CN116411061A - Application of E3 ligase RNF99 in negative regulation of TLR-mediated inflammatory immune response - Google Patents

Application of E3 ligase RNF99 in negative regulation of TLR-mediated inflammatory immune response Download PDF

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CN116411061A
CN116411061A CN202310038858.6A CN202310038858A CN116411061A CN 116411061 A CN116411061 A CN 116411061A CN 202310038858 A CN202310038858 A CN 202310038858A CN 116411061 A CN116411061 A CN 116411061A
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rnf99
expression
tab2
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张猛
张�杰
张澄
张运
考春雨
高飞
曹磊
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Shandong University
Qilu Hospital of Shandong University
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Qilu Hospital of Shandong University
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Abstract

The invention provides an application of E3 ligase RNF99 in negative regulation of TLR-mediated inflammatory immune response, belonging to the technical fields of biological medicine and molecular biology. According to the invention, the E3 ubiquitin ligase RNF99 is used as a feedback regulation factor, so that TLRs-mediated macrophage inflammatory immune response can be negatively regulated, and the immune response can be further participated in the progress of mouse endotoxemia and colonitis. Mechanistically, it was found that RNF99 can specifically bind to TAB2, promoting ubiquitination modification at position K48 of lysine at position K611, thereby mediating its degradation via the proteasome pathway. In particular, E3 ligase RNF99 modulates TAB2 degradation via ubiquitin-proteasome pathway, thereby affecting the formation of the protein kinase TAK1-TAB1-TAB2 complex and inflammatory response, and thus has potential practical value.

Description

Application of E3 ligase RNF99 in negative regulation of TLR-mediated inflammatory immune response
Technical Field
The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to application of E3 ligase RNF99 in negative regulation of TLR-mediated inflammatory immune response.
Background
The information disclosed in the background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Activation of the innate immune system requires pattern recognition receptors (PRPs) to recognize pathogen-associated molecular patterns (PAMPs), whereas macrophages are the primary effector cells. Toll-like receptors (TLRs), the most recently discovered and studied pattern recognition receptor, are a bacterial sensor that serves as an interface between the external environment and the cellular response. Once the TLRs recognize conserved microbial related motifs, macrophages secrete pro-inflammatory factors such as TNF- α, IL-1β and IL-6, which in turn activate further inflammatory signals. Abnormal TLRs signaling can lead to abnormal inflammatory responses and many related immune diseases, including sepsis and Inflammatory Bowel Disease (IBD). It is a pathological syndrome characterized by persistent excessive inflammation and immunosuppression, with high morbidity and mortality. TLR agonists have shown great potential as antibacterial drugs and vaccine adjuvants, while TLR antagonists are being developed as agents and drugs that inhibit immune responses. Past studies have shown that TLR4 can be a therapeutic target for sepsis. Despite the great knowledge of the key pathogenesis of TLR-mediated human diseases, there is a lack of entirely effective clinical therapies. Therefore, it is important to investigate the regulatory mechanism of TLR signaling to avoid aberrant activation of TLR signaling.
TLRs initiate signaling cascades by recruiting MyD88 or tri when stimulated by ligands such as Lipopolysaccharide (LPS) (TLR 4 ligand), R848 (TLR 7 ligand), and LTA (TLR 2 ligand). MyD88 triggers the formation and activation of the TAK1-TABs complex, leading to phosphorylation of nuclear factors NF-. Kappa.B and MAPKs. Eventually eliciting immune and inflammatory responses, fever, endotoxemia and shock. Studies have shown that TAK1 plays an important role in tumor necrosis factor receptor, interleukin-1 receptor I, and TLR-mediated NF- κB and MAPKs activation. Activation of TAK1 requires proteins (TAB 1, TAB2 and TAB 3) that bind to TAK1, whereas the TAK1-TAB complex plays an important role in innate immune and inflammatory responses. Although the TAK1-TAB complex has been widely studied, the role of individual proteins and their mechanism of activating molecules in different cell types remain to be elucidated.
Intracellular signals are covalently linked to protein substrates through different ubiquitin and are involved in numerous cellular molecular processes. In recent years, there has been growing evidence that E3 ligases are involved in the regulation of innate immunity and inflammation by modifying proteins associated with TLRs signaling pathways. For example, the E3 ligase Nrdp1 may modify MyD88 and TBK1 by ubiquitination, thereby down-regulating TLR-mediated expression of pro-inflammatory cytokines. The E3 ligase TRIM38 promotes k48 link ubiquitination of TRIF and negatively regulates TLR3 mediated IFN- β expression. TRIM38 can also promote ubiquitination of k48 links, degradation of NAP1, and inhibition of IRF3 activation, thereby negatively regulating TLR/RLR mediated signaling pathways. RNF99 is a new member of the TRIM family of E3 ligases, and is currently less studied. Studies have shown that RNF99 can act as a tumor suppressor in the brain by ubiquitination modification and stabilization of p53 through its E3 ligase activity. Meanwhile, studies have shown that RNF99 can negatively regulate NF-. Kappa.b-mediated transcriptional activity and proliferation of HeLa or NIH3T3 cells, but lacks detailed mechanisms and targets for ubiquitination modification. Currently, the function and mechanism of action of RNF99 in different diseases is still to be further studied.
There is a great deal of evidence that post-translational modifications of TAK1 and TAB play an important role in regulating TAK1 activation. Ubiquitination is by far the most widely studied post-translational modification, in addition to phosphorylation. Studies have shown that ubiquitin-proteasome systems play a critical role in the assembly of the TAK1-TAB complex and the activation of TAK 1. However, the studies on degradation of TAB2 have focused mainly on the lysosomal pathway. It has been reported that the E3 ligases TRIM38, TRIM 30. Alpha., TRIM22 and RNF4 can target TAB2 and degrade TAB2 via the lysosomal pathway. However, it is not clear whether the proteasome pathway is involved in the degradation of TAB2. Therefore, it is important to further explore the specific E3 ubiquitin ligase promoting TAB2 degradation in the proteasome pathway and to research the molecular mechanism of the enzyme affecting TAB2 ubiquitination and related TAK1 kinase activity, and to determine the physiological significance of the enzyme in inflammatory diseases such as endotoxemia, colonitis and the like.
Disclosure of Invention
In view of the shortcomings in the prior art, it is an object of the present invention to provide the use of E3 ligase RNF99 in the negative modulation of TLR-mediated inflammatory immune responses. According to the invention, the E3 ubiquitin ligase RNF99 is used as a feedback regulation factor, so that TLRs-mediated macrophage inflammatory immune response can be negatively regulated, and the immune response can be further participated in the progress of mouse endotoxemia and colonitis. Mechanistically, it was found that RNF99 can specifically bind to TAB2, promoting ubiquitination modification at position K48 of lysine at position K611, thereby mediating its degradation via the proteasome pathway. In particular, E3 ligase RNF99 modulates TAB2 degradation via the ubiquitin-proteasome pathway, thereby affecting the formation of the protein kinase TAK1-TAB1-TAB2 complex and inflammatory response. Based on the above results, the present invention has been completed.
Specifically, the technical scheme of the invention is as follows:
in a first aspect of the invention there is provided the use of a substance which detects the expression level of the E3 ubiquitin ligase RNF99 encoding gene and/or the expression product of the RNF99 encoding gene in the preparation of a product for screening, diagnosis (co-diagnosis), detection, monitoring or prediction of the progression of inflammatory disease.
In a second aspect of the invention, there is provided a product for screening, diagnosing (co-diagnosing), detecting, monitoring or predicting the progression of an inflammatory disease comprising detecting transcription of an RNF99 encoding gene in a subject based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; or detecting the expression of the subject E3 ubiquitin ligase RNF99 based on an immunoassay method.
The product may be a kit.
In a third aspect of the invention there is provided the use of a substance which promotes the expression and/or activity of an RNF99 encoding gene and its expression products in at least one of the following a 1) to a 4):
a1 Promoting K48 ubiquitination modification and proteasome degradation of the K611 site of the TAB2 protein, thereby inhibiting the formation of a TAK1/TABs complex and the activation of TAK1 kinase or preparing a product promoting the K48 ubiquitination modification and proteasome degradation of the K611 site of the TAB2 protein, thereby inhibiting the formation of the TAK1/TABs complex and the activation of the TAK1 kinase;
a2 Products that inhibit the activation of NF- κB and MAPKs signaling pathways or inhibit the activation of NF- κB and MAPKs signaling pathways;
a3 Inhibiting or producing products of inflammatory cytokine production in macrophages induced by TLRs ligands;
a4 For the preparation of a product for the prophylaxis and/or treatment of inflammatory diseases.
Wherein, the liquid crystal display device comprises a liquid crystal display device,
in the a 1), the TAK1/TABs complex is specifically a TAK1-TAB1-TAB2 complex.
In the a 3), the inflammatory cytokines include, but are not limited to, TNF- α, IL-6 and IL-1β.
In the a 4), the inflammatory diseases include TLR-related inflammatory diseases including, but not limited to, bacterial (G) - ) Infectious diseases, endotoxemia and colitis.
The product may be a drug or an experimental reagent that may be used for basic research. For example, the product is used for constructing inflammation-related cells, tissues or animal models, and research on TLR-mediated inflammation-related reactions and the like by regulating and controlling the expression of RNF99, and has good practical application value.
Meanwhile, as described above, the product can also be developed into a medicament, which is probably an important strategy for improving TLR related inflammatory diseases such as endotoxemia, refractory colitis and the like in the future.
The beneficial technical effects of one or more of the technical schemes are as follows:
the above technical scheme finds gram-negative bacteria (G - ) The expression of E3 ligase RNF99 in the peripheral blood mononuclear cell TLRs ligand-stimulated macrophages of infected patients was significantly reduced, suggesting a regulatory role for RNF 99. The above technical scheme is then used for the first time with RNF99 knockout mice (RNF 99-/-) and bone marrow transplanted mice, demonstrating the protective effect of RNF99 on LPS-induced infectious shock and Dextran Sulfate Sodium (DSS) -induced colitis. In vitro experiments show that RNF99 deletion significantly promotes macrophage TLR-mediated inflammatory cytokine expression, activating NF- κb and MAPK pathways. Mechanically, in macrophages and HEK293 cell lines stably transfected with TLR4, RNF99 interacts with TAB2 via the ubiquitin-proteasome pathway and mediates its degradation, further regulating TLR-mediated inflammatory responses.
Taken together, the above technical scheme suggests that RNF99 in macrophages has important physiological implications in regulating TLR-mediated inflammatory responses. The method provides new insight for TLRs signal transduction and a new method for preventing and treating bacterial infection, endotoxin shock and other inflammatory diseases, and therefore has good potential practical application value.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 shows that gram negative bacterial (G-) infection and TLRs stimulated reduced macrophage RNF99 expression in examples of the present invention. (A) Relative levels of RNF99 mRNA after different time points of stimulation of PMs by LPS (n=5-6), R848 (n=7), or LTA (n=6). (B) Representative WB images of RNF99 in PMs after various time periods of LPS, R848 or LTA stimulation. (C) Relative levels of tnf- α, il-6 and il-1β mRNA in peripheral blood mononuclear cells of healthy and G-infected patients (n=15). (D) Peripheral blood mononuclear cell RNF99 mRNA relative levels (n=15) in healthy and G-infected patients.
FIG. 2 shows that RNF99 knockout significantly aggravates TLR-mediated endotoxemia and acute colitis in the examples of the present invention, sex-and age-matched RNF99+/+ and RNF 99-/-mice were intraperitoneally injected with LPS or PBS. (A) H & E staining and histopathological analysis of mouse lung sections. Scale bar = 50 μm. (B) four groups of lungs were stained for representative CD68 and analyzed quantitatively. (C) ELISA analysis of TNF- α, IL-6 and IL-1β in serum of LPS stimulated RNF99+/+ and RNF 99-/-mice (n=8). (D) Survival of RNF99+/+ and RNF99-/-mice after LPS intraperitoneal injection. Sex and age matched RNF99+/+ and RNF 99-/-mice were given DSS drinking water for 7 days, or as controls (n=8-10). (E) Daily body weight, (F) stool softness, (G) bleeding changes. (H, I) day 7 photographs were taken to measure the colon length of RNF99+/+ and RNF 99-/-mice. (J, K) observing histopathological changes in colon tissue following H & E staining. Scale bar = 50 μm. (L) ELISA analysis of TNF- α, IL-6 and IL-1β levels (n=8) in serum of DSS treated mice.
FIG. 3 shows that RNF99 in the examples of the present invention modulates TLRs mediated inflammatory diseases in macrophages, and that RNF99+/+ mouse bone marrow cells matched to sex and age are depleted after irradiation. Transplantation from RNF99+/+ or RNF 99-/-mouse donors, respectively. The intraperitoneal injection of LPS establishes an endotoxemia model after bone marrow reconstruction of mice. Schematic of bone marrow transplantation (RNF99+/+ →RNF99+/+). (B) Mice with RNF99+/+ or RNF99+/+ reconstructed bone marrow cells were stained for H & E in lung sections following LPS induction. Scale bar = 50 μm. (C) ELISA analysis of TNF- α, IL-6 and IL-1β levels in serum of mice after LPS treatment (n=8). And constructing an acute colitis model after mouse bone marrow reconstruction by adopting DSS. (D) Clinical and pathological changes induced by DSS following RNF99+/+ mice engrafting to RNF99+/+ or RNF 99-/-. (E) DSS-induced changes in colonic length in colitis mice. (F) DSS induces colon histopathological changes in mice. (G) ELISA analysis of TNF- α, IL-6 and IL-1β levels in serum of DSS treated mice (n=8).
FIG. 4 shows the generation of macrophage inflammatory cytokines and activation of signaling pathways mediated by the loss of forward regulatory TLRs from RNF99 in an embodiment of the present invention. Wherein (a) ELISA analysis of TNF- α, IL-6 and IL-1β following LPS induction in RNF99+/+ or RNF 99-/-mouse pms (n=6). (B) RT-PCR and ELISA analysis of RNF99+/+ or RNF99-/-mouse PMs for TNF- α, IL-6 and IL-1β after R848 induction (n=6). (C) RT-PCR and ELISA analysis of LTA-induced TNF- α, IL-6 in PM of RNF99+/+ or RNF 99-/-mice (D, E) LPS, R848 or WB analysis of IκB- α, P65, ERK, JNK and P38 phosphorylation levels in PMs of RNF99+/+ or RNF 99-/-mice after LTA stimulation.
FIG. 5 shows the production of macrophage inflammatory cytokines and activation of signaling pathways mediated by the overexpression of RNF99 negatively-regulated TLRs in examples of the present invention. (A) ELISA analysis (empty vector, RNF99-WT or RNF 99-DeltaRing) overexpression and expression of TNF- α, IL-6 and IL-1β in PMs after LPS stimulation. (B-D) immunoblot analysis (empty vector, RNF99-wt or RNF99- ΔRing) showed altered levels of phosphorylation of IκB- α, P65, ERK, JNK and P38 following LPS, R848 or LTA stimulation.
FIG. 6 is a graph showing that RNF99 affects the formation of TAK1-TAB1-TAB2 complexes by promoting TAB2 degradation in an embodiment of the present invention. (A) Representative images of WB assays after cotransfection of IKK- α, IKK- β, IKK- γ, TAB1, TAB2, TBK1, IRAK-4, TRAF6, TAK1 with empty vector or RNF99 in 293-TLR4 cells. (B) Representative images of WB assays for TAK1, TRAF6, TAB1, TAB2, IRAK-1 and IRAK-4 in RNF99+/+ or RNF 99-/-mouse PMs. (C) Effect of different types of Myc-RNF99 (WT,. DELTA.Ring and C94/97A) on HA-TAB2 expression in 293-TLR4 cells. (D) Myc-RNF99 in 293-TLR4 cells affects expression of HA-TAB2 under DMSO, MG132, bortezomib, NH4Cl or chloroquinone pretreatment. (E) Expression of pTAK1 after LPS stimulation in RNF99+/+ or RNF 99-/-mouse PMs. (F) Changes in TAB1 binding to TAK1 following LPS stimulation in RNF99+/+ or RNF 99-/-mouse PMs.
FIG. 7 illustrates the interaction of RNF99 with TAB2 in an embodiment of the present invention. (A) CO-IP analysis of Flag-TAB2 and GFP-RNF99 interactions in LPS-treated 293-TLR4 cells. (B) CO-IP analysis of TAB2 and RNF99 interactions in LPS-treated PMs. (C) GFP-TAB2 and Flag-RNF99 co-localize in LPS-treated 293-TLR4 cells. (D) Schematic representation of RNF99 (WT) and its truncated mutant construction. (E) CO-IP analysis of the interaction of RNF99 truncation mutants with GFP-TAB2 in 293-TLR4 cells.
FIG. 8 shows ubiquitination of position K48 of the K611 site of TAB2 by RNF99 in an embodiment of the present invention. (A) CO-IP analysis of Flag-TAB2 ubiquitination modifications by Myc-RNF99 under different types of UB (WT, K48, or K63) expression in 293-TLR4 cells. (B) Analysis of GFP-TAB2 ubiquitination modified CO-IP in 293-TLR4 cells transfected with HA-UB-K48 and Flag-RNF99 (WT), flag-RNF 99-. DELTA.Ring or Flag-RNF99 (C53A/C56A) plasmids. (C) TUBE analysis of TAB2 ubiquitination modifications following LPS stimulation in RNF99+/+ or RNF 99-/-mouse PMs. (D) CO-IP analysis of Myc-RNF99 ubiquitination modifications of Flag-RNF99 point mutants (K522R, K611R, K653R and K656R) in 293-TLR4 cells. (E) Analysis of Myc-RNF99 degradation of Flag-RNF99 Point mutants (K522R, K611R, K653R, K656R, K653/656R) in 293-TLR4 cells.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention will now be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not intended to be limiting in any way. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.
As previously mentioned, RNF99 is a novel member of the TRIM family of E3 ligases, whose function and mechanism of action in different diseases has yet to be studied further.
In view of this, in a typical embodiment of the present invention, there is provided the use of a substance that detects the expression level of the E3 ubiquitin ligase RNF99 encoding gene and/or the expression product of the RNF99 encoding gene in the preparation of a product for screening, diagnosis (co-diagnosis), detection, monitoring or prediction of progression of inflammatory disease.
Experiments show that after stimulation by TLR4 ligand-lipopolysaccharide LPS, mRNA and protein levels of RNF99 are significantly down-regulated. Similar results were obtained with TLR7/8 ligand-R848 and TLR6 ligand lipoteichoic acid LTA treatment. It was shown that RNF99 may be a feedback regulator of TLR-mediated innate immune responses. Meanwhile, the detection finds that compared with a healthy subject, G - The decrease in RNF99 levels in infected patients suggests that its expression in monocytes may be closely related to bacterial infection. And further demonstrates that RNF99 has important physiological implications in regulating TLR-mediated inflammatory responses.
The RNF 99-encoding gene and its expression product may be of human or non-human mammalian (e.g., mouse, etc.); the expression product of the RNF 99-encoding gene may obviously be E3 ubiquitin ligase RNF99.
The inflammatory disease may be in particular a TLR-related inflammatory disease including, but not limited to, bacterial (G - ) Infectious diseases, endotoxemia and colitis.
In yet another embodiment of the invention, a product for screening, diagnosing (aiding diagnosis), detecting, monitoring or predicting the progression of an inflammatory disease is provided comprising detecting transcription of an RNF99 encoding gene in a subject based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; or detecting the expression of the subject E3 ubiquitin ligase RNF99 based on an immunoassay method.
In yet another embodiment of the invention, transcription of the RNF 99-encoding gene of the subject may be detected using techniques including, but not limited to, liquid phase hybridization, northern hybridization methods, miRNA expression profiling chips, ribozyme protection analysis techniques, RAKE methods, in situ hybridization; the subject's E3 ubiquitin ligase RNF99 expression is detected using a protocol including but not limited to ELISA, colloidal gold test strips, and protein chips.
The subject may be a human or a non-human mammal (e.g., a mouse).
The product may be a kit.
In yet another embodiment of the invention, there is provided the use of a substance that promotes increased expression and/or activity of an RNF99 encoding gene and its expression products in at least one of the following a 1) -a 4):
a1 Promoting K48 ubiquitination modification and proteasome degradation of the K611 site of the TAB2 protein, thereby inhibiting the formation of a TAK1/TABs complex and the activation of TAK1 kinase or preparing a product promoting the K48 ubiquitination modification and proteasome degradation of the K611 site of the TAB2 protein, thereby inhibiting the formation of the TAK1/TABs complex and the activation of the TAK1 kinase;
a2 Products that inhibit the activation of NF- κB and MAPKs signaling pathways or inhibit the activation of NF- κB and MAPKs signaling pathways;
a3 Inhibiting or producing products of inflammatory cytokine production in macrophages induced by TLRs ligands;
a4 For the preparation of a product for the prophylaxis and/or treatment of inflammatory diseases.
Wherein, the substances which promote the increase of the expression and/or activity of the RNF 99-encoding gene and the expression product thereof include, but are not limited to, substances which up-regulate the expression and/or promote the activity of the RNF 99-encoding gene based on the gene-specific Mimics technology; such as short hairpin RNA (shRNA) of synthetic RNF99, or a promoter or lentivirus for up-regulating RNF99 expression; and may also include compound accelerators.
Wherein, the liquid crystal display device comprises a liquid crystal display device,
in the a 1), the TAK1/TABs complex is specifically a TAK1-TAB1-TAB2 complex.
In the a 3), the inflammatory cytokines include, but are not limited to, TNF- α, IL-6 and IL-1β.
In the a 4), the inflammatory diseases include TLR-related inflammatory diseases including, but not limited to, bacterial (G) - ) Infectious diseases, endotoxemia and colitis.
The product may be a drug or an experimental reagent that may be used for basic research. For example, the product is used for constructing inflammation-related cells, tissues or animal models, and research on TLR-mediated inflammation-related reactions and the like by regulating and controlling the expression of RNF99, and has good practical application value.
Of course, substances that inhibit the expression and/or decrease in the expression of the RNF99 encoding gene and its expression products may exacerbate the occurrence of inflammatory diseases mediated by TLRs (e.g., endotoxemia and colitis). Therefore, substances which inhibit the expression and/or decrease of the RNF 99-encoding gene and its expression products (such as substances which knock out the RNF 99-encoding gene based on the CRISPR/Case9 technology) can be used for the construction of models of endotoxemia and colitis animals (such as mice, etc.), thereby conducting related scientific researches.
Meanwhile, as described above, the product can also be developed into a medicament, which is probably an important strategy for improving TLR related inflammatory diseases such as endotoxemia, refractory colitis and the like in the future.
According to the invention, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.
The pharmaceutically inactive ingredients may be carriers, excipients, diluents and the like which are generally used in pharmacy. Further, the composition can be formulated into various dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injectable solutions according to a usual method.
The medicament may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a blending agent, a surfactant, a dispersing agent, or an antifoaming agent.
The medicament may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a virus, a microcapsule, a liposome, a nanoparticle or a polymer, and any combination thereof. The delivery vehicle for the pharmaceutically acceptable carrier may be, in particular, a liposome, a biocompatible polymer (including natural and synthetic polymers), a lipoprotein, a polypeptide, a polysaccharide, a lipopolysaccharide, an artificial viral envelope, an inorganic (including metallic) particle, and a bacterial or viral (e.g., adenovirus, baculovirus, retrovirus, etc.), phage, cosmid, or plasmid vector.
In yet another embodiment of the invention, the medicament of the invention may be administered to the body in a known manner. For example, by intravenous systemic delivery or local injection into the tissue of interest. Administration is optionally via oral, intravenous, transdermal, intranasal, mucosal, or other delivery methods; in particular, the medicaments of the present invention may be administered by means of bone marrow transplantation, which is exemplified in detail in the specific embodiments of the present invention, such administration being via single or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.
In yet another embodiment of the present invention, the subject to be administered can be human or non-human mammal, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The test methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions.
Examples
1. Material method
Animals and cells
RNF 99-/-mice in the context of C57BL/6J were produced by beijing Shang Lide biotechnology company using CRISPR/Cas9 technology. The PCR fragment amplified from the tail end genomic DNA was sequenced using the following primers, and the genotyping of the deficient mice was determined as Forward 5'-tgcctacaacttccagaatgc-3' and Reverse 5'-CATCTAAGTGCCTCGCTTCC-3'. Mice were backcrossed to C57BL/6J mice for >10 passages prior to the experiment. Animals were randomly grouped using a random number table, and littermates from RNF 99-/-mice were used in the experiments to avoid potentially conflicting results. From our preliminary experiments, we set the significance level (α) to 0.05,1- β to 80% to determine the sample size. The exact number of each group is contained in the respective graphical illustration.
PMs were obtained from RNF 99-/-mice and their control mice (RNF99+/+). HEK293TLR4 cells were purchased from invitogen. Cells were cultured in DMEM (Gibco, USA) to which 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) were added.
Collection of human blood samples
Blood samples were collected from patients who were confirmed to be infected with gram-negative (G-) bacteria by menstrual blood culture. The pathogenic bacteria include 15 strains including 5 strains of Pseudomonas aeruginosa, 4 strains of Acinetobacter baumannii, 3 strains of Escherichia coli, 1 strain of Bacteroides fragilis, 1 strain of Enterobacter cloacae and 1 strain of Burkholderia cepacia. At the same time, blood samples were collected from healthy control individuals, and blood was negative for bacterial infection. Whole blood and serum samples were collected separately. Peripheral blood mononuclear cells of whole blood samples were labeled with Fitc against cd14 + The staining was followed by sorting and further real-time quantitative PCR (RT-PCR) analysis. All surveys were conducted as per the declaration of helsinki and were approved by the ethical committee of the Qilu hospital at Shandong university and informed consent was obtained for all participants.
LPS-induced mouse endotoxin
Endotoxin studies used age-matched (8 week old) RNF99+/+ and RNF 99-/-male and female mice were intraperitoneally injected with LPS (40. Mu.g/g) or PBS as control groups. Every 12 hours, a part of mice are monitored for survival, after 4 hours, the rest of mice are sacrificed to observe lung tissue changes, and after blood collection, the serum TNF-alpha, IL-6 and IL-1 beta levels are measured by adopting corresponding ELISA kits.
DSS-induced colitis in mice
In inflammatory colitis study, age-matched (8 week old) RNF99+/+ and RNF 99-/-male and female mice were fed 3.5% (w/v) DSS drinking water for 6-7 days, respectively, to establish a colitis model. Body weight, stool consistency, and the presence of fecal occult blood were monitored and scored every 24 hours. The last day after mice were sacrificed peripheral blood was collected, the total colon length was measured, colon tissue near the rectum was collected for hematoxylin and eosin (H & E) staining, and pathological changes were observed.
Bone marrow transplantation
Each recipient mouse (8 weeks old) received 2 (4 hours apart) 5Gy (total 10 Gy) of radiation 1 week before and 4 weeks after transplantation and received antibiotic-containing drinking water (polymyxin B sulfate, 6000U/ml and neomycin, 100 μg/ml). Femur and tibia bone marrow cells of 8-week-old RNF99+/+ and RNF 99-/-mice were transplanted with 5X 10 cells via tail vein, respectively 6 Individual bone marrow cells were given to recipient mice. Mice recovered 6 weeks after transplantation, hematopoietic cells were reconstituted, and further modeled.
Histological examination
RNF99+/+ and RNF99-/-mice colon and lung were fixed with 4% paraformaldehyde for 24h, paraffin embedded and 5 μm sectioned. H & E staining was performed using a staining kit (Solarbio, beijing, china) according to the manufacturer's instructions. Then, histological examination was performed with an optical microscope. Lung injury was scored according to alveolar septum thickening, bleeding and inflammation. The severity of colitis was analyzed according to the extent of lesions studied in the past.
Reagents and antibodies
LPS (Escherichia coli, 055:B5) was purchased from Sigma-Aldrich (MO, USA). R848 and LTA were purchased from InvivoGen. The agonist concentration used was 100ng/ml LPS, 10. Mu.g/ml R848, 10. Mu.g/ml LTA. Dextran sodium sulfate (DSS, 36-50 kDa) was purchased from MP biomedical corporation. MG132 (T2154) and Bortezomib (T2399) were purchased from Topscience, chloroquine (HY-17589A) and ATP (HY-B2176) were purchased from MCE. Lipofectamine 3000 reagent (Invitrogen, USA) was used for transient transfection of 293-TLR cells. Antibodies to P-P65 (Ser 536) (3033), P65 (8242), phospho-JNK (Thr 183/Tyr 185) (4668), JNK (9252), P-P44/42MAPK (Erk 1/2) (Thr 202/Tyr 204) (4370), P44/42MAPK (Erk 1/2) (4695), P-P38 MAPK (4511), P-IkB alpha (Ser 32) (2859), ikB alpha (4814), TAB1 (3226), TAB2 (3745), TAK1 (5206), pTAK1 (Thr 184/Tyr 187) (8), IRAK1 (4504), IRAK4 (4363), GFP (2956) and GAPDH (2118) were from CST. The anti-IgG antibody (AB 172730), anti-ubiquitin antibody (AB 134953), anti-k 48-ubiquitin antibody (AB 140601) and anti-k 63 ubiquitin antibody (AB 179434) were all from Abcam. Antibodies targeting RNF99 (SAB 2102558) and Flag (F1804) were from Sigma-Aldrich. Myc (TA 150121) and HA tag (TA 180128) antibodies are both from origin. Protein A/G PLUS (sc-2003) was from Santa Cruz Biotechnology (CA, USA), anti-FLAG (M2) affinity gel (A2220) and 3xFLAG peptide (F4799) was from Sigma-Aldrich. E1, ubcH5a, ubiquitin (WT), ubiquitin (K48) and ubiquitin (K63) were purchased from Boston Biochem (USA).
RT-PCR analysis
Total RNA was extracted from PMs or BMDMs using RNeasy Mini kit (Qiagen, 74106, germany) according to the manufacturer's instructions and reverse transcribed using Prime Script RT kit (047 a, taKaRa, japan). Reverse transcription product amplification was performed using a LightCycler480 instrument (LightCycler 480, switzerland) and SYBR Green PCR Master Mix (Roche, switzerland) under conditions of denaturation at 95℃for 10min, and 40 cycles of amplification (95℃15s,55℃15s,72℃20 s). Results were normalized to beta-actin levels, with 2 -ΔΔCt The method calculates the relative change. The primer sequences are as follows, mouse beta-actin: 5'-CCACACCCGCCACCAGTTCG-3' and 5'-TACAGCCCGGGGAGCATCGT-3'; mouse tnf- α:5'-CCCTCACACTCAGATCATCTTCT-3' and 5'-CCCTCACACTCAGATCATCTTCT'; mouse il-6:5'-AGTTGCCTTCTTGGGACTGA' and 5'-TCCACGATTTCCCAGAGAAC-3'; mousil-1 beta: 5'-ACCTTCCAGGATGAGGACATGA-3' and 5'-AACGTCACACACCAGCAGGTTA-3'; mouse RNF99:5'-TGAACAACGCCCTCC-3' and 5'-GCCGCTCTACAACCA-3'; human RNF99:5'-GGGAACTCAGGCAAGACTCA-3' and 5'-CCCCTCGGATGTCCACTACT-3'.
ELISA
TNF- α, IL-6 and IL-1β levels in samples collected from mouse blood or cell culture supernatants were detected using ELISA kits purchased from R & D Systems. TNF-alpha, IL-6 and IL-1β levels in human serum collected from healthy and G-infected individuals were determined using ELISA kits purchased from DAKEWE Biotechnology (Shenzhen, china).
CO-immunoprecipitation (CO-IP), immunoblotting (Western blot, WB) analysis and ubiquitination analysis
Cells used for immunoblot analysis were lysed in Cell Lysis (Sigma-Aldrich, USA) containing protease inhibitor and centrifuged at 12 000x g for 10 min. The BCA protein assay kit (Thermo Fisher Scientific, USA) was used to determine the concentration of the supernatant and let it equal in all samples. For Co-IP, the total cell extract was lysed using an IP buffer containing a protein inhibitor and centrifuged at 12 000Xg for 10 min. The supernatant was collected and incubated with protein A/G plus agaros and the corresponding antibodies or anti-flag (M2) affinity gel overnight. Then washed five times with IP buffer and eluted with 3xFlag peptide. The samples were then boiled in protein loading buffer (DL 101-02,Tans Gene,Beijing) and subjected to SDS-PAGE. WB was performed using whole cell lysates and IP samples. Proteins were transferred to PVDF membrane after 10% SDS-PAGE separation, blocked by BSA. Then incubated overnight with the appropriate antibodies, then with secondary enzyme-labeled antibodies for 1h. The strips were tested using ECL kit (Merck Millipore, USA).
In the ubiquitination experiment, cells were lysed in RIPA lysis buffer and immediately boiled in the presence of 2% (vol/vol) SDS for 10 min. The lysate was then diluted with lysis buffer until the SDS concentration was reduced to 0.1% for further immunoprecipitation examination.
TUBE detection
After LPS stimulation, PMs were lysed in lysis buffer containing protease inhibitors and centrifuged. The supernatant was added to TUBE beads (WT, K48 or K63) and incubated at 4℃for 2h. After 4 washes of TBST, endogenous ubiquitin was eluted and analyzed by immunoblotting.
Laser confocal detection
293-TLR4 cells were transfected with GFP-TAB2 and Flag-RNF99 overexpression plasmids. After 24h, cells were fixed with 4% paraformaldehyde and blocked with 5% bovine serum albumin for 30 min. Cells were then incubated with the indicated primary antibody overnight, followed by incubation of cells with the fluorescent dye-conjugated secondary antibody. Meanwhile, igG negative control is established as primary antibody control and solvent PBS control, and the antibody is verified, and false positive results are eliminated. Nuclei were stained with DAPI (Sigma-Aldrich, USA) and cells were observed with a Leica laser scanning confocal microscope.
Adenovirus infection
Adenovirus of RNF 99-DeltaRing and RNF99-C94/97A was transfected after construction by Boshang Biotech company (Shanghai, china) and PMs were transfected with 5. Mu.g/ml polybrene for 48h at MOI=600.
Statistical analysis
Each experiment was repeated at least three times. All data were analyzed using GraphPad Prism 8 software (GraphPad, san Diego, CA, USA) and expressed as mean ± Standard Error (SEM). First, shapiro-Wilk was used to verify whether the data obeyed normal distribution. For univariate data belonging to a normal distribution, statistical differences between the two groups were analyzed using an unpaired two-trained Student's t-tests. Statistical differences between the univariate groups were analyzed by one-way variance analysis and then post-hoc inspection using Dunnett's or Tukey's. Statistical analysis between the Two variable groups was performed using Two-way ANOVA analysis and Tukey or Sidak post hoc tests. For univariate data not belonging to normal distribution, multiple comparisons were made using the Kruskal-Wallis test and the Dunnett's post hoc test. In all statistical comparisons, p values <0.05 were considered statistically significant, and p values that were not significant were not shown.
2. Experimental results
TLRs stimulation and gram-negative bacilli (G-) infection significantly reduced RNF99 expression in macrophages
Abdominal Primary Macrophages (PMs) were extracted from wild-type (WT) C57BL/6J mice, and after stimulation with TLR4 ligand-LPS, mRNA (FIG. 1A) and protein (FIG. 1B) levels of RNF99 were significantly down-regulated. Similar results were obtained with TLR7/8 ligand-R848 and TLR6 ligand LTA treatments. This further suggests that RNF99 may be a feedback regulator of TLR-mediated innate immune responses.
LPS is a TLR4 ligand, a key component of the G-bacterial cell wall. We collected peripheral blood from G-infected and healthy persons, and sorted CD14+ monocytes by flow cytometry, and examined that G-infected patients had significantly elevated levels of TNF- α, IL-6, and IL-1β mRNA compared to healthy subjects (FIG. 1C). However, RNF99 levels were reduced (fig. 1D), suggesting that its expression in monocytes may be closely related to bacterial infection.
Effect of RNF99 knockout on TLRs-mediated endotoxemia and acute colitis
To clarify the role of RNF99 in TLR-mediated innate inflammatory responses, we constructed RNF99 knockout mice (RNF 99-/-) using CRISPR/Case9 technology, treated mice with TLR ligands, built in vivo models of LPS-induced endotoxemia and lung injury, and studied TLR-related molecular mechanisms and potential treatments of inflammatory-related lung injury. Inflammatory cell infiltration and interstitial pneumonia were significantly exacerbated in the lungs of RNF 99-/-mice after LPS stimulation (fig. 2a, b), and secretion of TNF- α, IL-6 and IL-1β was also significantly up-regulated in serum of RNF 99-/-mice compared to WT littermate control (RNF 99 +/+) (fig. 2C). RNF 99-/-mice died earlier and showed higher mortality under LPS lethal stimulation (figure 2D). These data indicate that RNF99 negatively regulates endotoxin-mediated inflammatory diseases in vivo.
The abnormal intestinal flora induces activation of macrophage TLRs signals and mediates inflammatory responses, and is an important event in the development and progression of IBD. DSS-induced colitis is a classical inflammatory model for studying the mechanism of IBD. Thus, we further investigated the effect of RNF99 deletion on DSS-mediated inflammatory responses. The results showed that the clinical manifestations of RNF 99-/-mouse colitis were significantly worse compared to RNF99+/+ mice, mainly as weight loss (FIG. 2E), soft stool (FIG. 2F), exacerbation of hematochezia (FIG. 2G), and shortening of the mouse colon (FIG. 2H, I). H & E staining of colon sections showed increased mucosal destruction and increased inflammatory cell infiltration (fig. 2j, k). In addition, the expression of proinflammatory factors in peripheral blood of mice was significantly up-regulated (fig. 2L). Overall, RNF99 deficiency significantly aggravates the severity of TLR-mediated endotoxemia and acute colitis.
RNF99 in macrophages is involved in the modulation of inflammatory diseases mediated by TLRs
To further demonstrate that RNF99 in macrophages plays a key role in TLR mediated inflammatory disease, we transplanted bone marrow cells of RNF99+/+ or RNF99-/-mouse donors into irradiated RNF99+/+ mice (RNF99+/++ to RNF99+/+ or RNF99-/- →RNF99+/+) (FIG. 3A). The chimeric mice were then stimulated with LPS to induce endotoxemia or to induce acute colitis with DSS. Compared to rnf99+/+ - > rnf99+/+ mice, RNF99-/- > rnf99+/+ mice showed severe lung lesions on H & E stained lung sections (fig. 3B), with increased pro-inflammatory factor production following LPS stimulation (fig. 3C). Compared with RNF99+/+ →RNF 99-/-mice after LPS stimulation, RNF 99-/-mice also show pulmonary inflammation exacerbates and pro-inflammatory factor production increases. After DSS induced colitis, clinical and pathological changes in mice showed that RNF99-/- > rnf99+/+ mice exhibited increased weight loss, soft stool, hematochezia (fig. 3D), shortened colon (fig. 3E), disrupted mucosa, increased inflammatory cell infiltration (fig. 3F), and increased production of pro-inflammatory factors (fig. 3G) compared to rnf99+/++ rnf99+/+ mice. These results indicate that mice bearing RNF 99-/-bone marrow cells are more sensitive to LPS-induced endotoxin and DSS-induced colitis. Taken together, RNF99 in macrophages plays an important role in combating TLR-induced inflammatory diseases. TLRs mediated production of inflammatory cytokines in RNF 99-deficient and aggravated macrophages
The role of RNF99 as an important E3 ubiquitin ligase in macrophages in TLRs-mediated immune responses is not yet clear. To illustrate this, we extracted PMs from RNF99+/+ and RNF 99-/-mice, respectively, and treated with different TLR ligands such as LPS, R848, and LTA, showed that RNF 99-/-mice were significantly upregulated after stimulation with LPS (fig. 4A), R848 (fig. 4B), and LTA (fig. 4C) compared to RNF99+/+ mice.
TLR-mediated production of inflammatory factors such as TNF- α, IL-6 and IL-1β is primarily dependent on activation and transduction of NF- κb and MAPKs. PMs and BMDMs from RNF99+/+ and RNF 99-/-mice were extracted and treated with LPS, R848 or LTA. The levels of phosphorylation of iκb- α, P65 were significantly elevated in RNF 99-/-mouse PMs (fig. 4d, e) compared to RNF99+/+ mice and MAPK activation marker ERK, JNK, P. The results demonstrate that macrophage RNF99 depletion enhances TLRs-induced activation of NF- κb and MAPK signaling pathways.
Reverse regulation of TLRs mediated macrophage inflammatory cytokine production and activation of signaling pathway by RNF99 overexpression
RNF99, a member of the TRIM family, has Ring, coil-coil, B-box and Filamin domains, where Ring domain is critical for its function as an E3 ubiquitin ligase. To further elucidate the role of RNF99 in TLR mediated inflammatory cytokine production, we constructed adenovirus plasmids expressing wild-type RNF99 (RNF 99-WT), ring deletion mutant (delta-Ring) and point mutant (C94/97A). Both the delta-Ring and the C94/97A mutants lost the function of the E3 ubiquitin ligase. In LPS-treated mice PMs, RNF99 overexpression was found to significantly alleviate TLR-mediated inflammatory factor secretion (fig. 5A), as well as NF- κb and MAPKs signaling (fig. 5B-D). Overexpression of delta-Ring and C94/97A did not have this effect, suggesting an important role for E3 ubiquitin ligase activity of RNF 99. These results further indicate that RNF99 can negatively regulate TLR-mediated inflammatory cytokine production, and that this effect is closely related to its E3 ubiquitin ligase function.
RNF99 affects formation of TAK1-TAB1-TAB2 complex by promoting TAB2 degradation
To elucidate the regulatory mechanisms by which RNF99 induces production of pro-inflammatory cytokines by TLRs and activation of NF-. Kappa.B-MAPK signaling pathways, we explored target molecules for RNF99 in TLRs-mediated signaling pathways. We co-transfected 293-TLR4 cell lines with IKK- α, IKK- β, IKK- γ, IRAK-1, IRAK-4, TRAF6, TAK1, TAB2 and RNF99 and treated with LPS showed that RNF99 overexpression significantly reduced TAB2 protein levels, but had no effect on expression of other pathway proteins (fig. 6A). Likewise, after LPS stimulated RNF99+/+ and RNF 99-/-mice PMs, TAB2 expression alone was increased, while expression of other pathway proteins was unchanged (FIG. 6B). These results indicate that TAB2 may be the target of RNF99 in the TLRs pathway. To further elucidate the effect of RNF99 on TAB2 expression, we overexpressed TAB2 with RNF99 in 293-TLR4 cells. As a result, it was found that the expression level of TAB2 decreased correspondingly with the overexpression of RNF99, and that the degradation of the RNF99 by TAB2 was lost when the delta-Ring and C94/97A mutants of RNF99 were co-transfected with TAB2, indicating that the degradation of TAB2 by RNF99 was dependent on its E3 ubiquitin ligase function (FIG. 6C). To further investigate how RNF99 down-regulates TAB2 we used proteasome inhibitors (MG 132 and bortezomib) and lysosomal pathway inhibitors (chlor oquinone and NH 4 Cl). The results showed that degradation of TAB2 by RNF99 disappeared after MG132 or bortezomib treatment. However, chloroquinone or NH 4 This degradation was unchanged after Cl treatment (fig. 6D. These results indicate that RNF99 degradation of TAB2 was dependent on the proteasome pathway.
The TAK1-TABs complex is critical for TLR-mediated signaling, while TAB2 plays a critical role in this process. We further explored the effect of RNF99 on TAK-TABs complexes and found that in RNF 99-/-mouse PMs, increased TAB2, LPS-induced TAK phosphorylation was significantly enhanced (fig. 6E). Meanwhile, after RNF99 knockout, TAK1 binding to TAB1 was significantly increased (fig. 6F). These results confirm that RNF99 affects the formation of TAK1-TABs complexes. RNF99 interaction with TAB2
Our previous results show that RNF99 specifically mediates degradation of TAB2, suggesting that TAB2 may be a direct target for RNF 99. To perfect the regulatory mechanism, we further demonstrated the binding of RNF99 to TAB2 in 293-TLR4 cells (fig. 7A). Furthermore, the interaction between RNF99 and TAB2 was also confirmed by endogenous CO-IP in PMs (FIG. 7B). After overexpression of RNF99 and TAB2 in 293-TLR4 cells, immunofluorescent staining of cells was performed and confocal laser microscopy showed significant co-localization of RNF99 and TAB2 in cells (fig. 7C). The above data indicate that there is a close interaction between RNF99 and TAB 2.
To determine the fragments responsible for the interactions in RNF99, we generated a series of truncation mutants based on protein structure (fig. 7D). We found that coil-coil in RNF99 is a TAB2 binding domain (FIG. 7E). Taken together, our findings indicate that TAB2 is a target for RNF99 in the signaling pathways of TLRs.
RNF99 mediates K48-ubiquitination modification of K611 site of TAB2
Protein ubiquitination modification is closely related to ubiquitin-proteasome degradation pathways. After demonstrating that RNF99 mediates degradation of TAB2 via the proteasome pathway, and that this degradation is dependent on E3 ubiquitin ligase activity, we further explored whether RNF99 can mediate ubiquitin modification of TAB 2. Ubiquitin molecules contain 7 lysine sites (K6, K11, K27, K29, K33, K48 and K63). Ubiquitination at positions K48 and K63 is the two most studied ubiquitination types, ubiquitination at position K48 is often reported to be involved in proteasome degradation. We overexpressed Myc-RNF99 and Flag-TAB2 with different types of ubiquitin (WT, K48 and K63) in cells. The results indicate that RNF99 can significantly promote ubiquitination of wild-type (WT) and K48 of TAB2, but has no effect on the ubiquitination level of K63 (fig. 8A), whereas RNF99 without E3 ubiquitin ligase function does not affect the ubiquitination level of K48 of TAB2 (fig. 8B). After LPS treatment of PMs in RNF99+/+ and RNF99-/-mice, the level of ubiquitination modification at endogenous TAB 2K 48 was significantly reduced in RNF99-/-mice (FIG. 8C).
Previous TAB2 ubiquitination histology studies predicted that the lysines at positions 522, 611, 653 and 656 of TAB2 could be ubiquitinated. However, the E3 ligase that regulates these site ubiquitination modifications has not been discovered. To elucidate which lysine sites on TAB2 can be ubiquitinated by RNF99, we constructed mutant over-expression plasmids of different lysine sites of TAB2 (K522R, K611R, K653R and K656R) and CO-transfected them with Myc-RNF99 and ubiquitin in 293-TLR4 cells, and CO-IP experiments found that RNF99 lost ubiquitination modification function for K611R mutant, indicating that K611 is the site of ubiquitination modification of TAB2 by RNF99 (FIG. 8D). In addition, after mutation of K611 in TAB2, degradation of TAB2 by RNF99 disappeared (FIG. 8E). The ubiquitinated modified lysine sites were identical to the degradation sites. These results indicate that RNF99 promotes degradation of its proteasome pathway by ubiquitination modification of lysine 611 of TAB2, thereby playing an important role in the innate immune response by inhibiting NF- κb and MAPK signaling pathways.
Taken together, we demonstrate that RNF99 is a key regulator of TLR signaling pathways. RNF99 down regulates pro-inflammatory factor production by promoting K48 ubiquitination modification and proteasome degradation at the TAB 2K 611 site, inhibiting the formation of TAK-TABs complex and activation of downstream NF-. Kappa.B and MAPKs signaling pathways. Our studies for the first time demonstrate that RNF99 may be a promising diagnostic biomarker and therapeutic target for the treatment of TLR-related inflammatory diseases.
The invention is not a matter of the known technology.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.

Claims (10)

1. The use of a substance that detects the expression level of the E3 ubiquitin ligase RNF99 encoding gene and/or the expression product of the RNF99 encoding gene in the preparation of a product for screening, diagnosing (aiding diagnosis), detecting, monitoring or predicting the progression of inflammatory diseases.
2. The use according to claim 1, wherein the RNF 99-encoding gene and its expression products are of human and non-human mammalian origin; the expression product of the RNF99 coding gene is E3 ubiquitin ligase RNF99.
3. The use according to claim 1, wherein the inflammatory disease is in particular a TLR-related inflammatory disease, including bacterial infection disease, endotoxemia and colitis.
4. A product for screening, diagnosing (co-diagnosing), detecting, monitoring or predicting the progression of an inflammatory disease, characterized in that it comprises detecting transcription of a RNF99 encoding gene in a subject based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; or detecting the expression of the subject E3 ubiquitin ligase RNF99 based on an immunoassay method.
5. The product of claim 4, wherein the substance is specifically a substance for detecting transcription of the RNF 99-encoding gene in a subject using a method comprising liquid phase hybridization, northern hybridization, miRNA expression profiling chip, ribozyme protection assay technology, RAKE method, in situ hybridization; detecting the expression condition of the RNF99 of the ubiquitin ligase of the subject by adopting ELISA, colloidal gold test strips and protein chips;
the subjects are human and non-human mammals (including mice);
the product is a kit.
6. Use of a substance that promotes increased expression and/or activity of an RNF99 encoding gene and its expression product in at least one of the following a 1) -a 4):
a1 Promoting K48 ubiquitination modification and proteasome degradation of the K611 site of the TAB2 protein, thereby inhibiting the formation of a TAK1/TABs complex and the activation of TAK1 kinase or preparing a product promoting the K48 ubiquitination modification and proteasome degradation of the K611 site of the TAB2 protein, thereby inhibiting the formation of the TAK1/TABs complex and the activation of the TAK1 kinase;
a2 Products that inhibit the activation of NF- κB and MAPKs signaling pathways or inhibit the activation of NF- κB and MAPKs signaling pathways;
a3 Inhibiting or producing products of inflammatory cytokine production in macrophages induced by TLRs ligands;
a4 For the preparation of a product for the prophylaxis and/or treatment of inflammatory diseases.
7. The use according to claim 6, wherein the agent that promotes increased expression and/or activity of the RNF 99-encoding gene and its expression products comprises an agent that upregulates expression and/or promotes activity of the RNF 99-encoding gene using a gene-specific mic-based technique; further comprising artificially synthesizing short hairpin RNA of RNF99, or a promoter or lentivirus for up-regulating RNF99 expression; compound promoters are also included.
8. The use according to claim 6, wherein in a 1) the TAK1/TABs complex is in particular a TAK1-TAB 2 complex;
in said a 3), said inflammatory cytokines include TNF- α, IL-6 and IL-1β;
in the a 4), the inflammatory disease includes TLR-related inflammatory disease, further including bacterial infection disease, endotoxemia and colitis.
9. The use according to claim 6, wherein the product is a pharmaceutical or experimental agent for use in basic research.
10. The use according to claim 9, wherein when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient;
the pharmaceutically inactive ingredients include pharmaceutically acceptable carriers, which are liposomes, biocompatible polymers (including natural and synthetic polymers), lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, inorganic (including metallic) particles, and bacterial or viral (including adenoviruses, baculovirus, retroviruses, etc.), phage, cosmids, or plasmid vectors;
The medicament is optionally administered via oral, intravenous, transdermal, intranasal, mucosal or bone marrow transplantation.
CN202310038858.6A 2023-01-12 2023-01-12 Application of E3 ligase RNF99 in negative regulation of TLR-mediated inflammatory immune response Pending CN116411061A (en)

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