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WO2013022262A2 - Récepteur de résistine humaine, et utilisation de celui-ci - Google Patents

Récepteur de résistine humaine, et utilisation de celui-ci Download PDF

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Publication number
WO2013022262A2
WO2013022262A2 PCT/KR2012/006269 KR2012006269W WO2013022262A2 WO 2013022262 A2 WO2013022262 A2 WO 2013022262A2 KR 2012006269 W KR2012006269 W KR 2012006269W WO 2013022262 A2 WO2013022262 A2 WO 2013022262A2
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human
lystine
protein
mfc
receptor
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Korean (ko)
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WO2013022262A3 (fr
WO2013022262A9 (fr
Inventor
박영배
김효수
권유욱
이사민
이현채
조영진
이상언
정준호
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Seoul National University Hospital
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Seoul National University Hospital
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Priority claimed from KR1020120086475A external-priority patent/KR101473526B1/ko
Publication of WO2013022262A2 publication Critical patent/WO2013022262A2/fr
Publication of WO2013022262A9 publication Critical patent/WO2013022262A9/fr
Publication of WO2013022262A3 publication Critical patent/WO2013022262A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to human lystine receptors. More specifically, a method of searching for a receptor of human ridgestin protein, a method of preventing or treating inflammatory disease and atherosclerosis using a modulator of expression or activity of a human ridgestin receptor, a pharmaceutical comprising a modulator of expression or activity of a human ridgestin receptor It relates to a composition.
  • Resistin is a cytokine first identified as a mediator of inducing insulin resistance in obese mice. It belongs to cysteine-rich proteins, also known as resistin-like molecules (RELMs), and is involved in the regulation of the inflammatory process. In addition, rat murine resistin is known to be involved in the development of obesity-mediated insulin resistance and type 2 diabetes.
  • RELMs resistin-like molecules
  • PBMC peripheral blood mononuclear cells
  • Human lystine is thought to be associated with the influx of other immune cells and the secretion of pro-inflammatory factors, and there is continuous evidence that it is associated with inflammatory diseases and atherosclerosis, with or without insulin resistance.
  • Lysistine which is present in both atherosclerotic lesions in mice and humans, is known to be an inflammatory marker of atherosclerosis in humans and is known to promote atherosclerosis by activating monocytes. Thus, human lystine is thought to be a key factor in controlling monocytes leading to the development of atherosclerosis.
  • NF- ⁇ B nuclear factor kappa B
  • the technical problem to be achieved by the present invention is to provide a method for the detection of human ridgestin receptor.
  • the present invention provides a method for preventing or treating inflammatory diseases and atherosclerosis by regulating the activity of a human lystine receptor detected by the above method, a pharmaceutical composition comprising an agent for regulating the expression or activity of a human zitin receptor.
  • the present invention comprises the steps of: a) producing a recombinant vector by cloning the expression vector mFc-human lystine recombinant DNA; b) transducing said recombinant vector into a cell line to express mFc human ridgetin fusion protein; c) culturing the expressed mFc human ridgestin fusion protein with the cells to form a complex of human ridgestin receptor; d) separating the human ridgestin receptor from the precipitate after immunoprecipitation of the complex; And e) identifying the isolated human ridgestin receptor.
  • the present invention comprises the steps of: a) producing a recombinant vector by cloning the expression vector of mFc-human ridgestin recombinant DNA, which binds the mFc gene to the N-terminal of the human ridgestin gene; b) transfecting said recombinant vector into HEK293F cells to express mFc human ridgetin fusion protein; c) purifying the expressed mFc human ridgestin fusion protein; d) incubating the purified mFc human lystine fusion protein with THP-1 cells (Human acute monocytic leukemia cells) to form a complex of human lystine receptors; e) immunoprecipitating the complex with mFc specific beads to obtain a precipitate; f) isolating human lystine receptors corresponding to 55 kDa size from the precipitate; And g) provides a method for searching for a human ridge
  • the present invention provides a method for treating or preventing a disease by administering to the subject a modulator of expression or activity of a human ridgestin protein receptor detected by the method.
  • the present invention is a method for screening a therapeutic agent for inflammatory diseases or atherosclerosis comprising the step of searching for a substance that modulates the expression or activity of the human ridgestin protein receptor discovered by the above method, wherein the human ridgestin protein receptor is CAP1 (Adenylyl It provides a method characterized in that the cyclase-associated protein 1).
  • the present invention also provides a pharmaceutical composition for the treatment and / or prophylaxis of inflammatory diseases or atherosclerosis comprising an agent for modulating the expression or activity of a human ridgestin protein receptor detected by the above method.
  • Figure 1 shows the structure and expression vector of recombinant DNA encoding the mFc-human lystine protein.
  • Figure 2 shows the Westin blot and Coomassie staining results for identifying the expressed mFc-human ridgetin protein.
  • Figure 3 shows the Westin blot results of protein complexes bound to mFc-human lysistine protein in THP-1, HUVEC, VSMC.
  • Figure 4 shows the results of an immunoprecipitation pulldown assay of THP-1 cell extracts and lystine for cloning human lystine receptors.
  • 5 is a conventional PCR and realtime PCR results showing the expression level of CAP1 in rabbit tissue cells.
  • Figure 6 shows the Western blot results and immunofluorescence staining showing the location of the CAP1 protein in the cells.
  • FIG. 7 shows flow cytometry results of THP-1 cells labeled with monoclonal antibodies of human CAP1.
  • Figure 8 shows the dual immunofluorescence results showing that the ligstine and CAP1 is in the same position.
  • Figure 9 shows the results of co-immunoprecipitation of human ligstin and CAP1.
  • Figure 10 shows the far western blotting results of human CAP1 and lystine.
  • FIG. 11 shows the results of an ELISA experiment to measure the competitive binding of mFc-human lystine protein and recombinant lystine to recombinant CAP1.
  • Figure 12 shows the results of FACS experiments to confirm the effect of recombinant CAP1 protein on the integrin- ⁇ 1 expression of lystine.
  • FIG. 13 is a schematic showing three major domains of human CAP1.
  • Figure 14 shows a comparison of the sequences of Thermus thermophilus HB8 (2PX7) and CAP1.
  • FIG. 15 is a final estimate of the poly-proline SH3 binding domain of CAP1 identified using Discovery Studio 2.5 (Accelrys Inc.).
  • 16 is a schematic diagram showing the structure of three deletion mutant genes of human CAP1.
  • FIG. 17 is a diagram showing relative mRNA expression levels of inflammatory cytokines induced by lystine after overexpressing the CAP1 mutant gene of FIG. 17 in THP-1 cells.
  • FIG. 17 is a diagram showing relative mRNA expression levels of inflammatory cytokines induced by lystine after overexpressing the CAP1 mutant gene of FIG. 17 in THP-1 cells.
  • Figure 18 shows the results of in vitro binding assays between rhResistin when each CAP1 mutant gene is expressed.
  • Figure 19 shows the change in the concentration of cAMP following the treatment with listerin.
  • Figure 20 shows the changes in the activity of PKA and NK- ⁇ B according to the treatment of listine.
  • FIG. 21 shows that the expression of inflammatory cytokines is increased following the treatment with lystine.
  • FIG. 22 shows the results of non-reducing SDS gel analysis of media using HUVEC lysates infected with adenovirus ridgestin.
  • Figure 23 is an experimental result showing the effect of the PKA inhibitors on lystine in the cytoplasm and cell nucleus.
  • FIG. 24 shows experimental results showing the effect of PKA inhibitors that inhibit the expression of cytokines induced by lystine.
  • 25 shows the effect of inhibiting CAP1 expression using CAP1 siRNA in THP-1 cells.
  • Figure 26 shows the change in cAMP concentration according to the inhibition of CAP1 expression.
  • Figure 27 shows the inhibition of PKA and NF- ⁇ B activity in accordance with the inhibition of CAP1 expression.
  • Figure 28 shows the inhibition of the expression of inflammatory cytokines according to the inhibition of CAP1 expression.
  • 29 shows overexpression of CAP1 by adenovirus.
  • Figure 30 shows the change in cAMP concentration according to CAP1 overexpression.
  • Figure 31 shows the increase in the activity of PKA and NF- ⁇ B according to CAP1 overexpression.
  • Figure 33 is a schematic diagram showing the pathway by which CAP1 acts as a functional receptor of lizithine.
  • 34 is a schematic diagram illustrating a transwell migration assay method.
  • FIG. 36 shows the results of transwell migration assay that monocytes overexpressing CAP1 significantly increased their migration toward lystine than monocytes that decreased CAP1 expression.
  • FIG. 37 shows a result of a vertical collagen gel invasion assay showing that monocytes overexpressing CAP1 significantly increased migration and migration through the collagen layer toward ligistine than monocytes with reduced CAP1 expression.
  • Figure 38 is a graph of the results of vertical collagen gel invasion assay showing that the degree of migration toward lystine was significantly increased than monocytes that reduced CAP1 expression.
  • Figure 44 is a schematic diagram showing that adenylyl cyclase-associated protein 1 (CAP1) acts as a receptor for human lystine.
  • CAP1 adenylyl cyclase-associated protein 1
  • CAP1 mediates intracellular signal transduction and induction of biological effects in adipose tissue inflammation.
  • CAP1 Addenylyl cyclase-associated protein 1
  • the present invention provides a method for searching for a human lystine protein receptor, comprising the steps of: a) constructing a recombinant vector by cloning mFc-human lystine recombinant DNA into an expression vector; b) transducing said recombinant vector into a cell line to express mFc human ridgetin fusion protein; c) culturing the expressed mFc human ridgestin fusion protein with the cells to form a complex with the human ridgetine receptor; d) separating the human ridgestin receptor from the precipitate after immunoprecipitation of the complex; And e) identifying the isolated human ridgestin receptor.
  • the mFc-human lystine recombinant DNA is capable of binding the mFc gene to the N-terminal human lystine gene
  • the expression vector includes but is not limited to pcDNA3.1.
  • the cell line of step b) may include HEK293F cells, but is not limited thereto.
  • the method of the present invention may further comprise the step of purifying the expressed mFc human ridgetin fusion protein.
  • the cells may include, but are not limited to, human acute monocytic leukemia cell (THP-1) cells.
  • THP-1 human acute monocytic leukemia cell
  • it may comprise culturing the mFc human lystine fusion protein with an anti-mFc-FITC secondary antibody.
  • the immunoprecipitation may be performed using beads specific for mFc, and the human ridgestin receptor is 55 kDa in size.
  • the present invention provides a method for screening a human ridgestin protein receptor comprising the following steps: a) Cloning an expression vector of mFc-human ridgestin recombinant DNA in which the mFc gene is bound to the N-terminus of the human ridgestin gene.
  • a recombinant vector To produce a recombinant vector; b) transfecting said recombinant vector into HEK293F cells to express mFc human ridgetin fusion protein; c) purifying the expressed mFc human ridgestin fusion protein; d) culturing the purified mFc human lystine fusion protein with THP-1 cells (Human acute monocytic leukemia cells) to form a complex with the human lystine receptor; e) immunoprecipitating the complex with mFc specific beads to obtain a precipitate; f) isolating human lystine receptors corresponding to 55 kDa size from the precipitate; And g) confirming the isolated human lystine receptor by mass spectrometry.
  • THP-1 cells Human acute monocytic leukemia cells
  • the present invention provides a pharmaceutical composition for preventing and / or treating an inflammatory disease or atherosclerosis, wherein the agent expresses or modulates the expression of the human ridgestin protein receptor detected by the above method.
  • the present invention also provides a method of preventing and / or treating an inflammatory disease or atherosclerosis by administering the pharmaceutical composition to a subject.
  • an individual means a subject in need of treatment of a disease, and more specifically, a human, or a non-human primate, a mouse, a rat, a dog, a cat, a horse, a cow, and the like. Mean mammal.
  • the pharmaceutically effective amount in the present invention may be variously adjusted in the range depending on the weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease of the patient. Do.
  • the preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. However, preferably, it is administered at 0.001 to 100 mg / kg body weight per day, more preferably 0.01 to 30 mg / kg body weight. Administration may be administered once a day or may be divided several times.
  • the pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes.
  • the method of administration is not limited, and may be administered by oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine dural, or intra cerbroventricular injection.
  • the pharmaceutical composition of the present invention may be prepared in various pharmaceutical formulations, and there is no limitation in the form of the preparation.
  • the inventors have cloned the mFc-human lystine protein in order to easily detect the binding complex of the ridgestine protein with the lystine receptor, and thought that the receptor protein can be searched through the selection of the complexes bound to the mFc-human ridgestine protein. It was.
  • the present inventors produced mFc-human lystine recombinant DNA, cloned it into an expression vector, and produced the mFc-human lystine recombinant protein by transducing the recombinant vector into a cell line and expressing the recombinant DNA.
  • the receptor that binds the mFc-human lystine protein was identified, and mass spectrometry revealed that the receptor was CAP1 (Adenylyl cyclase-associated protein 1) (see FIGS. 1 to 4). .
  • the inventors have demonstrated that extracellular human ridgestin binds to the monocyte plasma membrane to the CAP1 protein detected by the method by Western blot, immunofluorescence staining and flow cytometry (see FIGS. 6 and 7). .
  • CAP1 which is identified as a functional receptor of human lystine, is divided into three domains in structural and functional terms (see FIG. 13).
  • CAP1 protein is thought to be in the oligomer structure of dimers, the amino terminal domain of CAP1 may interact with itself, and the carboxyl terminus may exist in parallel or anti-parallel dimer form. Since there is essentially a poly-proline domain in the center of the protein, both models allow the poly-proline SH3 interacting domain to bind freely to the target protein.
  • homology modeling is the simplest and most reliable way to predict molecular structure. The basis of this modeling is based on the fact that proteins of similar sequence tend to fold into similar structures. In general, to make a useful model, sequences must be 30% identical and proteins with 25% sequence identity are folded into similar structures.
  • the present inventors attempted to elucidate the signal transduction pathways of lystine, and the concentration of cAMP, protein kinase A (PKA) and nuclear factor kappa B (NF- ⁇ B) activity and expression of inflammatory cytokines according to the addition of human lystine. Confirmed that this is reduced
  • lystine is important for a successful response in the regulation of macrophage function.
  • Lizzystin has a unique disulfide-dependent multiple binding structure, and human ligithine also forms oligomers, as in rat ridgeins, and is present in human serum as predominantly 660 kDa or higher oligomers, 45 kDa trimers. Oligomeric forms of human lystine are biologically more active and have a stronger effect on the stimulation of inflammation-inducing cytokines.
  • the present inventors were able to predict the oligomer form of adenovirus ridgestin through non-reduced SDS gel analysis of the medium using the lysate of UUVEC (Human Umbilical Vein Endothelial Cells) infected with adenovirus ridgestin. Shows the ratio of total protein percent of ligstine oligomers to all ridgeins in monomeric or dimeric form.
  • UUVEC Human Umbilical Vein Endothelial Cells
  • PKA inhibitors abolish the activity of NF- ⁇ B induced by lystine, show a new link to the signaling of cAMP / PKA axis and NF- ⁇ B, and increase the expression of cytokines in response to lizithine. It is said to reduce.
  • the present inventors regulated the activity of human ligstin by inhibiting the expression of CAP1 using CAP1 siRNA (small interfering RNA) in order to reveal the signaling pathway of the ligstin-CAP1 complex and that CAP1 is a functional receptor of human ligstin.
  • Inhibition of the expression of CAP1 decreased the concentration of cAMP, protein kinase A (PKA) and nuclear factor kappa B (NF- ⁇ B) activity, and inflammatory cytokine expression.
  • PKA protein kinase A
  • NF- ⁇ B nuclear factor kappa B
  • the present inventors have revealed a signaling pathway by the interaction of human ligstin with its receptor, CAP1 protein, and confirmed the interaction of human stigstin with CAP1 protein in vitro and in vivo.
  • the structure and expression vector of the recombinant DNA encoding the mFc and the human Lizzy stin protein are shown in FIG. 1.
  • a PCR primer comprising a HindIII and XhoI sequences (SEQ ID NO: 1.
  • MFc-coupled human lystine recombinant DNA was prepared by PCR (polymerase chain reaction) using GAGTGGGAGA-3 ′) and Taq poylmerase (polymerase).
  • the recombinant DNA amplified by the PCR was cut with HindIII and XhoI restriction enzyme, cloned into the expression vector pcDNA3.1 using T4 DNA ligase, and heat shock at 42 ° C. for 1 minute 30 seconds.
  • the transformed E. coli DH5 ⁇ was plated on a LB (Luria Bertani) agar plate containing ampicillin, incubated in a 37 ° C. incubator for 16-24 hours, and then grown on E. coli DH5 ⁇ on LB (Luria Bertani) plate. Plasmid mini-prep was performed from coli DH5 ⁇ colony.
  • HEK293-F cells had a cell density of 70-80%.
  • mFc human lystine recombinant DNA cloned into the expression vector pcDNA3.1 and PEI, a transduction inducing substance were mixed and put into HEK293-F cells. After culturing the cells for a certain time, only the cell culture solution was separated and concentrated.
  • the concentrated cell culture solution was passed through a column filled with mFc-specific beads (CaptureSelect Multi Species affinity matrix), and then only the mFc human lystine recombinant protein bound to the bead was elution buffer (0.1M Glycine). -HCl, pH2.8).
  • THP-1 human acute monocytic leukemia cells
  • HAVEC Human Umbilical Vein Endothelial Cells
  • VMSCs human vascular smooth muscle cells
  • protein electrophoresis SDS-PAGE; Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis
  • PVDF Polyvinylidine fluoride
  • mFc-specific anti-mFc-HRP secondary antibody anti-mouse Fc specific peroxidase conjugate
  • the whole protein was extracted from THP-1 cells, and then reacted with mFc human lystine recombinant protein at 4 ° C. for one day.
  • the mFc-specific agarose bead was mixed with THP-1 cell protein and mFc human lystine protein complex, followed by immunoprecipitation for 1 day at 4 ° C, followed by centrifugation to remove the supernatant.
  • RIPA radioimmunoprecipitation assay
  • the supernatant was subjected to protein electrophoresis (SDS-PAGE; Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis) using an 8% separating acrylamid gel, and the gel was stained with coomassie brilliant blue R250, and the results are shown in FIG. 4.
  • SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis
  • CAP1 is expressed anywhere in rabbit tissue cells, but its expression level is different in each tissue, particularly in peripheral blood mononuclear cells (PBMCs) of rabbits, and human lystine It is shown to be similar to the expression pattern of.
  • PBMCs peripheral blood mononuclear cells
  • CAP1 protein In general, the location of CAP1 protein is species specific and recent studies have shown that CAP1 is associated with THP-1 cell membrane.
  • the CAP1 protein was found in the cell membrane, and the CAP1 protein was dispersed throughout the cytoplasm through immunofluorescence staining, and particularly concentrated in the human monocyte cell membrane. I could see that it was.
  • FIG. 7 shows the results of sorting THP-1 cells with human CAP1 monoclonal antibody by flow cytometry, suggesting the possibility of concentration dependent stimulation of ligstin to transfer CAP1 to the cell membrane.
  • non-stimulated control THP-1 cells and THP-1 cells recovered by treatment with human recombinant resistin protein at different concentrations were recovered, and the control cells were treated with isotype control antibody, which is the origin of the primary antibody, and human recombinant reisistin.
  • Protein-stimulated THP-1 cells were reacted with a mixture of human CAP-1 monoclonal antibodies. After washing the cells with FACS buffer, the cells were reacted with anti-mouse IgG Alexa 488 fluorescent secondary antibody capable of recognizing human CAP-1 monoclonal antibody, and THP- with human CAP-1 monoclonal antibody was attached using FACS machine. 1 cells were sorted.
  • THP-1 cells were treated with human recombinant lystine, the cells were attached onto a slide glass, and then blocked and permeabilized the cells with PBS solution containing 0.5% Triton X-100 and 1% BSA.
  • THP-1 cells were double-stained with human ligsteine and human CAP-1 antibodies, and THP-1 cells stained with the primary antibody gave different colored fluorescence to recognize ligsteine and CAP-1.
  • confocal microscopy confirmed whether lithstein and CAP-1 were stained at the same position in THP-1 cells, and the results are shown in FIG. 8.
  • FIG. 8 shows that the recombinant human lystine-treated THP-1 cells were labeled with double immunofluorescence to show that lystine and CAP1 are in the same position. Lysistine and CAP1 strongly indicate that they are in the same position by exhibiting similar distribution and similar fluorescence intensity.
  • the whole protein was extracted from THP-1 cells, and the extracted protein was immunoprecipitated with anti-human CAP-1 antibody at 4 ° C. for one day.
  • mouse IgG which is the origin of the anti-human CAP-1 antibody
  • Protein G agarose bead was added to the THP-1 protein and the anti-human CAP-1 antibody complex, followed by reaction, followed by centrifugation to remove the supernatant leaving only the bead.
  • 1X electrophoresis sample buffer was added to the bead, and the mixture was boiled at 100 ° C. for 5 minutes, and centrifuged again to obtain only the supernatant except the bead.
  • the gel was transferred to the PVDF membrane.
  • the membrane was reacted in denaturing / renaturing buffer and then blocked with PBST containing 5% skim milk for 1 hour at room temperature.
  • FIG. 10 The far western blotting results are shown in FIG. 10. As shown in FIG. 10, CAP1 was identified where mFc-human lystine protein is located.
  • recombinant CAP1 protein was prepared by diluting each well with photoresist. Each plate was incubated with an amount of mFc-human lystine and the binding of recombinant CAP1 to the mFc-human lystine was measured.
  • ELISA Enzyme-linked immunosorbent assay
  • recombinant CAP-1 protein was coated (coating) at the same concentration on a 96-well plate.
  • MFc human lystine recombinant protein was applied to wells coated with CAP-1 protein at 0.05, 0.5, and 1 ug.
  • Each well was treated with increasing concentrations of recombinant human lystine protein, followed by incubation for 2 hours.
  • the TMB Tetramethylbenzidine
  • the cells were cultured by treatment with recombinant CAP-1 protein after overexpression of human lystine in THP-1 cells by using Lizestin adenovirus.
  • the THP-1 cells were recovered, and the control cells were reacted with isotype control antibody, which is the origin of the primary antibody, and with human integrin- ⁇ 1 antibody, mixed with THP-1 cells that overexpressed the Ligestin adenovirus.
  • isotype control antibody which is the origin of the primary antibody
  • human integrin- ⁇ 1 antibody mixed with THP-1 cells that overexpressed the Ligestin adenovirus.
  • the number of THP-1 cells with integrin- ⁇ 1 antibody was measured using a FACS machine.
  • CAP-1 is a receptor for lystine
  • the recombinant CAP1 acts as a neutralizing antibody against lystine. It inhibited the binding of ligsteine to human monocytes and also inhibited the expression of integrin- ⁇ 1 by peripheral blood mononuclear cells (PBMCs), which occurs in response to ligstin stimulation.
  • PBMCs peripheral blood mononuclear cells
  • CAP1 is known as a multifunctional molecule containing domains involved in actin binding, adenylyl cyclase association in yeast, SH3 binding and oligomer formation.
  • Homogeneity modeling is the simplest and most reliable way to predict molecular structure. The basis of this modeling is based on the fact that proteins of similar sequence tend to fold into similar structures. In general, to make a useful model, sequences must be 30% identical and proteins with 25% sequence identity are folded into similar structures.
  • the proline-rich domain of CAP1 is 20% identical and 32% similar to the nucleotide sequence of cytidyly transferase derived from Thermus thermophilus HB8 (2PX7) (FIG. 14).
  • FIG. 15 shows a final estimate of the poly-proline SH3 binding domain of CAP1 identified using Discovery Studio 2.5 (Accelrys Inc.).
  • an adenylyl cyclase (AC) binding domain deletion mutation AC binding domain [BD] deletion
  • an actin binding domain deletion mutation AC binding domain [BD]
  • a mutant (SH3actin BD deletion) recombination vector was deleted in which both the SH3 binding domain and the actin binding domain were deleted.
  • the structure of these genes is shown in FIG.
  • Each CAP1 mutant gene was overexpressed in THP-1 cells and the proinflammatory cytokine secretion was induced by Lizzystin delivered through an adenovirus vector and then measured.
  • binding assay of rhResistin was performed after CAP1 mutation in vitro. His overtaking CAP1 mutagenesis was overexpressed in 293A cells, then treated with rhResistin and whole cell extracts were immunoprecipitated with anti-His antibody.
  • the change in the amount of cAMP caused by lystine was measured using a cAMP (cyclic AMP) analysis kit.
  • a cAMP cyclic AMP analysis kit.
  • the supernatant was obtained by lysing THP-1 cells stimulated with recombinant lystine.
  • a primary antibody solution for cAMP was added to a well of a cAMP measurement plate and coated (coating) at room temperature for 1 hour. After washing each well with washing buffer, supernatant from standard solution and THP-1 cells was added to the wells. After adding the cAMP conjugate to each well, it was incubated for 2 hours at room temperature. Substrate solution was added and reacted at room temperature for 30 minutes. Then, a stop solution was added and the reaction was stopped. After measuring absorbance at 450 nm using a microplate reader, the results are shown in FIG. 19.
  • the present inventors evaluated whether protein kinase A (PKA) and nuclear factor kappa B (NF- ⁇ B) activity is altered by lystine. Specifically, THP-1 cells were stimulated with recombinant lystine protein or overexpressed human lystine with lystine adenovirus, and then cytosol and nuclear proteins were separated from each cell.
  • PKA protein kinase A
  • NF- ⁇ B nuclear factor kappa B
  • Lizzystin was shown to increase both PKA and NF- ⁇ B activity.
  • the ridgeins expressed by adenoviruses showed a more potent effect than the recombinant ridgesteen.
  • CDNA was synthesized at 42 °C using 1 ⁇ g total RNA, oligo-dT primer, dNTP mix, Rnase inhibitor, RTase.
  • conventional PCR was performed using inflammatory cytokine gene specific primers, MgCl 2 , dNTP mix, and Taq polymerase. The PCR products were electrophoresed in agrose gel to express each gene. Confirmed. In addition, real time PCR was performed for each gene using SYBR green.
  • inflammatory cytokines such as integrin- ⁇ 1 and IL-6 (interleukin 6), TNF- ⁇ (tumor necrosis factor- ⁇ ), IL-1 (interleukin 1) It can be seen that it is promoted by Lizzystin and becomes more pronounced in Lizzystin expressed by adenovirus.
  • lystine is important for a successful response in the regulation of macrophage function.
  • Lizzystin has a unique disulfide-dependent multiple binding structure, and human ligithine also forms oligomers, as in rat ridgeins, and is present in human serum as predominantly 660 kDa or higher oligomers, 45 kDa trimers. Oligomeric forms of human lystine are biologically more active and have a stronger effect on the stimulation of inflammation-inducing cytokines.
  • FIG. 22 shows the ratio of total protein percent of ligstine oligomers to all ridgeins in monomeric or dimeric form.
  • THP-1 cells were pretreated with a PKA inhibitor (KT5720), and then THP-1 cells were stimulated with lystine adenovirus. Cytosol and nuclear proteins were extracted and Western blot was performed. PK (protein kinase A) and NF- ⁇ B (nuclear factor kappa B) using p-VASP (Ser157) and p50 and p65 antibodies The result of confirming the change of is shown in FIG.
  • the PKA inhibitor abolishes the activity of NF-kB induced by lystine and shows a new connection to the signaling of cAMP / PKA axis and NF-kB.
  • THP-1 cells were pretreated with PKA inhibitor and then stimulated with THP-1 cells with L. adenovirus.
  • Cells were recovered and total RNA was extracted with Trizol solution.
  • CDNA was synthesized at 42 ° C using 1 ⁇ g total RNA, oligo-dT primer, dNTP mix, Rnase inhibitor, and RTase.
  • real time PCR was performed for each gene using cytokine gene specific primers and SYBR green, and the results are shown in FIG. 24.
  • PKA inhibitors also reduce the increase in the expression of cytokines in response to lystine.
  • the results of the experiments provide evidence that the effect of cAMP on PRIS is influenced by the release of lystine-induced cytokine in macrophage lines.
  • CAP1 is a functional receptor in the biological sense.
  • Protein and RNA were extracted from THP-1 cells that inhibited the expression of human CAP-1 gene with CAP-1 gene specific siRNA.
  • Western blot was performed to confirm the change of protein kinase A (PKA) and nuclear factor kappa B (NF- ⁇ B) using p-VASP (Ser157) antibody and p50 and p65 antibodies.
  • cDNA was synthesized from extracted RNA. After that, the effects of CAP-1 gene inhibition were confirmed through real time PCR for each gene using gene-specific primers and SYBR green, and the results are shown in FIGS. 25 to 28.
  • FIG. 25 shows the inhibitory effect of CAP1 expression using specific siRNAs in THP-1 cells. According to FIG. 26, it can be seen that when siRNA is used to inhibit CAP1 expression, an increase in cAMP concentration caused by lystine is reduced.
  • CAP1 siRNA significantly reduces PKA and NF-kB activity.
  • siRNA target CAP1 reduces the expression of cytokines produced against lystine. have.
  • CAP1 acts as a receptor for lystine, and the interaction of lystine and CAP1 can regulate NF-kB related to the increase of cAMP, PKA activity and transcription of inflammatory cytokines. have.
  • Figure 33 is a schematic diagram showing how the CAP1 protein acts as a functional receptor of lizithine.
  • transwell migration asssay see FIGS. 34 to 36
  • vertical collagen gel invasion assays were performed (Fig. 37 and See FIG. 38).
  • THP-1 cells overexpressing CAP1 had a stronger tendency to migrate toward ligstin, while cells with reduced CAP1 expression had attenuated a tendency to migrate towards lizstin.
  • the response of macrophages or THP-1 cells to lystine is CAP1-dependent.
  • mice that are not expressed in the case of lystine but instead overexpress human ligstin have a greater inflammatory response in white adipose tissue when high-fat diets are administered. Therefore, in order to confirm whether monocytes can regulate the inflammatory response by regulating CAP1 expression, the following experiments were performed using mice that do not express lystine but instead express human ridgeins.
  • mice of 9-10 weeks of age expressing human lystine in monocytes / macrophages and not expressing rodent lystine were used, and CAP1 overexpression was induced using lentiviral vectors.
  • Retn ⁇ / ⁇ inbred mice were used as controls and all animals were fed a high fat diet (60% fat, D12492; Uni Faith, Inc.) for one month to induce white adipose tissue inflammation.
  • High-fat diet mice have increased expression of human ligsteine and monocyte chemotactic protein-1 (MCP-1) and CAP1, and the expression of CAP1 increases in proportion to the expression of MCP-1. The aspect was shown (see FIG. 39).
  • mice showed a greater number of macrophage deposits in white adipose tissue than the control group.
  • monocytes inhibited with CAP1 expression were administered to mice expressing human lystine, macrophage deposition in white adipose tissue (WAT) was significantly reduced.
  • WAT white adipose tissue
  • the results show a consistent trend with decreased inflammatory marker expression, including TNF- ⁇ (see FIG. 41). Macrophages infiltrated in WAT were observed by immunofluorescence staining.
  • monocytes migrated to inflammatory white adipose tissue, where monocytes overexpressing CAP1 (see FIG. 43) exhibited CAP1 expression. It can be seen that much more invaded WAT than the degraded monocytes (see FIG. 42).
  • Monocytes that regulate CAP1 expression are all green by GFP, which depends on the expression vector, and CD11b is a membrane protein (marked in red) that recognizes monocytes, so the cells that are simultaneously recognized are CAP1 expression-regulated monocytes.
  • CAP1 acts as a receptor for lystine in vivo and also acts as an important physiological regulator for regulating the inflammatory response of monocytes induced by lystine.
  • the present invention can be used for controlling the inflammatory effects of monocytes, diagnosing the molecular causes of vascular inflammation and atherosclerosis, and preventing and treating inflammatory diseases and atherosclerosis.

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Abstract

La présente invention concerne un récepteur de résistine humaine. Plus spécifiquement, la présente invention concerne un procédé pour rechercher un récepteur pour une protéine de résistine humaine, un procédé pour prévenir ou traiter des maladies inflammatoires et l'athérosclérose en utilisant un régulateur qui régule l'expression ou l'activité d'un récepteur de résistine humaine, et une composition pharmaceutique contenant un régulateur qui régule l'expression ou l'activité d'un récepteur de résistine humaine. Il est possible de séparer un récepteur qui se lie directement à la résistine de monocytes humains en utilisant le procédé pour rechercher un récepteur pour une protéine de résistine humaine de la présente invention, et il est attendu que la découverte du système de transmission de signal d'un récepteur de résistine contribue à la régulation d'effets inflammatoires d'un monocyte, au diagnostic de la cause moléculaire de l'inflammation des vaisseaux sanguins et de l'athérosclérose, et au développement d'un agent pour prévenir et traiter des maladies inflammatoires et l'athérosclérose.
PCT/KR2012/006269 2011-08-08 2012-08-07 Récepteur de résistine humaine, et utilisation de celui-ci Ceased WO2013022262A2 (fr)

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KR20180089253A (ko) * 2017-01-31 2018-08-08 서울대학교병원 Cap1로부터 유래된 폴리펩티드 및 이를 유효성분으로 포함하는 약학적 조성물

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GB0223272D0 (en) * 2002-10-08 2002-11-13 Oxford Glycosciences Uk Ltd A protein involved in therapy
KR20080045953A (ko) * 2006-11-21 2008-05-26 재단법인서울대학교산학협력재단 유방암, 폐암 또는 대장암 특이적인 마커, 상기 마커에대한 항체를 포함하는 키트 및 마이크로어레이, 및 상기마커를 이용한 유방암, 폐암 또는 대장암 예후 예측 방법
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KR20180089253A (ko) * 2017-01-31 2018-08-08 서울대학교병원 Cap1로부터 유래된 폴리펩티드 및 이를 유효성분으로 포함하는 약학적 조성물
KR102040974B1 (ko) * 2017-01-31 2019-11-06 서울대학교병원 Cap1로부터 유래된 폴리펩티드 및 이를 유효성분으로 포함하는 약학적 조성물

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