WO2002053591A1 - Cofacteur de recepteur nucleaire mammalien cf12 et ses procedes d'utilisation - Google Patents

Cofacteur de recepteur nucleaire mammalien cf12 et ses procedes d'utilisation Download PDF

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WO2002053591A1
WO2002053591A1 PCT/EP2001/015135 EP0115135W WO02053591A1 WO 2002053591 A1 WO2002053591 A1 WO 2002053591A1 EP 0115135 W EP0115135 W EP 0115135W WO 02053591 A1 WO02053591 A1 WO 02053591A1
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cofactor
nucleic acid
protein
cells
seq
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PCT/EP2001/015135
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English (en)
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David Jackson
Georg Casari
Jörg SUCKOW
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Lion Bioscience Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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

Definitions

  • the present invention is related to an isolated nucleic acid molecule coding for a cofactor of a mammalian nuclear receptor, a vector and a host cell comprising said nucleic acid molecule, a method of producing a polypeptide, an isolated polypeptide encoded by said nucleic acid molecule, a method for screening for agents which are capable of inhibiting or activating the cellular function of the cofactor, and a method for inhibiting or activating the cellular function of the cofactor.
  • Multicellular organisms are dependent on advanced mechanisms of information transfer between cells and body compartments.
  • the information that is transmitted can be highly complex and can result in the alteration of genetic programs involved in cellular differentiation, proliferation, or reproduction.
  • the signals, or hormones are often simple molecules, such as peptides, fatty acid, or cholesterol derivatives.
  • NR nuclear receptors
  • Orphan receptors may be indicative of unknown signaling pathways in the cell or may be nuclear receptors that function without ligand activation. The activation of transcription by some of these orphan receptors may occur in the absence of an exogenous ligand and/or through signal transduction pathways originating from the cell surface (Mangelsdorf, D. J. et al., The nuclear receptor superfamily: the second decade, Cell 83, 835-839, 1995).
  • a DNA-binding domain hereinafter referred to as "DBD” usually comprises two zinc finger elements and recognizes a specific Hormone Responsive Element hereinafter referred to as "HRE" within the promoters of responsive genes.
  • HRE Hormone Responsive Element
  • Specific amino acid residues in the “DBD” have been shown to confer DNA sequence binding specificity (Schena, M. & Yamamoto, K.R., Mammalian Glucocorticoid Receptor Derivatives Enhance Transcription in Yeast, Science, 241:965-967, 1988).
  • a Ligand-binding-domain hereinafter referred to as "LBD" is at the carboxy-terminal region of known NRs.
  • the LBD appears to interfere with the interaction of the DBD with its HRE.
  • Hormone binding seems to result in a conformational change in the NR and thus opens this interference (Brzozowski et al., Molecular basis of agonism and antagonism in the oestrogen receptor, Nature, 389, 753 - 758, 1997; Wagner et al., A structural role for hormone in the thyroid hormone receptor, Nature, 378, 690 - 697. 1995).
  • a NR without the HBD constitutively activates transcription but at a low level.
  • TAF transcription activation functions
  • Acidic residues in the amino-terminal domains of some nuclear receptors may be important for these transcription factors to interact with RNA polymerase. TAF activity may be dependent on interactions with other protein factors or nuclear components (Diamond et al., Transcription Factor Interactions: Selectors of Positive or Negative Regulation from a Single DNA Element, Science, 249:1266-1272 , 1990).
  • Certain oncoproteins e.g., c-Jun and c-Fos
  • GR glucocorticoid receptors
  • the receptors for estrogen and vitamins A and D, and fatty acids have been shown to interact, either physically or functionally, with the Jun and Fos components of AP-1 in the transactivation of steroid- or AP-1 regulated genes.
  • Coactivators or transcriptional activators are proposed to bridge between sequence specific transcription factors, the basal transcription machinery and, in addition, to influence the chromatin structure of a target cell.
  • Nuclear receptor modulators like steroid hormones affect the growth and function of specific cells by binding to intracellular receptors and forming nuclear receptor- ligand complexes. Nuclear receptor-hormone complexes then interact with a hormone response element (HRE) in the control region of specific genes and alter specific gene expression.
  • HRE hormone response element
  • the present invention relates to the identification of a novel cofactor of a mammalian nuclear receptor.
  • the present invention provides novel proteins, nucleic acids, and methods useful for developing and identifying compounds for the treatment of diseases and disorders such as metabolic disorders, immunological indications, hormonal dysfunctions and/or neurosystemic diseases and others not specifically mentioned here.
  • CF12 protein sequences for a novel cofactor and the nucleic acid sequences encoding this cofactor, which we call: CF12 .
  • the importance of this invention is manifested in the effects of the cofactor CF12 to modulate genes involved in cellular functions like regulation of metabolism and cell homeostasis, cell proliferation and differentiation, pathological cellular aberrations, or cellular defense mechanisms.
  • CF12 binds retinoic acid receptor (RAR) (NR1A) and thyroid hormone receptor (TR) (NR1B) in a ligand-dependent manner, but does not interact with retinoid X receptor (RXR) (NR2B) or steroid hormone receptors.
  • RAR retinoic acid receptor
  • TR thyroid hormone receptor
  • CF12 down-regulates transcriptional activation by binding to ligand- bound nuclear receptors.
  • ROR ⁇ protein-protein- interactions between CF12 and retinoid-related orphan receptor ⁇
  • CF12 expression is restricted to the central nervous systeme and could be confined to neurons in the dentate gyms of the hippocampus, the amygdala, thalamic and hypothalamic regions. CF12 is inter alia involved in the coregulation of nuclear receptor functions in brain.
  • the CF12 protein is useful for screening for (neuronal) nuclear receptors, thereby providing for agents which may influence the activity of such a receptor.
  • This nucleic acid sequence has a variety of uses. For example, it is useful for making vectors and for transforming cells, both of which are ultimately useful for production of the CF12 protein.
  • a homogenous composition comprising the cofactor protein.
  • the protein is useful for screening drugs for agonist and antagonist activity, and, therefore, for screening for drugs useful in regulating physiological responses associated with the cofactor according to the invention.
  • antagonists to the CF12 could be used to treat metabolic disorders, immunological indications, hormonal dysfunctions, neurosystemic diseases.
  • the proteins are also useful for developing antibodies for detection of the protein.
  • vectors such as plasmids, comprising the cofactor nucleic acid sequence that may further comprise additional regulatory elements, e.g., promoters,
  • transformed cells that express the cofactors
  • nucleic acid probes e.g., nucleic acid probes
  • antisense oligonucleotides e.g., agonists, (f) antagonists, and (g) transgenic mammals.
  • transgenic mammals e.g., transgenic mammals.
  • Further aspects of the invention comprise methods for making and using the foregoing compounds and compositions.
  • the present invention comprises, in part, a novel cofactor (CF12) of a mammalian nuclear receptor.
  • CF12 novel cofactor
  • Particularly preferred embodiments of this cofactor are those having an amino acid sequence substantially the same as SEQ ID NO. 3. (see also Fig. 1 ).
  • Fig. 1 a novel cofactor
  • the present invention also comprises the nucleic acid sequence encoding the cofactor CF12 which nucleic acid sequence is substantially the same as SEQ ID NO. 1 (see also Fig. 1 ) encoding a human cofactor as preferred embodiment and/or the complement thereof as shown in SEQ ID NO. 2. (see also Fig. 1 , here all nucleic acid sequences are written in 5'-3' orientation).
  • complement refers to the complementary strand of the nucleic acid according to the invention, thus the strand that would hybridize to the nucleic acid according to the invention.
  • complements refers to the complementary strand of the nucleic acid according to the invention, thus the strand that would hybridize to the nucleic acid according to the invention.
  • all DNA sequences herein are however written in 5'-3' orientation, thus the complements depicted (see also figures) are actually “reverse” complements (as also stated in the figures). For simplification purposes they are, however, often referred to simply as “complements”.
  • a protein "having an amino acid sequence substantially the same as SEQ ID NO 3" means a protein whose amino acid sequence is the same as SEQ ID NO 3 or differs only in a way such that at least 50% of the residues compared in a sequence alignment with SEQ ID NO. 3 are identical, preferably 75% of the residues are identical, even more preferably 95% of the residues are identical and most preferably at least 98% of the residues are identical
  • nucleic acid sequence encoding the protein as disclosed herein
  • a molecule having a nucleotide sequence substantially the same as SEQ ID NO 1 means a nucleic acid encoding a protein "having an amino acid sequence substantially the same as SEQ ID NO. 3 as defined above. This definition is intended to encompass natural allelic variations in the CF12 sequence.
  • Cloned nucleic acid provided by the present invention may encode CF proteins of any species of origin, including (but not limited to), for example, mouse, rat, rabbit, hamster, cat, dog, pig, primate, and human.
  • nucleic acids provided by the invention encode CF12 of mammalian, preferably mouse and most preferably human origin.
  • Nucleic acid hybridization probes provided by the invention are nucleic acids consisting essentially of the nucleotide sequences complementary to any sequence depicted in SEQ ID NO.1 and/or the complements thereof as shown in SEQ ID NO. 2, or parts thereof which are effective in nucleic acid hybridization
  • Nucleic acid hybridization probes provided by the invention are nucleic acids capable of detecting i.e. hybridizing to the gene encoding the polypeptide according to SEQ ID No: 3.
  • Nucleic acid probes are useful for detecting CF12 gene expression in cells and tissues using techniques well-known in the art, including, but not limited to, Northern blot hybridization, in situ hybridization, and Southern hybridization to reverse transcriptase - polymerase chain reaction product DNAs.
  • the probes provided by the present invention including oligonucleotide probes derived therefrom, are also useful for Southern hybridization of mammalian, preferably human, genomic DNA for screening for restriction fragment length polymorphism (RFLP) associated with certain genetic disorders.
  • RFLP restriction fragment length polymorphism
  • complementary means a nucleic acid having a sequence that is sufficiently complementary in the Watson-Crick sense to a target nucleic acid to bind to the target under physiological conditions or experimental conditions those skilled in the art routinely use when employing probes. It is understood in the art that a nucleic acid sequence will hybridize with a complementary nucleic acid sequence under highly stringent conditions as defined herein, even though some mismatches may be present. Such closely matched, but not perfectly complementary sequences are also encompassed by the present invention. For example, differences may occur through genetic code degeneracy, or by naturally occurring or man made mutations and such mismatched sequences would still be encompassed by the present claimed invention.
  • the nucleotide sequences of the nuclear cofactor SEQ ID NO:1 and/or its complement SEQ ID NO.2 can be used to derive oligonucleotide fragments (probes) of various length. Stretches of 17 to 30 nucleotides are used frequently but depending on the screening parameters longer sequences as 40, 50, 100, 150 up to the full length of the sequence may be used. Those probes can be synthesized chemically and are obtained readily from commercial oligonucleotide providers. Chemical synthesis has improved over the years and chemical synthesis of oligonucleotides as long as 100-200 bases is possible. The field might advance further to allow chemical synthesis of even longer fragments.
  • probes can also be obtained by biochemical de novo synthesis of single stranded DNA.
  • the nucleotide sequence of the nuclear receptor or its complement serve as a template and the corresponding complementary strand is synthesized.
  • a variety of standard techniques such as nick translation or primer extension from specific primers or short random oligonucleotides can be used to synthesize the probe (Sambrook, J., Fritsch, E.F. & Maniatis , T. Molecular cloning: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, 1989)).
  • Nucleic acid reproduction technologies exemplified by the polymerase chain reaction (Saiki, R.K. et al.
  • RNA probe In some cases one might also consider to use the nucleic acid sequence of the cofactor or its complement as a template to synthesize an RNA probe.
  • a promoter sequence for a DNA-dependent RNA polymerase has to be introduced at the 5'- end of sequence. As an example this can be done by cloning the sequence in a vector which carries the respective promoter sequence. It is also possible to introduce the needed sequence by synthesizing a primer with the needed promoter in the form of a 5' "tail". The chemical synthesis of a RNA probe is another option.
  • Appropriate means are available to detect the event of a hybridization.
  • labels and detection systems e.g. radioactive isotopes, fluorescent, or chemiluminescent molecules which can be linked to the probe.
  • methods of introducing haptens which can be detected by antibodies or " other ligands such as the avidin/biotin high affinity binding system.
  • Hybridization can take place in solution or on solid phase or in combinations of the two, e.g. hybridization in solution and subsequent capture of the hybridization product onto a solid phase by immobilized antibodies or by ligand coated magnetic beads.
  • Hybridization probes act by forming selectively duplex molecules with complementary stretches of a sequence of a gene or a cDNA.
  • the selectivity of the process can be controlled by varying the conditions of hybridization.
  • stringent conditions for the hybridization e.g. low salt in the range of 0.02 M to 0.15 M salt and/or high temperatures in the range from 50°C degrees centigrade to 70°C degrees centigrade.
  • Stringency can be further improved by the addition of formamide to the hybridisation solution.
  • stringent hybridization conditions are those where between 0.02 M to 0.15 M salt and/or high temperatures in the range from 50°C degrees centigrade to 70°C degrees centigrade are applied.
  • highly stringent hybridization conditions are those where between 0.02 - 0.3 M salt and 65°C degrees centigrade are applied for about 5 to 18 hours of hybridization time and additionally, the sample filters are washed twice for about 15 minutes each at between 60°C - 65°C degrees centigrade, wherein the first washing fluid contains about 0.1 M salt (NaCI and/or Sodium Citrate) and the second contains only about 0.02 M salt (NaCI and/or Sodium Citrate).
  • first washing fluid contains about 0.1 M salt (NaCI and/or Sodium Citrate) and the second contains only about 0.02 M salt (NaCI and/or Sodium Citrate).
  • the following conditions are considered to be highly stringent:
  • Unspecific hybridization products are removed by washing the reaction products repeatedly in 2 x SSC solution and increasing the temperature.
  • the nucleotide sequence of the cofactor CF12 or its complement can be used to design primers for a polymerase chain reaction. Due to the degeneracy of the genetic code the respective amino acid sequence is used to design oligonucleotides in which varying bases coding for the same amino acid are included. Numerous design rules for degenerate primers have been published
  • annealing temperature is often much lower than the standard annealing temperature of
  • PCR reaction products can be cloned. Either the PCR product is cloned directly, with reagents and protocols from commercial manufacturers ⁇ e.g. from Invitrogen, San Diego, USA). Alternatively, restriction sites can be introduced intro the PCR product via a 5'-tail of the PCR primers and used for cloning.
  • GENETIC VARIANTS GENETIC VARIANTS
  • Fragments from the nucleotide sequence of the cofactors or their complements can be used to cover the whole sequence with overlapping sets of PCR primers. These primers are used to produce PCR products using genomic DNA from a human diversity panel of healthy individuals or genomic DNA from individuals which are phenotypically conspicuous.
  • the PCR products can be screened for polymorphisms, for example by denaturing gradient gel electrophoresis, binding to proteins detecting mismatches or cleaving heteroduplices or by denaturing high- performance liquid chromatography. Products which display mutations need to be sequenced to identify the nature of the mutation. Alternatively, PCR products can be sequenced directly omitting the mutation screening step to identify genetic polymorphisms.
  • genetic variants are identified and are associated with a discrete phenotype, these genetic variations can be included in diagnostic assays.
  • the normal variation of the human population is of interest in designing screening assays as some variants might interact better or worse with a respective lead, i.e. therapeutic or potentially therapeutic substance (a pharmacodynamic application).
  • Polymorphisms or mutations which can be correlated to phenotypic outcome are a tool to extend the knowledge and the commercial applicability of the nucleotide sequences of the cofactor CF12 or its complement or its gene products, as variants might have a slightly different molecular behavior or desired properties.
  • Disease-causing mutations or polymorphisms allow the replacement of this disease inducing gene copy with a wild-type copy by means of gene therapy approaches and/or the modulation of the activity of the gene product by drugs.
  • DNA which encodes cofactor CF12 may be obtained, in view of the instant disclosure, by chemical synthesis, by screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by screening genomic libraries from appropriate cells, or by combinations of these procedures, as illustrated below.
  • oligonucleotide probes generated from the CF12 nucleotide sequence information provided herein. These oligonucleotides are in addition useful to isolate a full length cDNA from an appropriate cDNA library.
  • the design of these oligonucleotide probes and how to perform the screening procedure is well known by a person skilled in the art.
  • oligonucleotide primers derived from the 3 ' and 5 ' end of the CF12 nucleotide sequence information according to the present invention (SEQ ID. NO.1).
  • Probes may be labeled with a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with known procedures and used in conventional hybridization assays, as described in greater detail in the Examples below.
  • a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group
  • the CF nucleotide sequence may be obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers being produced from the CF12 nucleotide sequence provided herein, according to SEQ ID NO 1 and/or the complement thereof as shown in SEQ ID NO. 2, or parts thereof.
  • PCR polymerase chain reaction
  • nucleic acid according to the invention may be labeled, e.g. for use as a probe.
  • single and differential labeling agents and methods any agents and methods which are known in the art can be used provided that they do not significantly altering the stability or function of said primer in the DNA sequencing method of the present invention.
  • single and differential labels may consist of the group comprising enzymes such as ⁇ -galactosidase, alkaline phosphatase and peroxidase, enzyme substrates, coenzymes, dyes, chromophores, fluorescent, chemiluminescent and bioluminescent labels such as FITC, Cy5, Cy5.5, Cy7, Texas-Red and IRD40 (Chen et al. (1993), J. Chromatog. A 652: 355-360 and Kambara et al. (1992), Electrophoresis 13: 542-546), ligands or haptens such as biotin, and radioactive isotopes such as 3 H, 35 S, 32 P 125 l and 14 C.
  • enzymes such as ⁇ -galactosidase, alkaline
  • the CF12 nucleic acid or polypeptide may be synthesized in host cells transformed with a recombinant expression construct comprising a nucleic acid encoding the cofactor CF12 according to the invention.
  • a recombinant expression construct can also be comprised of a vector that is a replicable DNA construct. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. See, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Press, New York, 1989).
  • An expression vector comprises a polynucleotide operatively linked to a prokaryotic promoter.
  • an expression vector is a polynucleotide operatively linked to an enhancer-promoter that is a eukaryotic promoter, and the expression vector further has a polyadenylation signal that is positioned 3' of the carboxy-terminal amino acid and within a transcriptional unit of the encoded polypeptide.
  • a promoter is a region of a DNA molecule typically within about 500 nucleotide pairs in front of (upstream of) the point at which transcription begins (i.e., a transcription start site).
  • a vector contains a replicon and control sequences which are derived from species compatible with the host cell. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene).
  • a major function of an enhancer is to increase the level of transcription of a coding sequence in a cell.
  • enhancer-promoter means a composite unit that contains both enhancer and promoter elements.
  • An enhancer-promoter is operatively linked to a coding sequence that encodes at least one gene product.
  • An enhancer-promoter used in a vector construct of the present invention may be any enhancer-promoter that drives expression in a prokaryotic or eukaryotic cell to be transformed/transfected.
  • a coding sequence of an expression vector is operatively linked to a transcription terminating region.
  • RNA polymerase transcribes an encoding DNA sequence through a site where polyadenylation occurs.
  • An expression vector that comprises a polynucleotide that encodes the polypeptide of the cofactor CF12.
  • a polynucleotide is meant to include a sequence of nucleotide bases encoding a CF polypeptide sufficient in length to distinguish said segment from a polynucleotide segment encoding a non-cofactor polypeptide.
  • a polypeptide of the invention may also encode biologically functional polypeptides or peptides which have variant amino acid sequences, such as with changes selected based on considerations such as -the relative hydropathic score of the amino acids being exchanged.
  • variant sequences are those isolated from natural sources or induced in the sequences disclosed herein using a mutagenic procedure such as site-directed mutagenesis.
  • an expression vector of the present invention may contain regulatory elements for optimized translation of the polypeptide in prokaryotic or eukaryotic systems. These sequences are operatively located around the transcription start site and are most likely similar to ribosome recognition sites like prokaryotic ribosome binding sites (RBS) or eukaryotic Kozak sequences as known in the art (Kozak M., Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187- 208 (1999)).
  • RBS prokaryotic ribosome binding sites
  • Kozak sequences as known in the art
  • An expression vector of the present invention is useful both as a means for preparing quantities of the CF12 polypeptide-encoding DNA itself, and as a means for preparing the encoded CF12 polypeptide and peptides. It is contemplated that where cofactor polypeptides of the invention are made by recombinant means, one may employ either prokaryotic or eukaryotic expression vectors as shuttle systems.
  • a vector such as a plasmid, that incorporates a eukaryotic origin of replication.
  • a vector such as a plasmid, that incorporates a eukaryotic origin of replication.
  • the invention provides homogeneous compositions of mammalian cofactor polypeptides produced by transformed prokaryotic or eukaryotic cells as provided herein. Such homogeneous compositions are intended to be comprised of mammalian cofactor protein that comprises at least 90% of the protein in such homogenous composition.
  • the invention also provides membrane preparation from cells expressing the mammalian cofactors polypeptides as the result of transformation with a recombinant expression construct, as described here.
  • recombinant protein or coding sequence both also include tagged versions of the proteins depicted in SEQ ID NO. 3 and/or and fusion proteins of said proteins with any other recombinant protein.
  • Tagged versions here means that small epitopes of 3-20 amino acids are added to the original protein by extending the coding sequence either at the 5 ' or the 3 ' terminus leading to N-terminal or C-terminal extended proteins respectively, or that such small epitopes are included elsewhere in the protein.
  • fusion proteins where the added sequences are coding for longer proteins, varying between 2 and 100 kDa.
  • Fusion proteins are usually used to facilitate purification of recombinant proteins by specific antibodies or affinity matrices or to increase solubility of recombinant proteins within the expression host. Fusion proteins are also of major use as essential parts of yeast two hybrid screens for interaction partners of recombinant proteins.
  • EEF alpha Tubulin
  • B-tag QYPALT
  • E tag GAPVPYPDPLEPR
  • c- myc Tag EQKLISEEDL
  • Flag epitope DYKDDDDK, HA tag (YPYDVPDYA), 6 or 10 x His Tag
  • HSV QPELAPEDPED
  • Pk-Tag GKPIPNPLLGLDST
  • protein C EDQVDPRLIDGK
  • T7 MASMTGGQQMG
  • VSV-G YTDIEMNRLGK
  • Fusion proteines may include Thioredoxin, Glutathiontransferase (GST), Maltose binding Protein (MBP), Cellulose Binding protein, calmodulin binding protein, chitin binding protein, ubiquitin, the Fc part of Immunoglobulins, and the IgG binding domain of Staphylococcus aureus protein A.
  • vector constructs harboring a recombinant cofactor as set forth in SEQ ID NO. 1 and/or the complement thereof as of SEQ ID NO. 2 and/or are transformed or transfected into appropriate host cells.
  • a recombinant host cell of the present invention is transfected with a polynucleotide of SEQ ID NO. 1 and/or the complement thereof SEQ ID NO. 2).
  • Means of transforming or transfecting cells with exogenous polynucleotide such as DNA molecules are well known in the art and include techniques such as calcium- phosphate- or DEAE-dextran-mediated transfection, protoplast fusion, electroporation, liposome mediated transfection, direct microinjection and virus infection (Sambrook et al., 1989).
  • transfection mediated by either calcium phosphate or DEAE-dextran The most widely used method for transfection of eukaryotic cells is transfection mediated by either calcium phosphate or DEAE-dextran. Although the mechanism remains obscure, it is believed that the transfected DNA enters the cytoplasm of the cell by endocytosis and is transported to the nucleus. Depending on the cell type, up to 90% of a population of cultured cells may be transfected at any one time. Because of its high efficiency, transfection mediated by calcium phosphate or DEAE-dextran is the method of choice for studies requiring transient expression of the foreign nucleic acid in large numbers of cells. Calcium phosphate-mediated transfection is also used to establish cell lines that integrate copies of the foreign DNA, which are usually arranged in head-to-tail tandem arrays into the host cell genome.
  • protoplasts derived from bacteria carrying high numbers of copies of a plasmid of interest are mixed directly with cultured mammalian cells. After fusion of the cell membranes (usually with polyethylene glycol), the contents of the bacterium are delivered into the cytoplasm of the mammalian cells and the plasmid DNA is transported to the nucleus.
  • Protoplast fusion is not as efficient as transfection. for many of the cell lines that are commonly used for transient expression assays, but it is useful for cell lines in which endocytosis of DNA occurs inefficiently. Protoplast fusion frequently yields multiple copies of the plasmid DNA tandemly integrated into the host chromosome.
  • Electroporation may be extremely efficient and may be used both for transient expression of cloned genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA.
  • Liposome transfection involves encapsulation of DNA and RNA within liposomes, followed by fusion of the liposomes with the cell membrane. The mechanism of how DNA is delivered into the cell is unclear but transfection efficiencies may be as high as 90%.
  • Direct microinjection of a DNA molecule into nuclei has the advantage of not exposing DNA to cellular compartments such as low-pH endosomes. Microinjection is therefore used primarily as a method to establish lines of cells that carry integrated copies of the DNA of interest.
  • adenovirus vector-mediated cell transfection has been reported for various cells (Stratford-Perricaudet et al., 1992).
  • a transfected cell may be prokaryotic or eukaryotic, transfection may be transient or stable. Where it is of interest to produce a full length human CF12 protein, cultured mammalian or human cells are of particular interest.
  • the recombinant host cells of the present invention are prokaryotic host cells.
  • eukaryotic microbes such as yeast may also be used illustrative examples for suitable cells and organisms for expression of recombinant proteins are belonging to but not limited to the following examples: Insect cells, such as Drosophila Sf21 , SF9 cells or others, Expression strains of Escherichia coli, such as XL1 blue, BRL21 , M15, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula polymorpha and Pichia pastoris strains, immortalized mammalian cell lines such as AtT-20, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COSM6, COS-7, 293 and MDCK cells, BHK-21 cells, Att 20HeLa cells, HeK 294, T47 D cells and others.
  • Insect cells such as Drosophila Sf21
  • Expression of recombinant proteins within the scope of this invention can also be performed in vitro. This may occur by a two step procedure, thereby producing first mRNA by in vitro transcription of an apt polynucleotide construct followed by in vitro translation with convenient cellular extracts. These cellular extracts may be reticulocyte lysates but are not limited to this type.
  • In vitro transcription may be performed by T7 or SP6 DNA polymerase or any other RNA polymerase which can recognize per se or with the help of accessory factors the promoter sequence contained in the recombinant DNA construct of choice.
  • one of the recently made available one step coupled transcription/translation systems may be used for in vitro translation of DNA coding for the proteins of this invention.
  • TNT® T7 Quick System by Promega.
  • transfected cells are maintained for a period of time sufficient for expression of the recombinant cofactor proteins according to the invention.
  • a suitable maintenance time depends strongly on the cell type and organism used and is easily ascertainable by one skilled in the art. Typically, maintenance time is from about 2 hours to about 14 days. For the same reasons and for sake of protein stability and solubility incubation temperatures during maintenance time may vary from 20°C to 42 °C.
  • Recombinant proteins are recovered or collected either from the transfected cells or the medium in which those cells are cultured. Recovery comprises cell disruption, isolation and purification of the recombinant protein. Isolation and purification techniques for polypeptides are well-known in the art and include such procedures as precipitation, filtration, chromatography, electrophoresis and the like. In a preferred embodiment, purification includes but is not limited to affinity purification of tagged or nontagged recombinant proteins. This is a well established robust technique easily adapted to any tagged protein by one skilled in the art.
  • affinity purification of tagged proteins small molecules such as gluthathio ⁇ e, maltose or chitin, specific proteins such as the IgG binding domain of Staphylococcus aureus protein A, antibodies or specific chelates which bind with high affinity to the tag of the recombinant protein are employed.
  • specific proteins such as the IgG binding domain of Staphylococcus aureus protein A
  • antibodies or specific chelates which bind with high affinity to the tag of the recombinant protein are employed.
  • affinity purification of non-tagged proteins specific monoclonal or polyclonal antibodies, which were raised against said protein, can be used.
  • immobilized specific interactors of said protein may be employed for affinity purification. Interactors include native or recombinant proteins as well as native or artificial specific low molecular weight ligands.
  • the protein itself may be produced using chemical methods to synthesize any of the amino acid sequences according to the invention (SEQ ID No: SEQ ID NO. 3) or that is encoded by the nucleotide sequence according to the invention (SEQ ID NO. 1) and/or the complement thereof (SEQ ID NO. 2) or a portion thereof.
  • peptide synthesis can be performed using conventional Merrifield solid phase f-Moc or t-Boc chemistry or various solid-phase techniques (Roberge, J. Y. et al. (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer).
  • the newly synthesized peptide(s) may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
  • the amino acid sequences according to the invention i.e. SEQ ID NO. 3 or the sequence that is encoded by SEQ ID NO. 1 or any part thereof, may be altered during direct synthesis. and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
  • CF12 binds to retinoic acid receptor and thyroid hormone receptor in vivo.
  • CF12 is complexed with the receptor polypeptide or portions thereof.
  • Such complexes are particular suited for all forms of binding or screening assays (see also below).
  • such assays are performed with complexes of the receptor(s) associated with CF12.
  • the entire CF12 polypeptide is part of the complex but only a portion, e.g. a truncated fragment of the other polypeptide, is part of the complex.
  • the present invention concerns a method for identifying new inhibitory or stimulatory substances of the cofactor according to the invention, these substances may be termed as “candidate substances”. It is contemplated that this screening technique proves useful in the general identification of compounds that serve the purpose of inhibiting or stimulating cofactor activity.
  • the pharmaceutical agents to be screened can also be derived from chemical compositions or man-made compounds.
  • the candidate substances can could also include monoclonal or polyclonal antibodies, peptides or proteins, such as those derived from recombinant DNA technology or by other means, including chemical peptide synthesis.
  • the active compounds may include fragments or parts or derivatives of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive. We anticipate that such screens will in some cases lead to the isolation of agonists of nuclear receptors or cofactors, in other cases to the isolation of antagonists. In other instances, substances will be identified that have mixed agonistic and antagonistic effects, or affect nuclear receptors or cofactors in any other way.
  • the invention concerns the isolation of substances inhibiting the interaction of the cofactor protein and the mammalian nuclear receptor.
  • substances are useful for the development of drugs against diseases such as metabolic disorders, immunological indications, hormonal dysfunctions and/or neurosystemic diseases or related to defects in neuronal diseases.
  • Substances disrupting the interactions may be isolated by a variety of screening methods including the two hybrid system or the reverse two hybrid system (Lenna CA. and Hannink, M. 1996, Nucl. Acids Res. 24: 3341-3347), or any variation of cellular or cell free assays as described in this invention, as is obvious to anyone skilled in the art.
  • the binding of the cofactor protein and the mammalian nuclear receptor can be used to monitor the binding of a substance to one of the binding partners.
  • the substance which can be a small molecule such as a ligand to a nuclear receptor, will lead to a change in the allosteric conformation of the binding protein which in consequence leads to a loss of the interaction of the two proteins.
  • ligand-dependent protein-protein interactions one can design assays where the protein-protein interaction serves as a surrogate read-out for the binding of one of the proteins to small molecule ligand.
  • Any assay method which is useful for the measurement of protein-protein interactions can be used for such an indirect assay.
  • Such assay methods are well known in the art and include the methods described in this patent under "Cell free assays" and "Cell based assays”.
  • a recombinant cell line To identify a candidate substance capable of influencing the cofactor protein activity, one first obtains a recombinant cell line.
  • a reporter such as luciferase, fluorescent proteins such as green or red fluorescent protein
  • beta-galactosidase alpha- galactosidase
  • beta-lactamase beta-lactamase
  • chloramphenicol-acetyl-transferase beta-glucuronidase
  • the amount of reporter protein present reflects the activity of the cofactor.
  • This recombinant cell line is then screened for the effect of substances on the expression of the reporters, thus measuring the effect of these substances on the activity of the cofactor.
  • These substances can be derived from natural sources, such as fungal extracts, plant extracts, bacterial extracts, higher eukaryotic cell extracts, or even extracts from animal sources, or marine, forest or soil samples, may be assayed for the presence of potentially useful pharmaceutical agents. It will be understood that that the pharmaceutical agents to be screened may be derived from chemical compositions or man-made compounds.
  • the candidate substances can also include monoclonal or polyclonal antibodies, peptides or proteins, such as those derived from recombinant DNA technology or by other means, including chemical peptide synthesis.
  • the active compounds may include fragments or parts or derivatives of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive.
  • the assay can be performed by firstly bringing a suitable cell containing a reporter gene which transcription is influenced by the cofactors activity in contact with a compound and secondly monitoring the expression of the reporter gene to evaluate the effect of the compound on the activity of the cofactor.
  • assays are included where measuring the activity of di- or multimeric complexes of the cofactor and other proteins. Further included are assays aiming at the identification of compounds which specifically influence only the monomeric, homodimeric or homomultimeric form of the cofactor, or influencing only multimeric forms of the cofactor.
  • Such assays include measuring the effect of a compound on the cofactor in the absence of a binding partner, and measuring the effect of a compound on the cofactor in the presence of a binding partner.
  • One skilled in the art will find numerous more assays which are equally covered by the invention.
  • a cell line where the activity of another nuclear receptor determines the expression of a reporter can be obtained by generating an artificial promoter upstream of the reporter gene.
  • transgenic animals described in the invention can be used to derive cell lines useful for cellular screening assays.
  • Cell lines useful for such an assay include many different kinds of cells, including prokaryotic, animal, fungal, plant and human cells.
  • Yeast cells can be used in this assay, including Saccharomyces cerevisiae and Schizosacciiaromyces pombe cells.
  • the two hybrid assay relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins.
  • the method makes use of chimeric genes which express hybrid proteins.
  • a first hybrid gene comprises the coding sequence for a DNA-binding domain of a transcriptional activator fused in frame to the coding sequence for a cofactor.
  • the second hybrid protein encodes a transcriptional activation domain fused in frame to another gene, for example the mammalian nuclear receptor. If the cofactor and the receptor proteins are able to interact, they bring into close proximity the two domains of the transcriptional activator.
  • Suitable host cells for such assays include yeast cells, but also mammalian cells or bacterial cells.
  • Two hybrid systems using hybrid protein fusions with other proteins than transcription factors, including enzymes such as beta-galactosidase or dihydrofolate reductase may also be applied. These assays are useful both to monitor the effect of a compound, including peptides, proteins or nucleic acids on an interaction of a cofactor with a given binding partner, as well as to identify novel proteins or nucleic acids interacting with the cofactor.
  • Recombinant forms of the polypeptides according to SEQ ID NO. 3 can be used in cell-free screening assays aiming at the isolation of compounds affecting the activity of cofactors.
  • the cofactor polypeptides are brought into contact with a substance to test if the substance has an effect on the activity of the cofactor.
  • the detection of an interaction between an agent and a cofactor may be accomplished through techniques well-known in the art. These techniques include but are not limited to centrifugation, chromatography, electrophoresis and spectroscopy. The use of isotopically labeled reagents in conjunction with these techniques or alone is also contemplated. Commonly used radioactive isotopes include 3 H, 14 C, . 22 Na, 32 P, 33 P, 35 S, 45 Ca, 60 Co, 125 l, and 131 l. Commonly used stable isotopes include 2 H, . 13 C, 15 N, 1a O.
  • an agent binds to any of the cofactors of the present invention
  • the binding may be detected by using radiolabeled agent or radiolabeled cofactor.
  • radiolabeled agent or radiolabeled cofactor is utilized, the agent-cofactor complex may be detected by liquid scintillation or by exposure to x-ray film or phosho-imaging devices.
  • One way to screen for substances affecting cofactor activity is to measure the effect of the substance on the binding affinity of the cofactor to other proteins or molecules, such as activators or repressors, DNA, RNA, other proteins, antibodies peptides or other substances, including chemical compounds known to affect receptor activity or to a nuclear receptor itself.
  • Assays measuring the binding of a protein to a ligand are well known in the art, such as ELISA assays, FRET assays, bandshift assays, plasmon-resonance based assays, scintilllation proximity assays, fluorescence polarization assays, alpha screen assays.
  • a mixture containing a cofactor polypeptide, effector and candidate substance is allowed to incubate.
  • the unbound effector is separable from any effector/cofactor complex so formed.
  • One then simply measures the amount of each e.g., versus a control to which no candidate substance has been added). This measurement may be made at various time points where velocity data is desired. From this, one determines the ability of the candidate substance to alter or modify the function of the cofactor.
  • TLC thin layer chromatographic methods
  • HPLC high-density polyethylene glycol
  • spectrophotometric gas chromatographic/mass spectrophotometric or NMR analyses.
  • Another method of separation is to immobilize one of the binding partners on a solid support, and to wash away any unbound material. It is contemplated that any such technique may be employed so long as it is capable of differentiating between the effector and complex, and may be used to determine enzymatic function such as by identifying or quantifying the substrate and product.
  • a " screening assay in which candidate agent binding of cofactors is analysed can include a number of conditions. These conditions include but are not limited to pH, temperature, tonicity, the presence of relevant other proteins, and relevant modifications to the polypeptide such as glycosylation or lipidation. It is contemplated that the cofactors can be expressed and utilized in a prokaryotic or eukaryotic cell.
  • the host cell expressing the cofactors can be used as a whole or the cofactor can be isolated from the host cell.
  • the cofactor can be membrane bound in the membrane of the host cell or it can be free in the cytosol of the host cell.
  • the host cell can also be fractionated into sub-cellular fractions where the cofactor can be found. For example, cells expressing the cofactor can be fractionated into the nuclei, the endoplasmic reticulum, vesicles, or the membrane surfaces of the cell.
  • pH is preferably from about a value of 6.0 to a value of about 8.0, more preferably from about a value of about 6.8 to a value of about 7.8, and most preferably, about 7.4.
  • temperature is from about 20°C degrees to about 50°C degrees more preferably, from about 30°C degrees to about 40°C degrees and even more preferably about 37°C degrees.
  • Osmolality is preferably from about 5 milliosmols per liter (mosm/L) to about 400 mosm/l, and more preferably, from about 200 milliosmols per liter to about 400 mosm/l and, even more preferably from about 290 mosm/L to about 310 mosm/L.
  • cofactors include sodium, potassium, calcium, magnesium, and chloride.
  • prosthetic groups small, non-peptide molecules, known as prosthetic groups may also be required.
  • Other biological conditions needed for cofactor function are well-known in the art.
  • proteins can be reconstituted in artificial membranes, vesicles or liposomes. (Danboldt et al.,1990).
  • the present invention contemplates that the cofactor can be incorporated into artificial membranes, vesicles or liposomes.
  • the reconstituted cofactor can be utilized in screening assays.
  • a cofactor of the present invention can be coupled to a solid support, e.g., to agarose beads, polyacrylamide beads, polyacrylic, sepharose beads or other solid matrices capable of being coupled to polypeptides.
  • a solid support e.g., to agarose beads, polyacrylamide beads, polyacrylic, sepharose beads or other solid matrices capable of being coupled to polypeptides.
  • Well-known coupling agents include cyanogen bromide (CNBr), carbonyldiimidazole, tosyl chloride, diaminopimelimidate, and glutaraldehyde.
  • a typical screening assay for identifying candidate substances one employs the same recombinant expression host as the starting source for obtaining the cofactor polypeptide, generally prepared in the form of a crude homogenate. Recombinant cells expressing the cofactor are washed and homogenized to prepare a crude polypeptide homogenate in a desirable buffer such as disclosed herein. In a typical assay, an amount of polypeptide from the cell homogenate, is placed into a small volume of an appropriate assay buffer at an appropriate pH.
  • Candidate substances such as agonists and antagonists, are added to the admixture in convenient concentrations and the interaction between the candidate substance and the cofactor polypeptide is monitored.
  • screening assays for the testing of candidate substances are designed to allow the determination of structure-activity relationships of agonists or antagonists with the cofactor, e.g., comparisons of binding between naturally- occurring hormones or other substances capable of interacting with or otherwise modulating the cofactor; or comparison of the activity caused by the binding of such molecules to the cofactor.
  • the polypeptides of the invention are crystallized in order to carry out x-ray crystallographic studies as a means of evaluating interactions with candidate substances or other molecules with the cofactor polypeptide.
  • the purified recombinant polypeptides of the invention i.e. of the cofactors according to the invention, when crystallized in a suitable form, " are amenable to detection of intra-molecular interactions by x-ray crystallography.
  • the structure of the polypeptides can be determined using nuclear magnetic resonance.
  • This invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of an agonist or antagonist drug identified by the method described herein and a pharmaceutically acceptable carrier.
  • Such drugs and carrier can be administered by various routes, for example oral, subcutaneous, intramuscular, intravenous or intracerebral.
  • the preferred route of administration would be oral at daily doses of.about 0.01 -100 mg/kg.
  • This invention provides a method of treating metabolic disorders, immunological indications, hormonal dysfunctions, neurosystemic diseases, wherein the abnormality is improved by altering the activity of the cofactor thereby influencing the binding affinity of the cofactor to the above mentioned receptors.
  • the invention also provides methods for treating diseases and conditions resulting from metabolic disorders, immunological indications, hormonal dysfunctions, neurosystemic diseases, or other diseases, which method comprises administering an effective amount of an agonist- or antagonist containing pharmaceutical composition described above.
  • the recombinant expression constructs of the present invention are useful in molecular biology to transform cells which do not ordinarily express the CF12 to express this cofactor upon transformation.
  • Such cells are useful as intermediates for making cellular preparations useful for cofactor binding assays, which are in turn useful for drug screening.
  • the recombinant expression constructs of the present invention are also useful in gene therapy. Cloned genes of the present invention, or fragments thereof, may also be used in gene therapy carried out by homologous recombination or site- directed mutagenesis. See generally Thomas & Capecchi, Cell 51 , 503-512 (1987); Bertling, Bioscience Reports 7, 107-112 (1987); Smithies et al., Nature 317, 230-234 (1985).
  • Oligonucleotides of the present invention are useful as diagnostic tools for probing cofactor gene expression in tissues.
  • tissues are probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiographic techniques, as explained in greater detail in the Examples below, to investigate native expression of this cofactor or pathological conditions relating thereto.
  • chromosomes can be probed to investigate the presence or absence of the CF12 gene, and potential pathological conditions related thereto, as also illustrated by the Examples below.
  • Probes according to the invention should generally be at least about 15 nucleotides in length to prevent binding to random sequences, but, under the appropriate circumstances may be smaller.
  • Another aspect of the invention includes antibodies specifically reactive with the proteins or any parts of the proteins according to the invention (SEQ ID NO. 3) and or polypeptides encoded by the nucleotide sequences of the cofactors or their complements (SEQ ID NO. 1 or SEQ ID NO. 2).
  • the term tauantibody refers to intact molecules as well as fragments thereof, such as Fa, F(ab).sub.2, and Fv, which are capable of binding the epitopic determinant.
  • immunogens derived from the polypeptide according to the invention and/or encoded by the nucleic acids according to the invention anti-protein/anti-peptide antiserum or monoclonal antibodies can be made by standard protocols (E. Howell & D. Lane. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory (1988)).
  • a polyclonal antibody is prepared by immunizing a mammal, such as a mouse, a hamster or rabbit with an immunogenic form of the cofactors according to the invention depending on which of these are desired) of the present invention, and collecting antisera from that immunized animal. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • fusion proteins As an immunizing antigen, fusion proteins, intact polypeptides or fragments containing small peptides of interest can be used. They can be derived by expression from a cDNA transfected in a host cell with subsequent recovering of the protein/peptide or peptides can be synthesized chemically (e.g. oligopeptides with 10-15 residues in length).
  • Important tools for monitoring the function of the cofactor gene according to the present invention i.e. encoded by a sequence according to SEQ ID NO. 1 are antibodies against various domains of the proteins according to the invention.
  • a given polypeptide or polynucleotide may vary in its immunogenicity. It is often necessary to couple the immunogen (e.g. the polypeptide) with a carrier. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal in the presence of an adjuvant, a non-specific stimulator of the immune response in order to enhance immunogenicity. The production of polyclonal antibodies is monitored by detection of antibody titers in plasma or serum at various time points following immunization. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. When a desired level of immunogenicity is obtained, the immunized animal may be bled and the serum isolated, stored and purified.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • antibody-producing ceils e.g. spleen cells
  • immunized animal preferably mouse or rat
  • immortalizing cells such as myeloma cells
  • myeloma cells e.g. spleen cells
  • myeloma cell e.g. murine NS-1 myeloma cell.
  • Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler & Milstein. Nature 256: 495-497 (1975)), the human B cell hybridoma technique (Kozbar et al.
  • the fused spleen/myeloma cells are cultured in a selective medium to select fused spleen/myeloma cells from the parental cells.
  • Fused cells are separated from the mixture of non-fused parental cells, for example, by the addition of agents that block the de novo synthesis of nucleotides in the tissue culture media.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single clone dilution in microtiter plates, followed by testing the individual clonal supematants for reactivity with an antigen-polypeptide.
  • the selected clones may then be propagated indefinitely to provide the monoclonal antibody in convenient quantity.
  • antibodies which specifically bind the polypeptides according to the invention and/or encoded by the nucleotide sequences of the cofactors or their complements provides an important utility in immunolocalization studies, and may play an important role in the diagnosis and treatment of diseases and disorders such as metabolic disorders, immunological indications, hormonal dysfunctions and/or neurosystemic diseases.
  • the antibodies may be employed to identify tissues, organs, and cells which express the cofactors.
  • Antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate cofactor protein levels in tissue or from cells in bodily fluid as part of a clinical testing procedure.
  • Monoclonal antibodies provided by the present invention are also produced by recombinant genetic methods well known to those of skill in the art, and the present invention encompasses antibodies made by such methods that are immunologically reactive with an epitope of a mammalian cofactor protein or peptide according to the invention.
  • the present invention encompasses fragments of the antibody that are immunologically reactive with an epitope of a cofactor protein or peptide. Such fragments are produced by any number of methods, including but not limited to proteolytic cleavage, chemical synthesis or preparation of such fragments by means of genetic engineering technology.
  • the present invention also encompasses single-chain antibodies that are immunologically reactive with an epitope of a cofactor protein or peptide made by methods known to those of skill in the art.
  • the invention also includes chimeric antibodies, comprised of light chain and heavy chain peptides immunologically reactive to an epitope that is a cofactor protein or peptide according to the invention.
  • the chimeric antibodies embodied in the present invention include those that are derived from naturally occurring antibodies as well as chimeric antibodies made by means of genetic engineering technology well known to those of skill in the art.
  • the present invention also encompasses one or more epitopes of a cofactor protein or peptide that is comprised of sequences and/or a conformation of sequences present in the cofactor proteins or peptide molecule.
  • epitopes may be naturally occurring, or may be the result of proteolytic cleavage of the cofactor proteins or peptides and isolation of an epitope-containing peptide or may be obtained by synthesis of an epitope-containing peptide using a method of genetic engineering technology and synthesized by genetically engineered prokaryotic or eukaryotic cells.
  • Antisense oligonucleotides are short single stranded DNA or RNA molecules which may be used to block the availability of the cofactor messengers). Synthetic derivatives of ribonucleotides or desoxyribonucleotides and/or PNAs (see above) are equally possible. These are potential candidate agents which may interact with the cofactor according to the invention.
  • sequence of an antisense oligonucleotide is at least partially complementary to the sequence of the cofactor of interest.
  • the complementarity of the sequence is in any case high enough to enable the antisense oligonucleotide to bind to the nucleic acid according to the invention or parts thereof (SEQ ID NO.
  • Antisense oligonucleotides can be conjugated to different other molecules in order to deliver them to the cell or tissue expressing any of the cofactor genes.
  • the antisense oligonucleotide can be conjugated to a carrier protein (e.g. ferritin) in order to direct the oligonucleotide towards the desired target tissue, i.e. in case of ferritin predominantly to the liver.
  • a carrier protein e.g. ferritin
  • Antisense expression constructs are expression vector systems that allow the expression - either inducible or uninducible - of a complementary sequence to the cofactor sequences according to the invention.
  • the potential possibility of such an approach has been demonstrated in many different model systems (von Ruden T, Gilboa E, Inhibition of human T-cell leukemia virus type I replication in primary human T cells that express antisense RNA, J Virol 1989 Feb;63(2):677-82; Nemir M, Bhattacharyya D, Li X, Singh K, Mukherjee AB, Mukherjee BB, Targeted inhibition of osteopontin expression in the mammary gland causes abnormal morphogenesis and lactation deficiency, J Biol Chem 2000 Jan 14;275(2):969-76; Ma L, Gauville C, Berthois Y, Millot G, Johnson GR, Calvo F Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line, Oncogene
  • an antisense expression construct can be constructed with virtually any expression vector capable of fulfilling at least the basic requirements known to those skilled in the art.
  • retroviral expression systems or tissue specific gene expression systems are preferred.
  • Microinjection still plays a major role in most gene transfer techniques for the generation of germ-line mutants expressing foreign DNA (including antisense RNA constructs) and is preferred embodiment of the present invention.
  • Ribozymes are either RNA molecules (Gibson SA, Pellenz C, Hutchison RE, Davey FR, Shillitoe EJ, Induction of apoptosis in oral cancer cells by an anti-bcl-2 ribozyme delivered by an adenovirus vector, Clin Cancer Res 2000 Jan;6(1):213- 22; Folini M, Colella G, Villa R, Lualdi S, Daidone MG, Zaffaroni N, Inhibition of Telomerase Activity by a Hammerhead Ribozyme Targeting the RNA Component of Telomerase in Human Melanoma Cells, J Invest Dermatol 2000 Fe ' b;114(2):259-267; Halatsch ME, Schmidt U, Botefur IC, Holland JF, Ohnuma T, Marked inhibition of glioblastoma target cell tumorigenicity in vitro by retrovirus- mediated transfer of a hairpin ribozyme against deletion-mutant epidermal growth factor receptor messenger RNA
  • the catalytic activity located in one part of the RNA (or DNA) molecule can be "targeted" to a specific sequence of interest by fusing the enzymatically active RNA molecule sequence with a short stretch of RNA (or DNA) sequence that is complementary to the cofactor gene transcript of interest.
  • a construct will, when introduced into a cell either physically or via gene transfer of a ribozyme expression construct find the corresponding cofactor sequence (our sequence of interest or also targeted in RNA) and bind via its sequence-specific part to said sequence.
  • the catalytic activity attached to the construct usually associated with a special nucleic acid structure (people distinguish so called “hammerhead” structures and "hairpin” structures), will then cleave the targeted RNA.
  • the targeted mRNA will be destroyed and cannot be translated efficiently, thus the protein encoded by the mRNA derived from cofactor will not be expressed or at least will be expressed at significantly reduced amounts.
  • the invention covers inducible ribozyme constructs (Koizumi M, Soukup GA, Kerr JN, Breaker RR, Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP, Nat Struct Biol 1999 Nov;6(11): 1062-1071).
  • the invention concerns the use of "bivalent" ribozymes (multimers of catalytically active nucleic acids) as described in (Hamada M, Kuwabara T, Warashina M, Nakayama A, Taira K, Specificity of novel allosterically trans- and cis-activated connected maxizymes that are designed to suppress BCR-ABL expression FEBS Lett 1999 Nov 12;461(1-2):77-85).
  • non-human transgenic animals grown from germ cells transformed with a CF12 nucleic acid sequence according to the invention and that express the cofactor according to the invention and offspring and descendants thereof.
  • transgenic non-human mammals comprising a homologous recombination knockout of the native cofactors, as well as transgenic non-human mammals grown from germ cells transformed with nucleic acid antisense to the nucleic acids of the invention and offspring and descendants thereof.
  • non- human transgenic animals in which the native cofactor has been replaced with the human ortholog are also encompassed by the invention.
  • Transgenic animals according to the invention can be made using well known techniques with the nucleic acids disclosed herein.
  • Leder et al. U.S. Patent Nos.4,736,866 and 5,175,383; Hogan et al., Manipulating the Mouse Embryo, A Laboratory Manual (Cold Spring Harbor Laboratory (1986)); Capecchi, Science 244, 1288 (1989); Zimmer and Gruss, Nature 338, 150 (1989); Kuhn et al., Science 269, 1427 (1995); Katsuki et al., Science 241 , 593 (1988); Hasty et al., Nature 350, 243 (1991 ); Stacey et al., Mol. Cell Biol. 14, 1009 (1994); Hanks et al., Science 269, 679 (1995); and Marx, Science 269, 636 (1995).
  • Such transgenic animals are useful for screening for and determining the physiological effects of the cofactor agonists and antagonist.
  • transgenic animals are useful for developing drugs to regulate physiological activities in which the cofactors participate.
  • the amino acid sequences of the present invention can be used for structural drug design.
  • Aim is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g. agonists, antagonists or inhibitors) in order to design drugs which are, for example, more active or stable forms of the polypeptide, or which, for example, enhance or interfere with the function of a polypeptide in vivo.
  • one first determines the three-dimensional structure of a protein of interest, i.e. the cofactor, by computer-modeling, x-ray crystallography or a combination of both approaches.
  • Fig. 1 shows CF12 DNA sequence (SEQ.ID.No. 1), CF12 DNA complement sequence (SEQ.ID.No. 2) and CF12 protein sequence (SEQ.ID.No. 3)
  • Plasmids containing the desired coding and control sequences employs standard ligation and restriction techniques that are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.
  • Site-specific DNA cleavage is performed by treatment with the suitable restriction enzyme (or enzymes) under conditions that are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog.
  • plasmid and/or DNA sequence is cleaved by one unit of enzyme in about 20 ⁇ l of buffer solution. Often excess of restriction enzyme is used to ensure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37°C are workable, although variations are tolerable.
  • nucleic acid may be recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymology 65, 499-560 (1980).
  • Transformed host cells are cells which have been transformed or transfected with recombinant expression constructs made using recombinant DNA techniques and comprising cofactor encoding sequences.
  • Preferred host cells for transient transfection are COS-7 cells.
  • Transformed host cells may ordinarily express one of the cofactor CF12, but host cells transformed for purposes of cloning or amplifying nucleic acid hybridization probe DNA need not express the cofactors. When expressed, the cofactor proteins will typically be located in the host cell membrane.
  • Cultures of cells derived from multicellular organisms are desirable hosts for recombinant nuclear receptor protein synthesis.
  • any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture.
  • mammalian cells are preferred. Propagation of such cells in cell culture has become a routine procedure. See Tissue Culture (Academic Press, Kruse & Patterson, Eds., 1973).
  • useful host cell lines are bacteria cells, insect cells, yeast cells, human 293 cells, VERO and HeLa cells, LMTK- cells, and WI138, BHK, COS-7, CV, and MDCK cell lines. Human 293 cells are preferred.
  • EXAMPLE 2 COFACTOR CF12 OR TISSUE LOCALIZATION:
  • a multiple tissue northern blot (Clontech, Palo Alto) is hybridized to a labeled probe.
  • the blot contains about 0.3 to 3 ⁇ g of poly A RNA derived from various tissues.
  • Hybridization may be carried out in a hybridization solution such as one containing SSC (see Maniatis et al, ibid) at an optimized temperature between 50°c and 70°C, preferably 65°C.
  • the filter may be washed and a film exposed for signal detection (see also: Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.(1989)).
  • RNA is obtained from various tissues and used to prepare cDNA expression libraries by using for example an Invitrogen kit (Invitrogen Corporation, San Diego).
  • Invitrogen kit Invitrogen Corporation, San Diego.
  • the chosen library may be screened under stringent condition (see definitions above) by using CF12 specific probes.
  • the cDNA insert of positive clones is subsequently sequenced and cloned in a suitable expression vector.
  • full length cofactor clones from various species are obtained by using RACE PCR technology.
  • suitable cDNA libraries are constructed or purchased.
  • the first strand cDNA is used directly in RACE PCR reactions using a RACE cDNA amplification kit according to the manufactures protocol (Clontech, Palo Alto). Amplified fragments are purified, cloned and subsequently used for sequence analysis.
  • genomic libraries (Clontech, Palo Alto) are screened with a receptor specific probe under stringent conditions. Positive clones are isolated and the complete DNA sequence of the putative receptor is determined by sequence analysis (Maniatis et al., Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.(1989)).
  • EXAMPLE 4 DETECTION OF MUTANT ALLELES OF THE GENE ACCORDING TO THE INVENTION AND THEIR UTILISATION FOR DIAGNOSTIC PURPOSES.
  • alteration of the wild-type cofactor gene is detected.
  • the method can be performed by detecting the wild-type cofactor gene and confirming the lack of cause of the disease as a result of the locus.
  • “Alteration of the wild-type gene” encompasses all forms of mutations including deletions, insertions and point mutations in the coding and non-coding regions. Deletions may be of the entire gene or of only a portion. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues and are not inherited in the germline. Germline mutations can be found in any of a body's tissue and are mostly inherited. Point mutational events may occur in regulatory regions, such as the promotor of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the cofactor gene product or to a decrease in mRNA stability or translation efficiency.
  • Applicable diagnostic techniques include, but are not limited to fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single stranded conformation analysis (SSCA), RNAse protection assay, allele-specific oligonucleotide (ASO), dot blot analysis, hybridization using nucleic acid modified with gold nanoparticles and PCR-SSCP, as discussed in detail further below.
  • FISH fluorescent in situ hybridization
  • direct DNA sequencing PFGE analysis
  • Southern blot analysis single stranded conformation analysis
  • SSCA single stranded conformation analysis
  • ASO allele-specific oligonucleotide
  • dot blot analysis hybridization using nucleic acid modified with gold nanoparticles and PCR-SSCP, as discussed in detail further below.
  • DNA microchip technology can be applied.
  • the presence of a disease due to a germline mutation of a cofactor can be ascertained by testing any tissue of the diseased human for mutations of the cofactor gene. For instance, a person who has inherited a germline mutation in the cofactor gene, especially one that will alter the interaction of the cofactor with the PXR protein, will be prone to develop a disease.
  • the presence of such a mutation can be determined by extracting DNA from any tissue of the body. For example, blood can be drawn and DNA extracted from blood cells and analyzed.
  • prenatal diagnosis of the disease will be possible by testing fetal cells, piacental cells or amniotic cells for mutations in the cofactor gene.
  • direct genomic DNA Sequencing either manual or by automated means can detect sequence variations of cofactor genes (Nucleic Acids Res 1997 May 15;25(10):2032-2034 Direct DNA sequence determination from total genomic DNA. Kilger C, Paabo S, Biol. Chem. 1997 Feb; 378(2): 99- 105, Direct exponential amplification and sequencing (DEXAS) of genomic DNA. Kilger C, Paabo S, DE 19653439.9 and DE 19653494.1).
  • SSCP single-stranded conformation polymorphism assay
  • SNPs single nucleotide polymorphisms
  • SNPs in coding or regulatory regions of genes which are thought to contribute to a disease physiology can have a direct impact on the phenotype, e.g. change a quantitative readout of disease physiology, for example the age of onset of heart attack.
  • Association and linkage studies with related individuals therefore provide an excellent means to test or verify a hypothesis on the functional impact of the gene of interest on disease physiology in vivo, in humans.
  • DNA samples can be prepared from normal individuals and from persons being affected by the disease and these samples can be cut by one or more restriction enzymes and applied to Southern analysis. Southern blots displaying hybridizing fragments differing in length from the control DNA when probed with sequences near or including the cofactor locus could indicate a possible mutation. If large DNA fragments are used it is appropriate to separate these fragments by pulsed field gel electrophoresis (PFGE).
  • PFGE pulsed field gel electrophoresis
  • Detection of point mutations may be accomplished by amplification, for instance by PCR, from genomic or cDNA and sequencing of the amplified nucleic or by molecular cloning of the cofactor allele and sequencing the allele using techniques well known in the art.
  • restriction fragment length polymorphism (RFLP) probes for the gene or surrounding marker genes can be used to score for alteration of an allele or an insertion in a polymorphic fragment.
  • RFLP restriction fragment length polymorphism
  • SSCP detects a band which migrates differently because the variation causes a difference in single strand, intra molecular base pairing.
  • the RNAse protection assay involves cleavage of the mutant fragment into two or more smaller fragments. By using DGGE variations in the DNA can be detected by differences in the migration rates of mutant compared to normal alleles in a denaturing gradient gel. In the mutS assay, the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences.
  • Mismatches are hybridised nucleic acid duplexes in which the two strands are not 100% complementary. Lack of total homology may be due to deletions, insertions, inversions or substitutions. Mismatch detection can be used to detect point mutations in the gene or the corresponding mRNA product.
  • mismatch cleavage method is the RNAse protection assay.
  • the method involves the use of a labeled ribonucleotide probe which is complementary to the wild-type sequence of the cofactor gene coding sequence.
  • the riboprobe and either mRNA or DNA isolated from the person are hybridised together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by the enzyme, it cleaves at the site of the mismatch.
  • RNA product when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNAse A, an RNA product will be seen which is smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA. If the riboprobe comprises only a fragment of the mRNA or the gene, it is advantageous to use a number of probes to screen the whole mRNA sequence for mismatches.
  • DNA probes can be used to detect mismatch mutations through enzymatic or chemical cleavage (Cotton et al., PNAS 85, 4397 (1988); Shenk et al., PNAS 72, 989 (1975); Novack et al., PNAS 83, 586 (1986)).
  • mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to match duplexes (Cariello, Human Genetics 42, 726 (1988)).
  • the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridisation.
  • DNA sequences of the cofactor gene which have been amplified by PCR may also be screened using allele specific probes. These probes are nucleic acid oligomers, each of which contains a region of the gene sequence harboring a known mutation. For instance, one oligomer could be about 25 nucleotides in length corresponding to a portion of the gene sequence. By using a number of such allele-specific probes, PCR amplification products can be screened to identify the presence of a previously discovered mutation in the gene.
  • Hybridisation of allele-specific probes with amplified cofactor sequences can be performed, for example, on a nylon filter. Under high stringency hybridisation conditions, the hybridisation of a particular probe should indicate the presence of the same mutation in the tissue as in the allele-specific probe.
  • the newly developed technique of nucleic acid analysis via microchip technology is also applicable to the present invention.
  • this technique thousands of distinct nucleotide probes are built up in an array on a silicon chip.
  • Nucleic acid to be analysed is fluorescently labeled and hybridised to the probes on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips.
  • This technique one can determine the presence of mutations or even sequence the nucleic acid being analysed or one can measure expression of a gene of interest.
  • This method is one of parallel processing of thousands of probes at once and can tremendously accelerate the analysis.
  • the most definite test for mutations in a candidate locus is to directly compare genomic cofactor sequences from patients with those from normal individuals. Alternatively one could sequence mRNA after amplification (for example by PCR) thereby eliminating the necessity of determining the exon structure of the respective gene.
  • Mutations from patients falling outside the coding region of the cofactor gene can be detected by examining the non-coding regions, such as introns and regulatory sequences within or near the genes. Early indications of mutations in non-coding regions could be for example the abundance or abnormal size of mRNA products in patients as compared to control individuals as detected by northern blot analysis.
  • Alteration of cofactor expression can be detected by any techniques known in the art. These include northern blot analysis, PCR amplification and RNAse protection. Diminished mRNA expression indicates an alteration in the wild-type gene sequence. Alterations of wild-type genes can also be detected by screening for alteration of cofactor protein. For example, monoclonal antibodies against cofactor protein can be used to screen a tissue. Lack of cognate antigen would indicate a mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant gene product. These kind of immunological assays could be done in any convenient format known in the art. These include western blots, immunohistochemical assays and ELISA assays.
  • Any means for detecting an altered cofactor protein can be used to detect alteration of the wild-type cofactor gene.
  • Functional assays such as protein binding determinations can be used.
  • assays can be used which detect the cofactor's biochemical function. Finding a mutant cofactor gene product indicates an alteration of the cofactor wild- type gene.
  • Mutant cofactor gene or gene product or a mutant protein can also be detected in other human body samples, such as serum, stool, urine and sputum.
  • Other human body samples such as serum, stool, urine and sputum.
  • the same techniques discussed above for detection of mutant genes or gene products in tissues can be applied to other body samples. By screening such body samples, a simple early diagnosis can be achieved for the disease) resulting from a mutation in the cofactor gene.
  • EXAMPLE 5 ISOLATION OF THE NUCLEAR RECEPTOR BINDING PARTNER OF THE COFACTOR PROTEIN BY USE OF THE YEAST TWO-HYBRID SYSTEM
  • a yeast two-hybrid assay is performed using methods such as described by Fields and Song Nature 340, pp245 (1989), Bartel et al., Biotechniques 14, pp920 (1993) and Lee et al. Nature 374 pp91-4 (1995).
  • a sequence encoding amino acids according to SEQ ID NO. 3 of CF12 is cloned into the vector pGBT9 (Clontech) in such way that, after transformation of the haploid yeast strain CG1945 (Clontech), a hybrid protein is expressed consisting of the DNA-binding domain (BD) of the Gal4 transcription factor fused N-terminally to all amino acids according to SEQ ID NO. 3 or parts thereof. .
  • CG1945 cells expressing the Gal4BD::PXR fusion protein are mated to cells of strain Y187 (Clontech) containing a library of Gal4 transcription activation domain (AD) fusion plasmids with human cDNA generated from a range of tissues, preferentially also testis tissue, inserted into the vector pACT2 (Clontech).
  • Libraries are preferentially purchased from Clontech Laboratories (MATCHMAKER human cDNA libraries) and include Cat.
  • HL4040AH (aorta), HL4041AH (chondrocytes), HY4004AH (brain), preferentially also HY4035AH (testis), HY4024AH (liver), HY4042AH (heart), HY4053AH (bone marrow) and HY4028AH (fetal brain).
  • the two-hybrid screens are essentially performed following the Clontech "Pretransformed Matchmaker Libraries User Manual" (PT3183-1 ): Transformed CG1945 and Y187 cells are mated in order to co-express the Gal4::PXR fusion protein and the Gal4AD fusion proteins encoded on the library plasmids within one cell. Interaction of the two hybrid proteins leads to activation of reporter gene transcription.
  • Cells are selected for interactions of CF12 with library proteins on medium lacking tryptophane, leucine and histidine and are further assayed for expression of ⁇ -galactosidase, encoded by the MEL1 reporter gene. Colonies that are positive for reporter gene activation are chosen for further analysis.
  • the DNA inserts of the library plasmids contained in these colonies are amplified by use of the polymerase chain reaction directly on the yeast colonies using oligonucleotide primer which hybridize on vector sequences flanking both sides of the insert. The identity of the insert is determined by standard DNA sequencing techniques.
  • EXAMPLE 6 A CELL BASED ASSAY FOR MEASURING THE BINDING OF THE COFACTOR TO THE NUCLEAR RECEPTOR.
  • the DNA sequence encoding the open reading frame of the cofactor (CF12) is transferred into the vector pVP16 (Clontech) to allow the expression of a fusion protein of the cofactor with the strong transactivation domain of the VP16 protein (of herpex simplex virus) in mammalian cells under the control of the strong CMV promoter.
  • the reporter the luciferase gene is cloned under the control of a minimal promoter containing a nuclear receptor-responsive DNA element.
  • This vector also expresses a second enzyme, e.g: beta-galactosidase, under the control of a constitutive promoter, to allow normalisation for transfection efficiency between experiments.
  • a third vector contains the nuclear receptor gene under the control of the strong CMV promoter.
  • CV-1 cells are then transiently transfected with different combinations of the three plasmids.
  • Transfection is done by standard methods, e.g. by use of the CalPhos Maximizer (Clontech, #8021-1 ,-2).
  • Interaction of the cofactor protein with the nuclear receptor will lead to a strong transactivation due to the attached VP16 domain of the cofactor fusion protein.
  • interaction of the cofactor with the nuclear receptor will result in increased luciferase activity.
  • inclusion of the cofactor VP16 will result in increased luciferase activity as compared to transfection of the nuclear receptor and the reporter alone.
  • extracts are prepared of the transfected cells 48 to 72 hours after transfection, and luciferase activity is determined.
  • beta- galactosidase activity is also determined.
  • Addition of substances known or suspected to influence the binding of the nuclear receptor to the cofactor CF12 respectively are added to the medium of the transfected cells. These substances are added at different time points prior to cell lysis, typically ranging between 18 hours to a five minutes before cell lysis. Luciferase activity is taken as a measure of the effect of these substances on the binding of the cofactor to the nuclear receptor. To avoid activation of the nuclear receptor by substances contained in the serum of the medium, charcoal-stripped serum has to be used for these experiments.
  • the DNA-binding domain of the nuclear receptor is replaced with the DNA-binding domain of the yeast GAL4 transcription factor.
  • the luciferase is expressed under the control of GAL4-responsive upstream activating sequences. Expression of luciferase again is an indication for binding of the cofactor CF7-VP16 fusion or CF8-VP16 fusion to the nuclear receptor-GAL4 fusion.
  • This setting is also referred to as the mammalian two hybrid system. A description of the experiment is found in the manual to the Mammalian MATCHMAKER Two-Hybrid Assay Kit from Clontech, # PT3002-1, catalogue #K1602-1.
  • the plasmid directing expression of the VP16-cofactor chimeric protein is replaced by a plasmid directing the expression of the native cofactor protein.
  • the native cofactor protein has an effect on the transcriptional activity of the nuclear receptor. This effect can then be measured by measuring the amount of luciferase produced. This effect can be both positive, leading to more luciferase produced, or negative, leading to the expression of less luciferase protein.
  • Substances activating nuclear receptors cause an exchange of the proteins bound to the. receptors, thus effecting the dissociation of some proteins and promoting the binding of other proteins.
  • nuclear receptor-activating compounds and nuclear receptor- inactivating compounds by monitoring the binding of the CF12 cofactor to the nuclear receptor.
  • stably transfected cell lines which contain copies of the two different expression constructs for the nuclear receptor and the CF12 cofactor as well as the reporter construct stably integrated into the chromosomes of the cells.
  • DNA sequences encoding the open reading frame of the cofactor and the nuclear receptor gene are each transferred separately into the vector pENTRY (Life Technologies) to allow efficient construction of a diverse set of expression constructs.
  • the open reading frame is then recombined into the vector pDEST17 for expression in E. coli strain BL21 as a fusion protein to a six-histidine tag induced by IPTG, as well as into the pDEST15 for expression as a fusion protein with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • the plasmids pDEST15, pDEST17and pENTRY are purchased from LIFE TECHNOLOGIES.
  • the open reading frame is introduced into the vector pLV-CBDgw for expression as a fusion protein with the calmodulin binding protein using recombinant baculoviruses as specified by the manufacturer (Becton Dickinson).
  • pLV-CBDgw is a derivative of the vector pLV1392 (Becton Dickinson) which is modified by the insertion of a calmodulin binding protein fragment, followed by the sequence required for recombinational cloning via the Gateway system (Life Technologies). Protein expression is induced and recombinant protein is purified by passage over a Ni- NTA-column, or a glutathione column or a calmodulin column, respectively.
  • a biotinylated (Biotintag Micro biotinylation Kit, Sigma) His-tagged nuclear receptor protein and the GST fusion of the CF12 cofactor are mixed at 0.2-5 ⁇ M.
  • Antibody to the GST protein is added which is labelled by the europium chelate at a concentration of 1-3 (typical 2.5) nM.
  • Streptavidin which is fluorescently labeled by covalent attachment of allophycocyanin is added at a concentration of 5-30 ⁇ g/ml (typical 10 ⁇ g/ml).
  • the europium chelate is stimulated by a flash of light (320nm) and, the emitted light is measured in a delayed (50-200 ⁇ s) time window for 300 to 600 ⁇ s after the flash at 615 nm (fluorescence of europium chelate) and 655nm (fluorescence of APC). Since APC is only excited by the light emitted by the europium chelate, a close proximity of the two different fluorophores is required for excitation. The strength of the APC signal, as well as the ratio of the signals from the two fluorophores (i.e. the ratio of the intensities of light emitted at 655 and 615nm) serves as a measure for the interaction of the two proteins.
  • Reaction buffers contain 20mM TrisHCI pH 7.9, 60mM KCI, 4mM MgCI 2 . Reaction volume is 25 ⁇ l.
  • the Wallac VictorV fluorimeter is used for the fluorimetric measurements.
  • the cofactor is used as a biotinylated His-tagged protein, and the nuclear receptor protein is used as fusion to GST.
  • the His-tagged proteins are replaced by the same proteins fused to the calmodulin binding protein. In the latter case, the detection of the interaction is via biotinylated calmodulin, which is in turn binding to APC-coupled streptavidin. Calcium has to be included in the buffer in the form of 4mM CaCI 2 , to allow complex formation between calmodulin and the calmodulin binding protein.
  • 293T cells grown on glass coverslips are transfected with 20 ⁇ g of vector containing hemagglutinin (HA)-tagged CF7/CF8 by the calcium phosphate precipitation method. Two days posttransfection, the cells are fixed for 30 min in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 1% nonfat dry milk in PBS. Cells are incubated with a 1 :75 dilution of anti-HA (monoclonal 12CA5) and anti-CF12 (polyclonal rabbit serum raised against GST- CF12) ; antibodies for 1 h in a humid chamber at 37°C.
  • anti-HA monoclonal 12CA5
  • anti-CF12 polyclonal rabbit serum raised against GST- CF12
  • rhodamine conjugated donkey anti-rabbit and fluorescein-conjugated donkey anti- mouse secondary antibodies are applied at 1 :200 dilution for 1 h at 37°C Cells are washed in PBS and mounted prior to photography.
  • C2C12 cells are harvested on days 0-4 of differentiation, and equal quantities of protein are separated by SDS-PAGE and transferred to nitrocellulose. Immunoblotting is performed with polyclonal anti-CF12 antibody at a 1 :400 dilution followed by detection with the ECL reagent system (Amersham). Immunoprecipitation assays are performed in HeLa, 293, and CV-1 cells transfected with both CF12 protein and RevErb (10 ⁇ g per 150-mm plate) or CF12. alone and lysed with NETN buffer (300 mM NaCI/0.5% Nonidet P-40) containing protease inhibitors.
  • NETN buffer 300 mM NaCI/0.5% Nonidet P-40
  • Immunoprecipitation reactions are then incubated with protein-A/G agarosebeads (Santa Cruz Biotechnology) for 1 h at 4°C. Samples are washed with NETN buffer, boiled, separated by SDS-PAGE, and transferred to nitrocellulose. Immunoblotting is performed with anti-HA antibody at 1 :500 dilution or anti-RevErb monoclonal antibody at 1 :400 followed by detection by ECL reagent.
  • GST fusion proteins are prepared as previously described in Zamir, I., Harding, H. P., Atkins, G. B., Horlein, A., Glass, C. K., Rosenfeld, M. G. & Lazar, M. A. (1996) Mol. Cell. Biol. 16, 5458-5465.
  • GST beads (50 ⁇ l) containing the fusion protein are incubated at room temperature in a buffer containing 50 mM KCI, 20 mM Hepes, pH 7.9, 2 mM EDTA, 0.1% Nonidet P-40, 10% glycerol, 0.5% nonfat dry milk, and 5 mM DTT.

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Abstract

L'invention concerne un nouveau cofacteur de récepteur nucléaire mammalien, dit CF12, ses séquences d'acides nucléiques isolées et ses protéines isolées. L'invention concerne également des procédés permettant d'isoler et/ou de produire ces acides nucléiques ou ces protéines, ainsi que des procédés d'utilisation dudit cofacteur, notamment pour inhiber ou activer sa liaison avec un récepteur nucléaire mammalien.
PCT/EP2001/015135 2000-12-30 2001-12-21 Cofacteur de recepteur nucleaire mammalien cf12 et ses procedes d'utilisation WO2002053591A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005090569A1 (fr) * 2004-03-24 2005-09-29 The Council Of The Queensland Institute Of Medical Research Cancer et acides nucleiques et proteines de testicule vsm1 et vsm2, et leurs utilisations

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2001055320A2 (fr) * 2000-01-31 2001-08-02 Human Genome Sciences, Inc. Acides nucleiques, proteines et anticorps
WO2002002610A2 (fr) * 2000-06-29 2002-01-10 Incyte Genomics, Inc. Molecules de circulation et de secretion

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WO2001055320A2 (fr) * 2000-01-31 2001-08-02 Human Genome Sciences, Inc. Acides nucleiques, proteines et anticorps
WO2002002610A2 (fr) * 2000-06-29 2002-01-10 Incyte Genomics, Inc. Molecules de circulation et de secretion

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Publication number Priority date Publication date Assignee Title
WO2005090569A1 (fr) * 2004-03-24 2005-09-29 The Council Of The Queensland Institute Of Medical Research Cancer et acides nucleiques et proteines de testicule vsm1 et vsm2, et leurs utilisations

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