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WO2008148349A1 - Utilisation de la bêta-arrestine 1 pour moduler la survie et l'auto-immunité des lymphocytes t - Google Patents

Utilisation de la bêta-arrestine 1 pour moduler la survie et l'auto-immunité des lymphocytes t Download PDF

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Publication number
WO2008148349A1
WO2008148349A1 PCT/CN2008/071180 CN2008071180W WO2008148349A1 WO 2008148349 A1 WO2008148349 A1 WO 2008148349A1 CN 2008071180 W CN2008071180 W CN 2008071180W WO 2008148349 A1 WO2008148349 A1 WO 2008148349A1
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cells
substance
protein
parrl
inhibitor
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PCT/CN2008/071180
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Chinese (zh)
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Gang Pei
Jingwu Zang
Yufeng Shi
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Shanghai Institutes For Biological Sciences, Cas
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to the field of biomedicine, and in particular to the use of an inhibitor of beta inhibitory protein 1 for the preparation of a disease associated with abnormal apoptosis of CD4+ T cells for the treatment or prevention of an object.
  • sputum cells and sputum cells constitute the acquired immune system to protect the body from pathogens.
  • the apoptosis of these cells is precisely regulated.
  • the thymus depending on the strength of the TCR signal, most of the sputum cells undergo apoptosis through negative selection and positive selection.
  • the surviving sputum cells enter the peripheral blood system to form a peripheral blood CD4 + and CD8 + T cell bank.
  • CD4 + T cells in peripheral blood maintain their homeostasis due to new cell input in the thymus and apoptosis of CD4 + T in peripheral blood (1, 2).
  • CD4 + T cells When CD4 + T cells are exposed to their specific antigen, the cells begin to divide and differentiate into functional CD4 + T cells. These functional CD4 + T cells need to be apoptotic in time after the foreign antigen is cleared, otherwise it will cause damage to the normal body.
  • the signaling pathways involved in the apoptosis of these functional CD4 + T cells are: activation-induced apoptosis (AICD) and activated cell apoptosis (ACAD).
  • AICD activation-induced apoptosis
  • ACAD activated cell apoptosis
  • apoptosis of CD4 + T cells is mainly mediated by two pathways (1, 6).
  • One is through cell surface receptors such as TNF and Fas. They primarily mediate apoptosis in activated CD4 + T cells. Activation of these receptors can induce apoptosis directly by recruiting and activating caspase (7_11).
  • the pathway that mediates apoptosis in CD4 + T cells corresponding to this pathway is the Bcl-2 family protein-mediated mitochondrial pathway.
  • This family of proteins includes two major classes of anti-apoptotic and pro-apoptotic molecules that synergistically regulate the integrity of the mitochondrial membrane and the release of pro-apoptotic proteins present in mitochondria (12, 13).
  • Bcl-2 family proteins mainly mediate spontaneous apoptosis of CD4 + T cells due to cytokine deficiency, stress or activation (1, 14-17).
  • Bcl-2 is the prototype protein of this protein family.
  • T cells are prone to apoptosis and leukopenia occurs when the mouse ages (18).
  • T cells are not sensitive to many apoptotic stimuli (15, 16), the body's immune response time is longer and the mice spontaneously develop symptoms of autoimmune disease (19).
  • the beta inhibitor protein family comprises beta inhibitory protein 1 (PaiTl), beta inhibitory protein 2 (p a ir2) and the like. They are signal molecules with multiple functions (20) that, on the one hand, mediate endocytosis of multiple receptors (21-24). On the other hand, they regulate these signaling pathways by binding to many signaling molecules, such as: Src family kinase activity, some MAPK signaling pathways (20, 25, 26). --arrestin 1 (gene number: NM-004041) is an important regulatory protein of GPCRs and is distributed in both cytoplasm and nucleus.
  • Parrl not only regulates the signaling pathway in the cytoplasm, but also directly regulates gene transcription in the nucleus (27). It shows that some functions of parrl are realized by affecting the expression of genes. Pairs are a class of ubiquitously expressed proteins, but their protein levels are relatively higher in nerve cells and immune cells, as reported in (28, 29) and in Figure 2A. Recent studies have found that arr2 negatively regulates Toll-like receptor signaling in primary immune cells. And another lab found that The incidence and condition of the rat in the asthma model was significantly lower than in the wild type (30, 31). But until now, people still have not found the function of parrl in immune cells. Summary of the invention
  • parrl is regulating the survival of CD4 + T cells and their homeostasis in vivo. This function of arrl is likely to be achieved through its intranuclear function, which promotes the level of acetylation of histone H4 and the expression of this gene in the nucleus.
  • the important physiological significance of parrl regulating CD4 + T cell survival is further elucidated in the examples of the present application: 1) In the experiment of EAE in mouse model of autoimmune demyelinating disease, ⁇ rr knockout mice are not sensitive to the induction of EAE.
  • the present invention provides a novel molecular mechanism for regulating the physiological functions of CD4 + T cells and provides a basis for utilizing this molecular mechanism to treat autoimmune diseases.
  • the present invention relates to the use of an inhibitor of ⁇ -inhibitor 1 in the preparation of a medicament, characterized in that the inhibitor of ⁇ -inhibitor 1 is selected from the group consisting of (i) a substance which inhibits the activity of ⁇ -inhibitor 1; (ii) A substance which inhibits transcription, translation or both of a gene encoding ⁇ inhibitory protein 1 for treating or preventing a disease associated with abnormal apoptosis of CD4 + T cells in a mammalian subject.
  • Another aspect of the present invention provides a pharmaceutical composition for treating or preventing a disease associated with abnormal apoptosis of CD4 + T cells in a subject, characterized in that the pharmaceutical composition comprises an effective amount of an inhibitor of ⁇ inhibitory protein 1 As an active ingredient, and a pharmaceutically acceptable carrier, wherein the inhibitor of ⁇ inhibitory protein 1 is selected from the group consisting of (i) a substance that inhibits ⁇ -inhibitory protein 1 activity; (ii) an inhibition of transcription of a gene encoding ⁇ inhibitory protein 1, Translation or the substance of both.
  • Still another aspect of the present invention provides a method of screening a candidate for a drug for treating or preventing a disease associated with abnormal apoptosis of CD4 + T cells, the method comprising: a) allowing the candidate substance to Cell contact expressing ⁇ -arrestin 1; b) identifying the expression level of ⁇ -arrestin 1 in the nucleus of the cell, and expressing the expression level with the ⁇ -arrestin in the nucleus of the cell not contacting the candidate substance 1 Comparison of expression levels; c) Selection of a substance that reduces the expression level of ⁇ -arrestin 1 in the nucleus as a disease drug for treating or preventing apoptosis associated with abnormal apoptosis of CD4+ T cells.
  • FIG. 1 shows that ⁇ -arrestin 1 positively regulates the homeostasis and survival of CD4 + T cells.
  • A Separation of ⁇ - ⁇ - And wild-type (WT) mouse spleen cells (top) and lymphocytes (bottom), flow cytometry analysis of CD4 and CD8 molecules expression on these cells.
  • the FACS map is representative of 10 mice per group. The numbers show the percentage of cells in each region.
  • B, C from CD4 + and CD8+ T cells were isolated from ferrHg and wild-type mice, and these cells were activated (C) with or without activation of antibodies to CD3 and CD28 (B). Subsequently, the cells are cultured in a simple medium.
  • the number of viable cells is obtained by excluding PI and Annexin V positive cells.
  • D, E ⁇ ⁇ fiarrltg and wild-type mice were intraperitoneally injected with 100 ⁇ g of SEB in each group. Blood was taken from the tail vein, and the amount of TCR ⁇ 8 1/2+ or ⁇ 6+ positive CD4 + and P CD8 + T in peripheral blood was obtained by flow cytometry. Data were obtained from three parallel experiments, * P ⁇ 0.05, ** P ⁇ 0.01 compared to the corresponding controls.
  • Figure 2 shows the expression of ⁇ -arrestin 1 in different tissues of mice and the development of lymphocytes in ferr j and mice.
  • A Western blotting method was used to detect the expression of Parrl and Pair2 in different tissues of mice, and actin as an internal reference for loading.
  • B Expression of CD3 and B220 in spleen cells and lymphocytes derived from ferr" and W mice (13-18 weeks) by flow cytometry, and the FACS map is representative of 5 mice per group. Percentage of cells in each region. The number of cells in the specific cell population in spleen cells (top) and lymphocytes (bottom) is also shown.
  • Figure 3 shows the effect of ⁇ -arsenin 1 on IL-2 secretion, Akt and Erk activation in CD4 + T cells.
  • Spleen CD4 + T cells were activated with appropriate amounts of antibodies to CD3 and CD28, and IL-2 levels in the culture medium were measured by ELISA 24 hours later. The data was derived from three parallel experiments.
  • ⁇ '- and wt mouse-derived spleen CD4 + T cells were activated or not activated with the corresponding amounts of antibodies to CD3 and CD28, and immunoblot assays detected phosphorylated (P-) Akt, (P-) Erk and All Akt, Erk levels. The figure is representative of two parallel experiments.
  • Figure 4 shows the expression of ⁇ -arrestin and ⁇ 300 and the transcriptional effects of ⁇ -arrestin 1 on the Bcl-2 family gene after different treatments.
  • A After transfecting the plasmid as shown in the Jurkat cells, the effect of these proteins on the expression of these proteins was examined by Western blotting.
  • B After transfecting the plasmid as shown in the Jurkat cells, the RT-qPCR assay detects the transcription levels of /-2, Bax, Bim., Sad and 3 ⁇ / ⁇ / to normalize the loading. Data were derived from three parallel experiments, ** P ⁇ 0.01 compared to the corresponding controls.
  • FIG. 5 shows that ⁇ -arrestin 1 promotes the expression of Bcl-2 in CD4 + T cells.
  • A Spleen CD4 + T cells were activated with antibodies to CD3 and CD28 for the corresponding time, and the expression of Parrl, Bcl-2 and Bax was analyzed by Western blotting. Actin is used as an internal reference for loading.
  • B, C, IT ⁇ A ⁇ ( ⁇ >, CD4+ T cells were isolated from the spleens of ferr (D) and wild type mice and activated for the corresponding time as described above. After the cells were sampled, the mRNA levels of Bcl-2 and P Bax and the protein levels of Bcl-2, Bax and Parrl were detected by RT-qPCR and immunoblotting, respectively.
  • mHPRT is an internal reference for RT-qPCR experiments
  • actin is an internal reference for immunoblot experiments. All immunoblots and RT-qPCR experiments were performed in parallel for more than three times, **P ⁇ 0.01 compared with the corresponding controls.
  • Figure 6 shows that ⁇ -arrestin 1 promotes the expression of Bd-2 by transcription and the effect of ⁇ -arrestin 1 on the CREB and F-kappa B reporter genes.
  • A After transfecting the plasmid as shown in the Jurkat cells, RT-qPCR and immunoblotting were used to detect the expression levels of So and So.
  • B, C After 33 hours of transfection of ⁇ a/ or ferr in Jurkat cells, use 0, 5, 10 ⁇ g of cycloheximide (CHX) (B) or 0, 2, 4 ⁇ actinomycin D (Act (C) Cells were treated for 15 hours, and RT-qPCR and Western blotting assays were used to detect and express Sax levels.
  • CHX cycloheximide
  • Act Act
  • HEK293 cells were individually transfected with CREB-luciferase reporter plasmid or co-transfected with different amounts of the ⁇ rr plasmid. Data are expressed in multiples relative to the control. 10 ⁇ of forskolin (FK) is a positive control. The data was derived from three parallel experiments.
  • FK forskolin
  • ⁇ In ⁇ 293 cells, the NF-kappa ⁇ -luciferase reporter plasmid was transfected with a different amount of ⁇ plasmid. Data are expressed in multiples relative to the control.
  • T F- ⁇ is a positive control. The data was derived from three parallel experiments. *P ⁇ 0.05, **P ⁇ 0.01 compared to the corresponding controls.
  • Figure 7 shows that ⁇ -arrestin 1 promotes the level of Bd-2 locus histone H4 acetylation in CD4 + T cells.
  • Spleen CD4 + T cells were activated with antibodies to CD3 and CD28, and cells were sampled at different times and analyzed by CHIP assay.
  • Histone H3 acetylation and H4 acetylation antibodies were used in the CHIP assay.
  • the sequences of the 3 ⁇ -2 and 3 ⁇ gene promoter regions were analyzed by qPCR. The data is normalized by the amount in the sample.
  • A CD4 + T cells of wt mice were used, and levels of histone H4 (left) acetylation and H3 (right) acetylation were analyzed.
  • B CD4 + T cells using parrltg and W mice.
  • C CD4 + T cells using ferr and W mice.
  • D The histone H3 acetylation and H4 acetylation levels of the Bcl-2 gene region in CD4 + T cells of ⁇ - and W mice were analyzed by CHIP-qPCR assay. The ratio of W and ferr histone acetylation levels at the same site is shown in the figure. "0" represents the transcription start site, and "u” represents the upstream of the transcription start site. Data were derived from three parallel experiments, ** P ⁇ 0.01 compared to the corresponding controls.
  • Figure 8 shows the role of p300 in the up-regulation of ⁇ -arrestin-1 transcription.
  • A-D Jurkat cells transfected with the plasmid shown in the figure were analyzed for acetylation levels of histones H4 (A, C) and H3 (B, D) by CHIP assay.
  • E, F Jurkat cells transfected with the plasmid shown in the figure were analyzed for transcription levels of Bd-2 (E) and Sax (F) by RT-qPCR. Data were derived from three parallel experiments, ** P ⁇ 0.01 compared to the corresponding controls.
  • Figure 9 shows that ⁇ -arrestin 1 promotes experimental autoimmune encephalomyelitis in mice.
  • ⁇ Induction of sputum in ⁇ -, ferrHg and ⁇ mice at 6-8 weeks, and the onset of the mice was observed daily after induction. The data is the sum of 4 experiments including 10 ⁇ -, 10 arrltg and 14 W mice.
  • B The spinal cord was removed from EAE mice, fixed and observed for inflammatory infection with hematoxylin blush and Luxol fast blue for the degree of demyelination of the spinal cord. The figure is typical of 3-4 mice.
  • C After 8 days of induction of EAE in mice, cell proliferation was detected by stimulating mouse spleen cells with MOG peptides.
  • Figure 10 shows that ⁇ -arrestin 1 is associated with multiple sclerosis disease.
  • ⁇ , ⁇ CD4+ T cells were isolated from peripheral blood of 14 patients with multiple sclerosis and 14 healthy subjects, and the transcription levels of ferr (A) and Sc/(B) were detected by RT-qPCR. *P ⁇ 0.05, **P ⁇ 0.01 compared to the corresponding controls.
  • C Expression of Parrl was interfered with a lentivirus containing ⁇ siRNA from MBP-specific CD4 + T cell clones derived from multiple sclerosis patients. Western blotting assays were used to detect the expression levels of ⁇ and 3 ⁇ -2. The figure is typical of three experiments.
  • Figure 11 shows the expression of ⁇ -arrestin 1 in CD4 + T cells after EAE induction, as well as CD4 + and
  • CD4 + T plays an important role in acquired immunity, and abnormal regulation of such apoptosis may lead to autoimmunity.
  • Parrl is a discovered protein that functions in the G protein-coupled receptor signaling pathway, and it also functions to directly regulate gene transcription in the nucleus.
  • the inventors found that parrl is regulating the survival of CD4 + T cells.
  • naive and activated CD4 + T are prone to apoptosis in both in vivo and in vitro experimental conditions compared to their wild type. Parrl regulates the level of acetylation of histone H4 in c-gene and affects its gene expression in CD4 + T cells.
  • the pathological severity of the ⁇ rr knockout mice was significantly lower than that of the wild type, while the pathological severity of the yferr transgenic mice was significantly increased.
  • the expression of ⁇ arr in peripheral blood CD4 + T cells was significantly higher in patients with multiple sclerosis than in normal subjects.
  • the expression of fiarrl siRNAi in CD4 + T cells from patient sources and reactive with autoantigens decreased its expression, and this type of CD4 + T cells showed a significant increase in apoptosis under cytokine stress, revealing that parrl regulates CD4 + T cells.
  • the first aspect of the invention relates to the use of an inhibitor of ⁇ -inhibitor 1 in the preparation of a medicament, characterized in that the inhibitor of ⁇ -inhibitor 1 is selected from the group consisting of (i) a substance which inhibits the activity of ⁇ -inhibitor 1; Ii) A substance which inhibits transcription, translation or both of a gene encoding ⁇ inhibitory protein 1 for use in the treatment or prevention of a disease associated with abnormal apoptosis of CD4+ T cells in a mammalian subject.
  • inhibitor of beta inhibitory protein 1 herein has a broad meaning including direct or indirect (eg, by reactive intermediates, metabolites, etc.) acting on beta inhibitory protein 1 to inhibit or reduce beta inhibitory protein 1
  • the biologically active substance specifically includes: (i) a substance that directly inhibits the biological activity of ⁇ inhibitory protein 1 by interaction at the protein level; (ii) a substance that inhibits transcription and/or translation of a gene encoding ⁇ inhibitory protein 1.
  • beta-inhibitor 1 refers to the activity of beta-inhibitor 1 to promote survival of CD4+ T cells, the activity of promoting Bcl-2 expression, or the promotion of histone acetylation in the Bcl-2 locus. Horizontal activity.
  • ⁇ inhibitory protein 1 herein includes not only the full-length sequence of ⁇ -arrestin 1 but also various variant forms, fragments, derivatives or analogs of the protein, as long as The variant forms, fragments, derivatives or analogs have the same or similar activities as described above. These variant forms include, but are not limited to;: a plurality of (preferably 1-10, more preferably 1-5, most preferably 1-3) amino acids relative to the amino acid sequence of the polypeptide.
  • the difference between these analogs or derivatives and the native protein may be a difference in amino acid sequence and/or a difference in the modified form that does not affect the sequence.
  • the substance which inhibits the activity of ⁇ inhibitory protein 1 is a substance which specifically binds to ⁇ inhibitory protein 1 and thereby inhibits its activity, for example, polyclonal antibodies and monoclonal antibodies specific for ⁇ inhibitory protein 1.
  • “specificity” refers to an antibody that binds to ⁇ -inhibitor 1 but does not recognize and bind to other unrelated antigen molecules.
  • various modified forms of the antibody, and fragments thereof such as Fab' or (Fab) 2 fragments and the like.
  • the antibodies can be prepared by a variety of techniques known to those skilled in the art, such as immunizing animals to produce polyclonal antibodies or hybridoma producing monoclonal antibodies.
  • other known agents other than antibodies are specific to beta inhibitor 1
  • the receptors and ligands which bind sexually are also within the scope of the "substance which inhibits the activity of ⁇ -inhibitor 1".
  • the substance which inhibits the activity of ⁇ inhibitory protein 1 is a compound having such an inhibitory effect known to those skilled in the art, for example, actinomycin, cycloheximide or a pharmaceutically acceptable salt thereof , derivatives or analogues.
  • G protein-coupled receptors are known to indirectly affect the activity of parrl by affecting the distribution of ⁇ ⁇ in the nucleus and cytoplasm.
  • Such agonists of G protein-coupled receptors include, for example, but are not limited to, agonists such as ⁇ opioid receptors and kappa opioid receptors, and the like. Therefore, agonists of these G protein coupled receptors are also included in the scope of the present invention.
  • Those substances which inhibit the transcription and/or translation of a gene encoding ⁇ -suppressor 1 mainly refer to affecting the expression of ⁇ -inhibitor 1 by indirectly acting on a gene of ⁇ -inhibitor 1 at a gene level, thereby inhibiting its activity.
  • Those substances usually nucleic acids.
  • the antisense sequence may typically be between 5 and 200, preferably between 10 and 100, more preferably between 20 and 50 bases in length.
  • Such antisense nucleic acid molecules can be synthesized chemically using natural nucleotides or physics designed to increase molecular biological stability or increase duplex formation with beta inhibitor 1 mRNA or beta inhibitor 1 gene. Various modified nucleotides for stability.
  • the antisense sequence can also be produced by biological methods, and the expression vector is introduced into the cell in the form of a recombinant plasmid, a phagemid or an attenuated virus, in which the antisense sequence is produced under the control of a highly effective regulatory region, Activity can be determined by the type of cell into which the vector is introduced.
  • those substances that inhibit transcription and/or translation of a gene encoding beta inhibitory protein 1 are small interfering RNA (siRNA).
  • siRNA small interfering RNA
  • 0 RNA interference is a newly discovered in vivo gene silencing phenomenon.
  • Small interfering RNA is a processed product of exogenous double-stranded RNA, which mediates RNA interference effects, recognizes specific mRNA, and silences homologous gene expression in cells.
  • siRNA small interfering RNA
  • a more effective siRNA has a size of 20-30 bases, more preferably 21-25 bases, and preferably has two prominent bases at the 3' end.
  • the sequence specificity of siRNA is very stringent, and a base mismatch with the target mRNA significantly impairs the effect of gene silencing.
  • siRNAs can generally design siRNAs using the following steps: (1) selection of siRNA target sites; (2) sequence homology analysis; (3) design of negative controls.
  • the designed siRNA can also be synthesized by chemical synthesis or in vitro transcription.
  • ⁇ -inhibitor 1 siRNA see Parruti, G. et al., J Biol Chem 268, 9753-61 (1993).
  • the substance that inhibits transcription and/or translation of a gene encoding beta inhibitory protein 1 is a ribozyme.
  • a ribozyme is a ribonucleic acid molecule that is capable of specifically pairing with a target RNA molecule, and then cleavage of the latter at a specific site to stop the production of the corresponding protein.
  • the hammerhead ribozyme is the smallest (about 40-50 nucleotides in length), which has the advantages of simple structure and simple design.
  • the DNA sequence can be directly chemically synthesized, and then ligated by recombinant DNA into a recombinant plasmid to produce the ribozyme, and then the specificity and efficiency of the designed ribozyme to the RNA substrate molecule can be determined in a test tube. Verification.
  • PCR can be used to amplify RNA (Saiki, et al. Science 1985; 230: 1350-1354).
  • the ribozyme gene is cloned into a plasmid, adenovirus, or retroviral vector.
  • nucleic acid sequence for inhibiting transcription and/or translation of a gene encoding ⁇ inhibitory protein 1
  • small interfering RNAs can be introduced into the subject cells using conventional techniques such as transformation, transfection, infection, and physical techniques such as electroporation and microinjection. Alternatively, it can be delivered by chemical methods such as DNA co-precipitation and incorporation into liposomes or in aerosol or lavage format.
  • Suitable viral vectors, plasmids or cloning vectors for transferring nucleic acid molecules are known in the art. Examples of suitable viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and DNA viral vectors, and the like. These techniques are well known to those skilled in the art.
  • the inhibitor of ⁇ -inhibitor 1 of the present invention can be used for treating a disease associated with abnormal apoptosis of CD4 + T cells in a subject.
  • Mammalian subjects to which the present invention is applicable include humans, domestic and farm animals, non-human primates, and zoos, playground animals, or pets such as dogs, horses, cats, cows, and the like.
  • the mammal is a human.
  • abnormal apoptosis of CD4 + T cells means that compared with CD4 + T cells in normal subjects
  • Apoptosis of CD4 + T cells refers specifically to the occurrence of CD4+ T cell apoptosis in peripheral blood.
  • Autoimmune diseases refer to diseases caused by autoantibodies or sensitized lymphocytes produced by the body that damage and damage their own tissues and cellular components, leading to tissue damage and organ dysfunction. The underlying mechanism leading to autoimmune diseases is the termination and destruction of immune tolerance. Autoimmune diseases often have the following characteristics: 1 The sensitized lymphocytes in which the high-valent self-antibody and/or self-organological components react can be measured in the blood of the patient. 2 autoantibodies and/or autosensitized lymphocytes act on tissues and cells in which the target antigen is located, causing pathological damage and dysfunction of the corresponding tissues and organs.
  • Autoimmune diseases suitable for treatment or prevention with the present invention include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, scleroderma, multiple sclerosis, myasthenia gravis, demyelination Disease, primary adrenal atrophy, chronic thyroiditis, type I diabetes, chronic non-specific ulcerative colitis, chronic active hepatitis, avian anemia and atrophic gastritis, autoimmune glomerulonephritis, pulmonary and renal hemorrhagic Syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, etc.
  • autoimmune diseases suitable for treatment or prevention with the present invention include demyelinating diseases, multiple sclerosis or type I diabetes.
  • the present invention also provides a pharmaceutical composition for treating or preventing a disease associated with abnormal apoptosis of CD4 + T cells in a mammalian subject, the pharmaceutical composition comprising an effective amount of an inhibitor of ⁇ inhibitory protein 1 as an activity Component to And a pharmaceutically acceptable carrier, wherein the inhibitor of ⁇ inhibitory protein 1 is selected from the group consisting of (i) a substance that inhibits ⁇ inhibitory protein 1 activity; (ii) a gene that inhibits ⁇ -inhibitor protein 1 transcription, translation, or both Substance.
  • compositions of the present invention comprise a substance which inhibits ⁇ inhibitory protein 1 as described in detail herein or which is identified by the method of the present invention.
  • the active substance may be administered alone, but usually together with a pharmaceutical carrier or the like, which may be selected according to the chosen route of administration and standard pharmaceutical practice.
  • the dose administered should be an "effective amount", that is, an amount that treats, alleviates or prevents the target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect.
  • Dosage dosage according to the use and known factors (such as the pharmacodynamic properties of the specific substance and its administration and route; the age, health and weight of the recipient; the nature and extent of the disease; the type of treatment, the frequency of treatment and the expectation The effect varies).
  • the precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the combination of therapeutic and/or therapeutic agents selected for administration. Therefore, it is useless to specify an accurate effective amount in advance. However, for a given symptom, routine experimentation can be used to determine the effective amount that the clinician can judge.
  • pharmaceutically acceptable carrier refers to a carrier or excipient for the administration of a therapeutic agent which is compatible with the inhibitor of the beta inhibitory protein 1 of the present invention, i.e., can be blended therewith without the usual circumstances. The effect of the pharmaceutical composition is greatly reduced.
  • sugars such as lactose, glucose and sucrose
  • starches such as corn starch and potato starch
  • cellulose and its derivatives such as Sodium carboxymethylcellulose, ethylcellulose and methylcellulose
  • scutellaria powder malt
  • gelatin talc
  • solid lubricants such as stearic acid and magnesium stearate
  • calcium sulphate vegetable oils, such as peanut oil , cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter
  • polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol
  • alginic acid emulsifiers such as Tween
  • wetting agents such as laurel Sodium sulfate
  • coloring agent such as flavoring agent; tablet, stabilizer; antioxidant
  • preservative pyrogen-free water
  • phosphatemulsaline solution such as lactol Sodium sulf
  • More than one substance described in detail herein or identified by the methods of the invention may also be used in combination with other agents. They may be administered simultaneously or in parallel as separate dosage forms, or as a physical composition of the therapeutic agents of the various components, either alone or in combination.
  • the substance of the present invention can be administrated by mouth, buccal, rectal, parenteral, topical, inhalation.
  • the substance can also be administered by controlled release or sustained release capsule systems and other drug delivery techniques.
  • the active substance may be combined with a pharmaceutically acceptable non-toxic, orally, inert carrier (such as lactose, starch, sucrose, glucose, methylcellulose, stearic acid) Magnesium, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, etc.; for oral administration of liquid dosage forms, the oral active substance can be combined with any pharmaceutically acceptable oral, non-toxic inert carrier (eg Ethanol, glycerin, water, etc. are combined. If desired, suitable binders, lubricants, disintegrants, and colorants can also be incorporated into the dosage form.
  • a pharmaceutically acceptable non-toxic, orally, inert carrier such as lactose, starch, sucrose, glucose, methylcellulose, stearic acid
  • any pharmaceutically acceptable oral, non-toxic inert carrier
  • Suitable binders include starch, gelatin, natural sugars such as glucosamine or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose. , polyethylene glycol, wax, etc.
  • Suitable lubricants for use in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • the disintegrant include starch, methyl cellulose, qiongyue, bentonite, xanthan gum and the like.
  • compositions of the present invention may also be administered in the form of liposome drug delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholine. Substances which are described in detail in the present invention and which are identified by the methods of the invention may also be coupled to soluble polymers which are orientable pharmaceutical carriers.
  • polymers examples include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl A methacrylamide-phenol, a polyhydroxyethylaspartamide phenol, or a polyethylene oxide-polylysine substituted with a palmitoyl group. These materials can also be coupled to biodegradable polymers used to control drug release. Suitable polymers include polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, poly ⁇ -caprolactone, polyhydroxybutyrate, polyorthoesters, polyacetals, polydihydropyrans Classes, polycyanoacrylates, hydrogel crosslinked or amphiphilic block copolymers. Suitable pharmaceutical carriers and methods of preparation are described in the Remington Medical Sciences Mack Publishing Company, which is a standard reference in the art.
  • Another aspect of the present invention also provides a method of screening a candidate for a drug for treating or preventing a disease associated with abnormal apoptosis of CD4 + T cells, the method comprising:
  • a substance which lowers the expression level of ⁇ -arrestin 1 in the nucleus is selected as a disease drug for treating or preventing abnormal apoptosis associated with CD4 + T cells.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, all of which are known to those skilled in the art.
  • T cells were labeled with a murine anti-human anti-CD4 antibody, and then isolated and purified using a goat anti-mouse IgG magnetic bead, a separation column, and an AutoMACS separation device, and CD4 + T cells having a purity greater than 90% were obtained by FACS. 3.
  • C57BL/6 mice were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. With C57BL/6
  • mice were presented by Dr. Robert J. Lefkowitz, Duke University Medical Center.
  • ⁇ a mice such as: Rapid xenograft tumor growth in beta-arrestinl transgenic mice due to the elevation of tumor angiogenesis Lin Zoul, 2, Rongxi Yangl, 3 and Gang Pei l described production, and backcrossed with C57BL/6 for 9 generations or more.
  • Animal feeding and operation are carried out in accordance with the regulations of the Experimental Animal Committee of the Shanghai Academy of Life Sciences of the Chinese Academy of Sciences. The animals used were housed in a pathogen free environment and the genome was identified prior to the experiment.
  • Mouse anti-Bcl-2 and Bax antibodies PE-conjugated antibodies against murine CD4, CD8, B220, and ⁇ 6 and FITC-conjugated antibodies against murine CD4, CD8, CD3 and ⁇ 8 were purchased from BD Biosciences the company.
  • Rabbit polyclonal antibodies against parrs (Al CT) were kindly provided by Dr. Robert J. Lefkowitz.
  • Antibodies against acetylated histone H4 and histone H3 were purchased from Upstate Biotechnology.
  • IRDyeTM 800CW conjugated anti-mouse IgG and anti-rabbit IgG purified antibodies were purchased from Rockland Corporation.
  • Anti-actin antibodies and reagents Actinomycin D and dexamethasone were purchased from Sigma.
  • Antibodies against Erk and anti-ser217/221 phosphorylated Erk are products of Cell Signaling, Inc., while anti-Akt and anti-ser473 phosphorylated Akt antibodies were purchased from Shanghai KANGCHEN Company. SEB is produced by Beijing Biotinge Biomedicine. Plasmids encoding ⁇ ga/, HA-parrl (described above) (29), HA-/rr2, arrlQ394L were constructed as previously described (32). / ⁇ 6/ ⁇ siRNA, pBS/U6 ⁇ rr2 siRNA and pBS/U6l non-specific siRNA plasmids were constructed as previously described (29).
  • the p300 wild type and its active region mutant (C/H1 deletion) plasmid was purchased from Upstate.
  • the human MBP small peptide was synthesized and purified by the Anderson Cancer Center Small Peptide Center Laboratory. The purity of the small peptide is greater than 90%.
  • the cells were resuspended in PBS buffer containing 1% BSA. Fluorescent antibodies such as CD4, CD8, CD3, B220, TCR Vp 8 and Vp 6 with the corresponding cell surface antigen are incubated in the recommended buffer for 4 hours at 4 degrees. The labeled cells were washed and analyzed by BD FACSAria instrument.
  • the spleen cells of the mouse were labeled with purified rabbit anti-mouse CD4 antibody or rabbit anti-mouse CD8 antibody, and then isolated and purified by goat anti-rabbit IgG magnetic beads, separation column and AutoMACS separation device, and obtained by FACS test.
  • the purity of T cells is greater than 95%.
  • the isolated and purified T cells were activated in a CD3 (CD3 ⁇ chain) monoclonal antibody culture dish coated with a large buccal mouse anti-mouse and a CD28 monoclonal antibody. Cultures were RPMI 1640 medium plus 10% fetal bovine serum, 2 mM L-glutamine, 5 mM 2-ME, and 100 units/ml penicillin.
  • Myelin matrix protein (MBP)-specific CD4 + T cells were cultured in RPMI1640 medium containing 20 g/ml MBP small peptide (33) and 100 U/ml human recombinant IL-2.
  • RNA in the experiment was extracted with TRIzol according to the Invitrogen guidelines.
  • oligoCdT primers and the superscript II system were used. All quantitative gene transcription experiments were performed by qPCR.
  • the qPCR system used mainly includes: Brilliant SYBR Green QPCR Master MIX and a Light Cycler detector (Stratagene).
  • the primers used were: murine Bc/-2-sense, 5'-TTC TCC TTC CAG CCT GAG AGC AA-3 ' (SEQ ID NO: 1), antisense, 5'-ATG ACC CCA CCG AAC TCA AAG-3 ( SEQ ID NO: 2)'; murine to-sense, 5'-AGG ATG CGT CCA CCA AG-3 ' (SEQ ID NO: 3), antisense, 5'-AAG TAG AAG AGG GCA ACC AC-3 ' (SEQ ID NO: 4); Mouse HPRT-se e, 5'-CCT GCT GGA TTA CAT TAA AGC ACT G-3 ' (SEQ ID NO: 5), antisense, 5'-TTC AAC ACT TCG AGA GGT CCT-3 ' (SEQ ID NO: 6); Murine HPRT cDNA input control; human HPRT- ⁇ e, 5'-CCT GCT GGA TTA CAT CAA AGC ACT G-3 ' (S
  • the transcriptional level of X Bcl-2 was done using the Tagman probe: Bcl-2-smse, 5'-CCT GTG GAT GAC TGA GTA CCT GAA-3 ' (SEQ ID NO: 19), antisense, 5 ' -CAG CCA GGA GAA ATC AAA CAG A-3 ' (SEQ ID NO: 20), Taqman probe, 5'-£AGG ATA ACG GAG GCT GGG ATG CCT TTP-3 ' (SEQ ID NO: 21).
  • amino acid residue sequence at position 35-55 of the MOG antigen used in EAE induction is:
  • Acute EAE induction was performed as follows: On the first day, subcutaneous injection of CFA containing 300 ⁇ ⁇ MOG peptide and 5 mg/ml heat-inactivated Mycobacterium tuberculosis H37Ra peptide chain (produced by BD), and tail vein injection 200 ng/ Pertussis toxin in mice.
  • mice On the third day, 200 ng/mouse of pertussis toxin was injected into the tail vein again. Mice that induced EAE were weighed and observed daily. According to the severity of the disease, the mice were scored, specifically: 0, without any symptoms of the disease; 1, the tail is weak; 2, - or two hind limbs squat; 3, two hind limb paralysis; 4, fore limb paralysis; 5, dying or dying.
  • T cells were maintained in serum-free RPMI 1640 medium. At the time point shown on the graph, using annexin
  • the -V-FLUOS Labeling Kit (Roche Molecular Biochemicals) obtained the percentage of viable cells by excluding PI and annexin V positive cells. In the experiment to detect the survival of GFP-positive cells, the PI alone was excluded. Positive cells gave a percentage of viable cells.
  • Tissues were removed from EAE mice that were induced for 20 days, fixed in 4% formalin, and embedded in paraffin. Then dye with Luxol fast blue or H&E. Finally, observation photographs were taken with an optical microscope. A total of 3-4 mice were spinal cords, and each mouse took 3 samples for quantitative demyelination and infection (34).
  • Chromatin immunoprecipitation was performed according to Upstate Biotech's CHIP kit.
  • the sequence of the specific gene promoter region in the loading and neutralizing immunoprecipitation complex was detected by qPCR.
  • the primers for the specific gene promoter region are designed to be in the base region of 1000 upstream to 500 downstream of the transcription start site. The results obtained were obtained by loading the amount in the precipitate using a loading gauge.
  • the promoters of the gene promoter region and the ⁇ 3 ⁇ 4/-2 gene locus are: mouse promoter: sense, 5'-GGC AAA CCC TCC CCC ACC ACC TC-3 ' (SEQ ID NO: 22), antisense, 5 '-CCA CCG GAC CGC TTC AGA CCT C-3 ' (SEQ ID NO: 23); murine ⁇ promoter, sense, 5'-GGG GAA ACA ACC AAC TCT GG-3 ' (SEQ ID NO: 24), antisense 5 '-CAT CAC TGC CGC TGC CTC T-3 ' (SEQ ID NO: 25); murine Bd-2 locus u25,000 sense, 5'-GCT GTT TAT CAG TTA GTG GGT C-3 ' (SEQ ID NO: 26), antisense 5'-GGG TCA GAA GTG GGA GTG-3 ' (SEQ ID NO: 27); u20,000 sense, 5' -TGC CAA GGT TAG CAG GAC-3 '
  • the [3 ⁇ ] thymidine incorporated in the DNA was detected by a ⁇ plate detector.
  • Jurkat and HEK293 cells were purchased from the American Type Culture Collection and were
  • RPMI1640 or MEM (Gibco-BRL) culture medium were transfected with Amaxa transfection kit, while HEK293 cells were transfected with calcium phosphate.
  • CD4 + T cells were activated with CD3 and CD28 antibodies for 24 hours as described above, and the supernatants were collected and assayed using the IL-2 ELISA kit according to the PIERCE guidelines. At the same time, purified and known concentrations of recombinant murine IL-2 were used as standard lines for quantification.
  • the nuclear fraction was extracted with some modifications as previously described (27). Specifically, the cells treated by various methods were washed and resuspended in 400 ⁇ l of hypotonic buffer for 10 minutes in an ice bath. Add 3 ⁇ 1 and 10% ⁇ -40 and mix and continue to ice bath for 5 minutes. After a brief centrifugation, the resulting precipitate is a crude extract of the nuclear component. This fraction was washed, resuspended in hypotonic buffer and shaken at 4 degrees for 1 hour. After centrifugation, the resulting supernatant is the nuclear component. 17. Reporter gene experiment
  • Clontech's pNF-kappa-B-Luc or pCREB-TA-Luc, and pRL-TK, and some of the plasmids shown in the figure were co-transfected into ⁇ 293 cells. After 36 hours of transfection, the activity of luciferase was detected using a dual luciferase reporter assay system. Cells treated with 10 ng/ml forskolin (FK) and 10 ng/ml rhTNF- ⁇ were used as positive controls for the experiments.
  • FK forskolin
  • Quantitative experimental data is expressed as an average of 3 ⁇ 4 ⁇ sem.
  • Bonferroni post-hoc to detect significant differences between the two sets of data for multiple sets of data or tow-tailed Student's t.
  • CD4 + T cells is apoptosis + T cells and thymus from entering the outer periphery of CD4 to CD4 + T cells maintained.
  • the number of murine + T cells and wild type (w t) mouse thymocytes CD4 CD8 + T cells was found not changed (FIG. 2C).
  • the inventors examined apoptosis of peripheral blood CD4 + T cells.
  • CD4 + T cells were isolated from / hrrr mice, ⁇ transgenic ( ⁇ rrHg) mice and wt mouse spleens, activated or not activated with anti-CD3 and anti-CD28 antibodies, and then cultured in serum-free and cytokine-free Culture in the base.
  • Figures 1B and 1C show that the viability of activated rrHg CD4 + T cells is significantly higher than that of wild type, while CD4 + T cells of ⁇ ⁇ - are significantly more likely than wild type in their naive or activated state. Death is more sensitive.
  • SEB can be specifically activated in the body
  • CD4 + ⁇ 8.1/2 ⁇ cells have no effect on CD4 + ⁇ 6.1 T cells (16).
  • Figures 1D and 1E The number of CD4 + Vp8.1/2 T cells in each genotype of mice increased significantly after three days of SEB injection, and decreased significantly after seven days of injection. During the next few days, it was observed that: ⁇ rrHg CD4 + Vp8.1/2 T cell apoptosis was significantly less than W, The CD4 + ⁇ 8.1/2 ⁇ cell apoptosis was increased in mice. These results indicate that Parrl also promotes the survival of CD4 + T cells in vivo.
  • IL-2 is an important cytokine (55-57) that promotes naive and activated CD4 + T cells.
  • CD4 + T cells When CD4 + T cells are activated in vitro, they produce large amounts of IL-2 ( 1 ) 0 , however, as shown in Figure 3A: The ability of CD4 + T cells to produce IL-2 is slightly stronger than its wt. Therefore, Parrl is unlikely to regulate the survival of these cells by affecting the production of IL-4 by CD4 + T cells. 2.parrl promotes the expression of Bcl-2
  • the molecular mechanism of Parrl promoting the survival of CD4 + T cells was further studied. Considering that activation of Akt (38) and Erk (39) promotes CD4 + T cell survival, and Parrl itself positively regulates Akt and Erk activity (20), therefore, whether it is due to Parrl upregulation of Akt or Erk Activity to promote the viability of CD4 + T cells.
  • the parl-'- and wt CD4 + T cells were activated in vitro as previously described, and their activity was detected using phosphorylated antibodies of Akt and Erk. As shown in Figure 3B, in naive and activated CD4+ T cells, The phosphorylation level of Erk; while the phosphorylation level of Akt is decreased in activated CD4 + T cells.
  • parrl can simultaneously promote the survival of naive and activated CD4 + T cells, and parrl does not affect the phosphorylation levels of Akt and Erk in naive CD4 + T cells, so it is speculated that parrl promotes the survival of CD4 + T cells.
  • Other mechanisms Affymetrix gene chip data showed that inhibition of parrl expression by siRNA can reduce the expression of anti-apoptotic molecule Bd-2, but does not affect the expression of other proteins in Bcl-2 family, such as Bim, Bax and Bcl-xl.
  • Bcl-2 is an important molecule regulating the survival of naive and activated T cells (1, 16), thus further detecting the expression of parrl and Bcl-2 in CD4 + T cells. After CD4 + T cells were activated as described above, the protein levels of ⁇ and Bcl-2 gradually decreased in the first two days, and increased on the third day after activation and subsequently maintained (Fig. 5A).
  • parrl and Bcl-2 When the expression of parrl and Bcl-2 was further detected during the period of 40 to 64 hours of activation, it was found that the level of parrl protein was earlier than that of Bcl-2 (Fig. 5A). Is it that parrl regulates the expression of Bcl-2 in CD4 + T cells?
  • the inventors simultaneously activated CD4 + T cells of y&rrHg mice and W, and detected mRNA and protein levels of parrl and Bc!-2. It was found that the level of parrl in the CD4 + T cells derived from ⁇ rrHg mice was significantly higher than that in the wild type after 48 hours of activation, and the mRNA and protein levels were also significantly increased at this time (Fig. 5B). .
  • arrl promotes histone acetylation in the locus
  • Another way parrl regulates gene expression is by up-regulating the level of acetylation of histone H4 in the promoter region of a specific gene (27).
  • the inventors found that the pattern of changes in the level of histone H4 acetylation in the promoter region of the gene was very similar to that of Bd-2 (Fig. 7A).
  • the level of histone H4 acetylation in the promoter region of the Bcl-2 gene gradually decreased after activation of CD4 + T cells, reached a minimum at 48 hours, and then increased again at 72 hours.
  • the level of H3 acetylation as a control protein for histone H4 acetylation levels did not change.
  • parrl also affects the level of histone H4 acetylation in the promoter region of Bd-2 gene.
  • the histone H4 acetylation level of the Bd-2 gene promoter region was significantly increased relative to the wild type (Fig. 7B).
  • the level of histone H4 acetylation in the promoter region of the gene remained at the lowest level equivalent to that in the wild type (Fig. 7C).
  • parrl affects the level of histone H4 acetylation in the locus region from 10,000 bp upstream to 186,000 bp downstream of the transcription initiation site (Fig. 7D). These experiments show that parrl is likely to upregulate gene expression by promoting histone H4 acetylation in the locus. 4. p300 is required for upregulating Bcl-2 expression in parrl
  • the level of acetylation of intracellular histones is regulated by histone deacetylase and histone acetylase.
  • histone deacetylase and histone acetylase.
  • Previous studies have found that parrl promotes acetylation of histone H4 by binding to the histone acetylase p300 (43) and recruiting it to the promoter region of a specific gene (27). Therefore, the inventors examined the role of p300 in up-regulating Sc/expression in ⁇ ⁇ . Consistent with the previous results: In Jurkat cells, parrl, but not parr2 or parrlQ394L, promoted histone H4 acetylation levels in the promoter region of the gene (Fig. 8A).
  • p300 also promotes histone H4 acetylation level and mRNA level in the promoter region of Bcl-2 gene, and this promotion can be further enhanced by co-expression parrl and inhibited by co-expression of rr siRNA (Fig. 8C). And 8E).
  • p300DN active mutant of p300 significantly reduced histone H4 acetylation and Sc/ ⁇ mRNA levels in the promoter region of the gene, and also blocked the promotion of parrl ( Figures 8C and 8E).
  • MOG35-55 to mobilize MOG-specific CD4 + T cells purified from spleen lymphocytes of EAE mice, and observed the survival of these CD4 + T cells under cytokine stress. Consistent with Figures 1B and 1C: MOG-specific CD4 + T cells from arrltg mice were significantly more viable than wild-type, whereas CD4 + T cells from mice were less viable ( Figure 9D). . These results indicate that parrl plays a positive regulatory role in the EAE model, which may be due to the ability to promote the viability of CD4 + T cells.
  • the immune system In mammals, the immune system is one of the most dynamic systems in the body.
  • the apoptosis of immune cells in the immune system is regulated by many extracellular and intracellular signals to maintain their normal physiological functions.
  • Parrl is a signal molecule with multiple functions and is involved in the regulation of many signal paths. In addition, it has the ability to directly regulate nuclear-specific gene transcription.
  • the inventors have found in the present invention that parrl specifically regulates the survival and homeostasis of CD4+ T cells, thereby affecting the acquired immune response of the body.
  • the activity of Akt is inhibited in activated CD4 + T cells, the ability of parrl to affect the survival of CD4 + T cells is mainly achieved by its intranuclear function.
  • parrl In either the naive or activated CD4+ T cell nucleus, parrl upregulates the level of histone H4 acetylation in the promoter region of the gene, thereby promoting gene expression. Further studies have found that parrl plays an important role in demyelinating diseases caused by autoimmunity and type I diabetes caused by streptozotocin. In demyelinating diseases caused by autoimmunity, the high expression of ⁇ rr in CD4+ T cells that specifically cause encephalitis promotes the viability of these cells in vivo. Therefore, the inventors' results indicate that parrl plays an important regulatory role in the survival of CD4+ T cells and autoimmune diseases in humans and mice.
  • Bcl-2 regulates cell survival by regulating the integrity of the mitochondrial membrane and the release of pro-apoptotic proteins present in mitochondria (12). In T cells, Bcl-2 must be maintained at an appropriate level, otherwise it will cause the breaking of T cell homeostasis and the confusion of immune response.
  • the regulation mechanism of Bcl-2 in T cells has not been well understood. The inventors found that parrl promotes the level of histone acetylation in the gene region and the transcription level of the ⁇ 3 ⁇ 4/-2 gene, whether in naive or activated CD4+ T cells.
  • parrl promoted T cell survival mainly in peripheral blood CD4 + but not CD8 + T cells or thymic T cells.
  • thymic T cells as shown in Figure 2A: the expression level of parrl is significantly lower than its expression level in spleen cells or lymph node cells. This difference in expression levels may be responsible for the small role of parrl in thymic T cells.
  • Figure 11B the difference in parrl expression levels in CD4 + and CD8 + T cells is not significant.
  • the effect of parrl on the survival of CD4 + and CD8 + T cells was significantly different.
  • parrl differs in the subcellular localization of CD4 + and CD8 + T cells. As shown in 11C and 11D, parrl is expressed in the nucleus at a higher level than CD8 + T cells in both naive and activated CD4+ T cells. height of. Therefore, it may be that the distribution of parrl in subcellular levels in CD4 + and CD8 + T cells results in a different effect of parrl on the survival of CD4 + and CD8 + T cells.
  • parrl and parr2 are recruited to the cell membrane when GPCR is activated; where they bind to phosphorylated GPCRs causing receptor endocytosis and signal termination (47).
  • parrl but not arr2
  • This result of the inventors is consistent with previous conclusions (27), which may be due to the different distribution of the two parr at the subcellular level.
  • the two parrs are different in this function, they can regulate the immune response from the animal level.
  • parrl has the function of directly regulating gene transcription in the nucleus; this function is affected by some GPCRs (27).
  • Nguyen K. and Miller BC were found to have expression of delta opioid receptor (DOR) (48).
  • DOR delta opioid receptor
  • mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75, 229-40 (1993).
  • Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling. Science 301, 1394-7 (2003).
  • Kang, J. et al. A nuclear function of beta-arrestinl in GPCR signaling: regulation of histone acetylation and gene transcription. Cell 123, 833-47 (2005).
  • the transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953-9 (1996).

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Abstract

L'invention concerne l'utilisation d'un inhibiteur de la bêta-arrestine 1 pour la fabrication d'un médicament pour la prophylaxie ou le traitement d'un trouble chez des mammifères associé à l'apoptose des lymphocytes T CD4+. L'inhibiteur de la bêta-arrestine 1 est choisi parmi (i) des composés inhibant l'activité de la bêta-arrestine (1) ; (ii) des composés inhibant la transcription et/ou la traduction du gène de la bêta-arrestine 1.
PCT/CN2008/071180 2007-06-04 2008-06-04 Utilisation de la bêta-arrestine 1 pour moduler la survie et l'auto-immunité des lymphocytes t WO2008148349A1 (fr)

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WO2021159093A1 (fr) * 2020-02-07 2021-08-12 University Of South Florida Inhibition de l'oligomérisation de la bêta-arrestine en tauopathie

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CN102816205B (zh) * 2011-06-09 2014-12-10 中国科学院上海生命科学研究院 β抑制蛋白1、其片段及其应用
CN105561297A (zh) * 2016-01-15 2016-05-11 中国人民解放军第二军医大学 巨噬细胞β-抑制蛋白-1在制备预防或治疗动脉粥样硬化药物中的应用

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WO2004103287A2 (fr) * 2003-05-14 2004-12-02 Duke University Modulation de la beta-arestine-2 pour le traitement de l'asthme allergique
US20060029985A1 (en) * 2004-08-03 2006-02-09 Hypatia Ltd. Diagnostic compositions containing an antibody to a beta-arrestin, methods utilizing the same and kits containing the same

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WO2004103287A2 (fr) * 2003-05-14 2004-12-02 Duke University Modulation de la beta-arestine-2 pour le traitement de l'asthme allergique
US20060029985A1 (en) * 2004-08-03 2006-02-09 Hypatia Ltd. Diagnostic compositions containing an antibody to a beta-arrestin, methods utilizing the same and kits containing the same

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021159093A1 (fr) * 2020-02-07 2021-08-12 University Of South Florida Inhibition de l'oligomérisation de la bêta-arrestine en tauopathie
US11896644B2 (en) 2020-02-07 2024-02-13 University Of South Florida Inhibition of beta-arrestin oligomerization in tauopathy

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