Fredericks et al. Identification of the G protein-coupled receptor kinase phosphorylation sites in the human β2-adrenergic receptor
Evanko et al. Interaction with Gβγ is required for membrane targeting and palmitoylation of Gαs and Gαq
Richardson et al. The α2A-adrenergic receptor discriminates between Gi heterotrimers of different βγ subunit composition in Sf9 insect cell membranes
Stewart et al. The structure of the Q69L mutant of GDP-Ran shows a major conformational change in the switch II loop that accounts for its failure to bind nuclear transport factor 2 (NTF2)
Gijsbers et al. Functional characterization of the non-catalytic ectodomains of the nucleotide pyrophosphatase/phosphodiesterase NPP1
Eide et al. Distinct but overlapping domains of AKAP95 are implicated in chromosome condensation and condensin targeting
CA2323725A1 (fr) Essais de recherche systematique de composes en interaction avec des proteines de canal cation, proteines de canal cation procaryotes mutantes, et utilisations correspondantes
Kameyama et al. Activation of a GTP‐binding protein and a GTP‐binding‐protein‐coupled receptor kinase (β‐adrenergic‐receptor kinase‐1) by a muscarinic receptor m2 mutant lacking phosphorylation sites
CA2223077A1 (fr) Homologue du recepteur de la thrombine
US20070020684A1 (en) Structure of a glucocorticoid receptor ligand binding domain comprising an expanded binding pocket and methods employing same
US20030203419A1 (en) Methods for screening and therapeutic applications of kinesin modulators
Wang et al. Identification and characterization of AGTRAP, a human homolog of murine Angiotensin II Receptor-Associated Protein (Agtrap)
WO2002090578A2 (fr) Procedes et elements de methylation d'histones
WO2002024867A2 (fr) Nouvelles compositions et nouvelles methodes pour le diagnostic et le traitement des lymphomes et des leucemies
US5741689A (en) Methods to inhibit serine kinase activity and to alter intersubunit binding activity of phosphatidylinositol 3-kinase, and serine kinase active sequence of the same
WO2003056032A1 (fr) Domaine de liaison de l'oligosaccharide de la sous-unite beta de l'amp kinase
JP2002514926A (ja) ヒト・アデニル酸シクラーゼのクローニングおよび特性評価
US6437113B1 (en) Suppressors of death domains
US20030157553A1 (en) Methods of assaying for G protein-coupled receptor ligands and modulators
CA2351874A1 (fr) Recepteur couple a la proteine g ressemblant au recepteur b4 de la leukotriene
WO2001067106A2 (fr) Methodes de dosage permettant d'identifier des ligands et des modulateurs de recepteurs couples a la proteine g
JP2002508393A (ja) ヒト核内受容体タンパク質ｎＮＲ７およびｎＮＲ７−１をコードするＤＮＡ分子
US7060463B2 (en) DNA molecules encoding Macaca mulatta androgen receptor

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP