Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/136—Screening for pharmacological compounds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the present invention relates to the field of diagnosis, prevention and treatment of obesity for weight loss, associated or not with hormonal disorders and also with diabetes in humans or animals.
• the invention aims more particularly to offer a new method of screening for compounds useful for treating or preventing obesity or weight loss, or diabetes in a human or animal subject, based on the use of the genes LEPROTLl and OB- RGRP and the trafficking of proteins encoded by these genes, respectively endospanine and OB-RGRP.
• OB-RGRP leptin receptor gene related protein
• the subject of the present invention is therefore a method of identifying compounds active on weight gain or loss or diabetes in a human or animal subject, consisting in measuring the effect of these compounds on (a) the expression of at least one of the LEPROTL1 and OB-RGRP genes or part thereof and / or (b) intracellular transport to the cell membrane and / or (c) the presence at the cell membrane of proteins and / or (d) the internalization from the membrane of proteins encoded by at least one of said genes or part of them.
• the method of the invention comprises (i) bringing a test compound into contact with cells expressing at least one of the LEPROTL1 and OB-RGRP genes or fragments thereof, then (ii) measuring the quantity of proteins encoded by at least one of said genes or part of these present at the membrane of said cells and / or their internalization.
• endospanine and / or OB-RGRP or part of these are marked so as to be able to be detected and thus carry out the measurement of the method of the invention.
• Any type of labeling can be envisaged in the context of the method of the invention, but labeling of the peptide tag type is preferred.
• the method of the invention can be implemented in vitro on a model of cells in culture or in vivo in an animal model.
• the method of the invention can also include measuring the expression and / or transport advantageously to the surface of the membrane of the OB-R cells, or even in the case of an animal model, measuring circulating leptin.
• the cellular or animal models used in the context of the method of the invention may be of the type either expressing LEPROTL1 and / or OB-RGRP or fragments thereof endogenously or genetically modified to express said LEPROTL1 and / or OB-RGRP or fragments thereof in the form of recombinant proteins.
• a human model such as Hela, 293 or HepG2 or murine such as 3T3 may be mentioned, primary cultures could also be used. Any type of cell can be used because most lines and tissues express endospanine and OB-RGRP.
• Examples of animal models naturally expressing LEPROTL1 and / or OB-RGRP include the mouse, the rat and the rabbit. However, an implementation of the invention is preferred on cells transformed to express all or part of one or less of the genes, LEPROTL1 and / or OB-RGRP. They are advantageously cells in culture, but the invention can also be implemented on animals obtained by transgenesis according to techniques described in the literature from LEPROTL1 and / or OB-RGRP or fragments thereof.
• Preferred for the implementation of the invention are cells in culture which have been genetically modified to express at least one of LEPROTL 1 and / or OB-RGRP or fragments thereof.
• sequences of the cDNA of human LEPROTL1 and of human and mouse endospanin are given in the sequence list in the appendix respectively under the numbers SEQ ID N0.1, SEQ ID NO.2 and SEQ ID NO.3.
• sequences of the human OB-RGRP cDNA and of the human and murine protein are given in the sequence list in the appendix respectively under the numbers SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
• SEQ ID NO.4 The sequences of the human OB-RGRP cDNA and of the human and murine protein are given in the sequence list in the appendix respectively under the numbers SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
• These genetically modified cells are prepared by techniques well known to a person skilled in the art using expression vectors.
• They can also be fragments or sequences modified by the addition, deletion or change of one or more nucleotides, of these genes when the protein encoded by its fragments or modified sequences can be transported to the cell membrane and advantageously be then internalized.
• a preferred form of the invention is to express a modified form of LEPROTL1, OB-RGRP or fragments thereof so as to produce labeled proteins.
• the labeling advantageously consists of a peptide tag.
• modified form of these genes is also understood to mean genes coding for truncated proteins, that is to say having a deletion of one or more amino acids, useful in particular for peptide labeling.
• endospanin and / or OB-RGRP comprise four transmembrane domains.
• a fragment and a modified form of LEPROTL1 or OB-RGRP codes for a modified protein comprising at least two of the four transmembrane domains and a peptide marker in the form of fusion proteins.
• endospanine and / or OB-RGRP marker or fragments thereof may be the same or different.
• peptide markers generally used, there may be mentioned:
• HA of sequence YPYDVPDYA (SEQ ID NO. 7) c-myc: sequence EQKLISEEDL (SEQ ID NO. 8) VSV-G t sequence YTDIEMNRLGK (SEQ ID NO. 9) His 6 ⁇ sequence HHHHHH (SEQ ID NO. 10) FLAG s sequence DYKDDDDK (SEQ ID 10. 11)
• the insertion of a peptide marker into the complete protein can be carried out at any level of the protein or fragment thereof as long as it does not modify the expression of the protein at the cell membrane and advantageously also its internalization.
• the reading frame must be integrated into an expression vector of the type, plasmid (pCI / pCIneo, Promega, pcDNA3, Invitrogen) or virus (adeno, retro, vaccinia) for expressions either transient or stable.
• plasmid pCI / pCIneo, Promega, pcDNA3, Invitrogen
• virus adeno, retro, vaccinia
• endospanine and / or OB-RGRP or part of these can be labeled and labeling of the peptide tag type is preferred.
• the measurement can then be carried out using an antibody directed against the peptide marker, integrated for example in one of the two lumenal / extracellular loops of endospanine, OB-RGRP or fragments thereof or in extension of these loops in the truncated forms of proteins, by bringing the antibody into contact with the cells.
• the method of the invention can also be implemented on unlabelled endospanin and / or OB-RGRP.
• Antibodies directed against one of the two lumen / extracellular loops of endospanine or of OB-RGRP make it possible to detect the endogenous protein by contacting the cells.
• the position of the polypeptides for obtaining these specific antibodies, preferably monoclonals, to identify endogenous endospanine and OB-RGRP at the membrane level are located between the amino acids in positions 25 to 35 and the amino acids in positions 82 to 100 of SEQ ID NO. 2 and SEQ ID NO.5.
• the measurement can then include the implementation of a secondary antibody according to techniques well known to one skilled in the art.
• - ELISA secondary coupled to peroxidase, alkaline phosphatase or another enzyme, quantifiable and automatable colored reaction, - Western Blot,
• Immunofluorescence fluorescent secondary antibody useful for measuring morphology.
• the effect of the compound to be tested according to the method of the invention can be achieved by comparing measurements on cells with and without contacting said compound. This effect can be measured after or during a determined time of bringing the cells into contact with said compound.
• the specificity of the compound to be tested with respect to a) the expression of at least one of the LEPROTL1 and OB-RGRP genes and / or (b) intracellular transport to the cell membrane e / or (c ) the presence at the cellular membrane of proteins and / or (d) internalization from the membrane, proteins encoded by at least one of said genes or part of these can be determined by measuring the effect of the treatment on other genes or proteins.
• the effect of the compound to be tested on the intracellular trafficking of endospanine and / or OB-RGRP is also measured using an antibody directed against any membrane protein surface, a part or a marker thereof.
• transferin receptor As a preferred example, mention may be made of the transferin receptor.
• the invention also relates to a pharmaceutical composition for the prevention or treatment of weight gain or loss or of diabetes comprising at least one compound capable of modifying the expression of at least one of the LEPROTL1 and OB-RGRP genes or part of these, and / or (b) intracellular transport to the cell membrane, e / or (c) the presence at the level of the cell membrane, e / or (d) internalization from the membrane, proteins encoded by at least one of said genes or part thereof.
• a compound is advantageously an antagonist or an agonist of at least one of the proteins endospanine and OB-RGRP.
• the invention also relates to the diagnosis of weight gain or loss or of diabetes in a human or animal subject consisting in measuring and advantageously in comparing with at least one control, the expression of at least one of the LEPROTL1 and OB genes -RGRP or part thereof, and / or (b) intracellular transport to the cell membrane, e / or (c) presence at the level of the cell membrane, and / or (d) internalization from the membrane, proteins encoded by at least one of said genes or part thereof.
• FIG. 1 represents the cDNA sequence of the LEPROTL1 gene where the reading frame is underlined.
• FIG. 2 represents the alignment of the sequences of endospanine and of human and murine OB-RGRP. The four transmembrane domains of these proteins are indicated in line 1.
• FIG. 3 represents HeLa cells, in double labeling in indirect immunofluorescence, expressing 1 endospanine-HA-B1, incubated in the presence of anti-HA mouse antibodies. Green fluorescence reveals internalized anti-HA antibodies (A and C). The red fluorescence reveals the endospanine, -HA-B1 and endogenous, in these cells (B and C).
• FIG. 4 represents the quantity of leptin receptor on the surface relative to the total quantity of cellular leptin receptor and normalized to 100% for control, of HeLa cells co-infected with two recombinant adenoviruses in order to express a constant quantity of OBRa and increasing amounts of endospanin (multiplicity of infection from 0 to 80).
• FIG. 5 represents the expression of the mRNA of OB-RGRP (left) and LEPROTL1 (right) in the MCF7 line under treatment with estradiol and progesterone by real-time PCR.
• Figure 6 shows the distribution and intensity of expression of mRNAs, OB-RGRP (left) and LEPROTL1 (right) in the brain and fetus of mice.
• FIG. 7 shows that the overexpression of the protein OB-RGRP induces a dose-dependent decrease in the expression on the surface of the leptin receptor.
• Example 1 Transport of a labeled version of endospanin from the membrane to the cytoplasm
• HA epitope (or 'tag') (9 amino acids t PYDVPDYAY) was introduced by site-directed mutagenesis in the first extracellular loop (B1) of endospanine, between residues Y30 and N31 ( Figure 1 and 2). Binding residues have been included on each side of the tag (GAS and GA, respectively).
• HeLa cells previously cultivated on glass slides were transfected with a plasmid allowing the expression of the protein thus labeled; endospanin- HA-B1. Twenty-four hours after transfection, the cells were incubated for two hours in culture medium containing an anti-HA monoclonal antibody. The cells were then rinsed and then incubated in culture medium devoid of antibodies.
• the cells were fixed in a 3% paraformaldehyde solution.
• the internalized anti-HA antibody was revealed using an anti-mouse IgG antibody labeled with alexa-488, after prior permeabilization of the cells by incubation in a solution containing triton X-100 at 0 , 1%.
• Endospanin was revealed using an anti-endospanin rabbit antiserum against the C-terminal dodecapeptide and by an anti-rabbit IgG antibody labeled with aexa-594. The preparations were observed on an immunofluorescence microscope.
• Example 2 Modulation of expression of the leptin receptor on the surface of cells by endospanine. 1) Methods.
• OB-Ra short isoform of murine OB-R
• human endospanin were constructed by homologous recombination.
• OB-R expressed by this viral vector has an HA epitope at its N-terminal end.
• HeLa cells were co-infected with the receptor expressing virus and increasing doses of endospanin expressing virus. After 24 hours of expression, the surface proteins were biotinylated at 4 ° C. The cells were then lysed and the cell lysates clarified by centrifugation.
• the relative levels of biotinylated receptors were quantified by immunoblot from the material purified by streptavidin-agarose beads and revelation by immunoblot after separation of the proteins by electrophoresis in polyacrylamide gel.
• the total receptors (surface + internal) were quantified in parallel by the same method from a tenth of each cell lysate.
• OB-Ra was revealed with an anti-HA antibody.
• the same immunoblots were then revealed thanks to an antibody directed against the transferrin receptor.
• the quantities of surface receptors were expressed by the ratio of the quantity of receptor in the biotinylated fraction to the quantity of receptor present in the total cell lysates.
• OB-R Surface expression of OB-R is affected by overexpression of endospanine.
• the overexpression of endospanine induces a dose-dependent decrease in the surface expression of OB-R (FIG. 4) without any impact on the surface expression of the transferrin receptor.
• G ⁇ PDH was studied by quantitative real-time PCR on the LightCycler device (Roche) in the human breast cancer line MCF7 whether or not subjected to treatment with either progesterone or estradiol.
• the total RNAs were prepared using the kits: QIAGEN, RNeasy®.
• the cDNA (2 ⁇ g of RNA in a volume of 25 ⁇ L), are obtained using the Reverse Transcriptase
• the oligonucleotides for real-time PCR are as follows.
• Example 4 Expression of LEPROTL1 and OB-RGRP in the brain and fetus of mice.
• LEPROTL1 mRNA
• MRNA OB-RGRP
• ACGAATTCACCGCCATGGCAGGCATC (SEQ ID NO. 20) and ACTCTAGACCACTGCTGCCAGCTGAAG (SEQ ID NO. 21).
• the two probes were hybridized on 20 ⁇ m adjacent coronal sections of the mouse brain at the level of the hypothalamus (A; bregma 0.82mm B; bregma 1.46mm) or 13.5-day-old fetus p.c. with the placenta (C).
• the auto-radiographic signals generated by in situ hybridization with the LEPROTL 1 probe are more intense than those observed with the OB-RGRP probe, at the same level of radioactivity and the same specific radioactivity.
• the expression of LEPROTL1 is similar in distribution and in abundance relative to the expression of OB-RGRP ( Figure 6).
• the two, OB-RGRP and LEPROTLl antisense probes hybridize with a defined number of structures, with a weak signal, widely distributed.
• the sense probe shows a weak and uniform signal (data not shown). Both mRNAs are widely expressed in the hippocampus, including the dentate gyrus (DG).
• DG dentate gyrus
• CP choroid plexus
• PVN paraventricular
• ARC arched
• VMH ventromedial
• LH lateral hypothalamus
• LEPROTL1 and OB-RGRP probes show a similar hybridization profile in the fetus and placenta sections. In embryonic tissues, LEPROTL1 is most strongly expressed in the developing brain and lungs, where signal enrichment is observed for the OB-RGRP probe.
• Example 5 Modulation of the expression of the leptin receptor on the surface of cells by the protein OB-RGRP.
• adenoviruses expressing a short isoform (oB-Ra) of the murine leptin receptor, on the one hand, and the human OB-RGRP protein were constructed by homologous recombination.
• the leptin receptor expressed by this viral vector has an HA epitope at its end N-terminal.
• HeLa cells were co-infected with the virus expressing the receptor and increasing doses of virus expressing the protein OB-RGRP.
• the surface expression of the leptin receptor was quantified by biotinylation of the surface proteins, lysis of the cells, precipitation of the biotinylated material by streptavidin-agarose beads and revelation by immunoblot after separation of the proteins by electrophoresis in polyacrylamide gel.
• the leptin receptor was revealed with an anti-HA antibody.
• a portion of the cell lysate was also subjected to the same analysis in order to determine the total quantity of receptors (surface + internal).
• the amounts of surface receptor were expressed by the ratio of the amount of protein in the biotinylated fraction to the amount of protein present in the total cell lysates.

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