Nol12 mutant fusion protein and application thereof

Abstract

The application discloses a NOL12 mutant fusion protein and application thereof. The NOL12 mutant fusion protein comprises a TAT cell-penetrating peptide, a flexible linker and a NOL12 mutant. Compared with the amino acid sequence of the wild-type NOL12, the lysine at the 50th position of the NOL12 mutant is mutated into an arginine. The NOL12 mutant fusion protein of the application can enter cells without a transfection reagent and is at least partially located in the nucleus, and under the condition of LPS combined with ATP-induced in-vitro sepsis-related inflammation pyroptosis, the NOL12 mutant fusion protein can reduce the expression level of NLRP3 protein, reduce the production of Caspase 1 p20 cleavage fragments, and reduce the level of GSDMD-NT cleavage fragments, which indicates that the NOL12 mutant fusion protein can reduce the NLRP3 / Caspase 1 / GSDMD-related hepatocyte pyroptosis signal.

Description

Technical Field

[0001] This invention relates to the fields of recombinant protein engineering and biomedicine, specifically to an NOL12 mutant fusion protein and its applications. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection. The liver is one of the most vulnerable organs in sepsis, and currently, there are no effective targeted therapies available clinically. Pyroptosis is a programmed inflammatory cell death process mediated by the gasdermin protein family, and recent studies have suggested that it plays an important role in the development and progression of sepsis-related liver injury.

[0003] The classic NLRP3 inflammasome activation follows a two-step signaling model: the first signal (Priming signal) is mediated by pathogen-associated molecules such as LPS via the TLR4 / NF-κB pathway, upregulating the expression of inflammasome components such as NLRP3 and pro-IL-1β; the second signal (Activation signal) is triggered by activators such as ATP and potassium efflux, inducing the assembly of NLRP3 protein and adaptor protein ASC to form the inflammasome complex, recruiting and promoting the cleavage of pro-Caspase 1 via proximity induction to produce active Caspase 1 (containing the p20 subunit). Active Caspase 1 cleaves GSDMD to release GSDMD-NT, which oligomerizes on the cell membrane to form pores, leading to the release of pro-inflammatory cytokines and necroptotic hepatocyte death. The production of the Caspase 1 p20 fragment can serve as an important indicator of Caspase 1-dependent inflammasome pathway activation; combined with changes in NLRP3 protein expression and GSDMD cleavage results, it can be used to evaluate the activation status of the NLRP3 / Caspase 1 / GSDMD-related hepatocyte pyroptosis pathway.

[0004] Targeting the NLRP3 / Caspase 1 / GSDMD signaling pathway has become an important research direction for the prevention and treatment of sepsis-related liver injury. Several small-molecule inhibitors of NLRP3 have been reported, among which MCC950 is the most widely studied selective inhibitor, affecting its ATPase activity and conformational changes by directly binding to the NACHT domain of the NLRP3 protein. However, the above-mentioned small-molecule inhibitors have the following limitations: (1) It mainly affects the conformational changes of NLRP3 at the protein level; (2) Some small molecule inhibitors have hepatotoxicity or other side effects; (3) No NLRP3-targeting inhibitors have been approved by the FDA for marketing.

[0005] Therefore, developing anti-pyroptosis protein drugs with novel mechanisms of action is of great value. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a NOL12 mutant fusion protein and its applications.

[0007] To achieve the above objectives, the technical solution designed by the present invention is as follows: The present invention provides a NOL12 mutant, wherein the amino acid sequence of the NOL12 mutant shown in SEQ ID NO: 1 is modified by replacing lysine at position 50 with arginine.

[0008] Furthermore, the amino acid sequence of the NOL12 mutant is shown in SEQ ID NO: 2.

[0009] The present invention also provides an NOL12 mutant fusion protein, wherein the NOL12 mutant fusion protein comprises a TAT membrane-penetrating peptide, a flexible linker, and the NOL12 mutant; The amino acid sequence of the TAT membrane-penetrating peptide is shown in SEQ ID NO: 3.

[0010] Furthermore, the amino acid sequence of the flexible linker is shown in SEQ ID NO: 4.

[0011] Furthermore, the amino acid sequence of the NOL12 mutant fusion protein is shown in SEQ ID NO: 5.

[0012] The present invention also provides a gene encoding the NOL12 mutant fusion protein, the nucleotide sequence of which is shown in SEQ ID NO: 6.

[0013] The present invention also provides a method for preparing the NOL12 mutant fusion protein, comprising the following steps: (1) The gene sequence of the NOL12 mutant fusion protein was synthesized and double-digested with NcoI and XhoI to obtain the gene fragment; (2) The pET28a(+) vector was double-digested with NcoI and XhoI to obtain the vector fragment; (3) The gene fragment and the vector fragment are ligated to obtain a recombinant plasmid; (4) The recombinant plasmid was transferred into Escherichia coli BL21(DE3) competent cells, and protein expression was performed by shaking culture. Then, the protein was subjected to sonication and Ni-NTA affinity chromatography to obtain the NOL12 mutant fusion protein.

[0014] The present invention also provides the application of the NOL12 mutant fusion protein described above in the preparation of a drug for sepsis-associated hepatocellular pyroptosis-related liver injury.

[0015] The present invention also provides a drug for sepsis-associated hepatocellular pyroptosis-related liver injury, the drug comprising the aforementioned NOL12 mutant fusion protein, wherein the concentration of the NOL12 mutant fusion protein in the drug is 10~100 μg / mL.

[0016] Furthermore, the concentration of the NOL12 mutant fusion protein in the drug is 50 μg / mL.

[0017] The principle of this invention: NOL12 (Nucleolar Protein 12) is a highly conserved, multifunctional RNA-binding protein (RBP) in eukaryotes. The human NOL12 encoding gene (UniProt ID: Q9UGY1) belongs to the RRP17 protein family. Its yeast homolog is Rrp17, its Drosophila homolog is Viriato, and its mouse homolog is Nop25. NOL12 protein consists of 213 amino acids with a theoretical molecular weight of 23–25 kDa. It carries nuclear localization signals (NLS) and nucleolar localization signals (NoLS), and under steady-state conditions, it is localized to multiple subcompartments, including the nucleolus (GC region of the granular component), nucleoplasm, and cytoplasmic GW / P bodies, reflecting its multifunctional characteristics in various cellular processes.

[0018] NOL12 plays a crucial role in ribosome biosynthesis. Human NOL12, as a component of the early 90S and pre-60S ribosomal subunits, participates in the cleavage and dissociation of ribosomal large and small subunit rRNA precursors at site 2, which is essential for ribosome maturation. NOL12 deficiency leads to impaired 45S pre-rRNA processing and a 20%–25% decrease in mature 28S and 5.8S rRNA levels. In plants, AtNOL12 also participates in the processing of the 27S large subunit rRNA precursor, and NOL12 deficiency significantly alters the expression of ribosomal proteins and genes related to ribosome biosynthesis. Under stress conditions such as heat stress and pathogen infection, the rRNA biosynthesis deficiency phenotype is further aggravated, suggesting that NOL12 links ribosome biosynthesis to stress response.

[0019] NOL12 also plays an important role in maintaining genome integrity and responding to DNA damage. Studies have shown that NOL12, along with DNA damage repair proteins Dhx9 and TOPBP1, co-localizes at replication arrest sites. NOL12 deficiency leads to increased levels of oxidized DNA in cells, subsequently triggering a rapid p53-independent ATR-Chk1-mediated apoptosis response and G1 / S phase arrest. NOL12 also co-localizes with 53BP1 at etoposide-induced DNA damage sites, strongly supporting its role in maintaining genome integrity during G1 phase. Interacting proteins of NOL12 include NONO, Dhx9, DNA-PK, and Stau1, further supporting its multiple functions in RNA metabolism and DNA maintenance.

[0020] NOL12 plays a crucial role in regulating nucleolar homeostasis and cellular senescence. NOL12 regulates nucleolar structure and homeostasis by maintaining the levels of key nucleolar proteins fibrillarin and nucleolin. NOL12 deficiency leads to RPL11-dependent p53 activation, triggering G2 phase arrest. In human primary fibroblasts, NOL12 deficiency drives nucleolar stress-mediated cellular senescence, and NOL12 expression levels decrease with donor age, suggesting that NOL12 is closely related to normal physiological aging processes.

[0021] NOL12 is endogenously expressed in the liver and is closely related to hepatocyte biological functions. Based on ENCODE consortium eCLIP experimental data, NOL12 exhibits abundant rRNA binding in HepG2 cells, and NOL12 protein in HepG2 cells can be verified by immunoprecipitation. Further studies revealed that NOL12 expression is significantly upregulated in hepatocellular carcinoma (HCC) tissues and cell lines (including HepG2 and Huh-7). High NOL12 expression is positively correlated with poor overall survival, high pathological grade, lymph node metastasis, and advanced clinical stage in HCC patients. NOL12 knockdown significantly inhibits HCC cell proliferation and metastasis, and NOL12 expression levels are associated with 12 types of tumor-infiltrating immune cells. These findings reveal that NOL12 has abundant endogenous expression and important biological functions in the liver.

[0022] The beneficial effects of this invention are: (1) The present invention found that under the in vitro sepsis-associated pyroptosis induced by LPS combined with ATP, the level of NOL12 protein decreased, while the proteasome inhibitor MG132 could partially reverse this decrease, suggesting that the protein stability of NOL12 under this condition is related to the proteasome-associated degradation process; the K50R point mutation can significantly improve the protein stability of NOL12 under this condition.

[0023] (2) The present invention fuses the TAT membrane-penetrating peptide with the mutant NOL12 (K50R) and links them together with the flexible linker GGGGSGGGGS, thereby improving the cell delivery ability of NOL12 and its protein stability under sepsis-associated inflammatory pyroptosis conditions, so as to deliver the NOL12 mutant fusion protein to the cell and at least partially locate it in the cell nucleus without transfection.

[0024] (3) This invention achieves soluble prokaryotic expression and Ni-NTA affinity purification of the NOL12 mutant fusion protein TAT-NOL12(K50R) by codon optimization of Escherichia coli (CAI=1.0, GC content 51.69%).

[0025] (4) This invention found that the NOL12 mutant fusion protein TAT-NOL12(K50R) can reduce NLRP3 protein expression, decrease the production of Caspase 1 p20 cleavage fragments, and reduce GSDMD-NT cleavage fragment levels in an in vitro sepsis-associated hepatocyte pyroptosis model induced by LPS and ATP, suggesting that it can alleviate NLRP3 / Caspase 1 / GSDMD-related hepatocyte pyroptosis signaling. Under non-inflammatory conditions, this NOL12 mutant fusion protein has little effect on basal NLRP3 protein expression, and TAT transmembrane peptide alone did not show a similar regulatory effect.

[0026] (5) Unlike existing small molecule inhibitors that directly act on NLRP3 protein, the protein intervention tool provided by this invention can reduce NLRP3 protein level and alleviate downstream Caspase 1 / GSDMD pyroptosis signal, and has the potential to be further developed into a sepsis-related hepatocellular pyroptosis intervention tool or candidate drug. Attached Figure Description

[0027] Figure 1 Figure 1 shows the changes in endogenous NOL12 protein levels in L02 cells under in vitro sepsis-associated pyroptosis induced by LPS and ATP, and the results of MG132 intervention. Figure 1 In the text, "+" indicates that the corresponding substance was added for treatment, and "-" indicates that the corresponding substance was not added for treatment; Figure 2 A comparison of protein stability between HA-NOL12 WT and HA-NOL12-K50R under in vitro sepsis-associated pyroptosis induced by LPS and ATP; Figure 3 This is a map of the recombinant plasmid pET28a(+)-TAT-NOL12(K50R); Figure 4The image shows the Western blot results of recombinant protein expression and purification (detected by His-tagged monoclonal antibody). Figure 5 Immunofluorescence images of the transmembrane and nuclear localization of the NOL12 mutant fusion protein TAT-NOL12(K50R); Figure 6 The Western blot results show the effects of the NOL12 mutant fusion protein TAT-NOL12(K50R) on LPS- and ATP-induced pyroptosis-related proteins in L02 hepatocytes. Figure 6 In the image, A shows the Western blot bands of NLRP3, full-length Caspase 1 and p20 cleavage fragment, full-length GSDMD protein and GSDMD-NT cleavage fragment (5 groups, β-actin as internal control); B~D are grayscale quantitative bar charts of NLRP3, Caspase 1 p20, and GSDMD-NT, respectively. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0029] Example 1: Design of the NOL12 mutant Based on the full-length amino acid sequence of NOL12 (UniProt ID: Q9UGY1) (SEQ ID NO: 1), a K50R point mutation was introduced, that is, the lysine at position 50 was mutated to arginine, to obtain the NOL12 mutant NOL12(K50R), whose amino acid sequence is shown in SEQ ID NO: 2.

[0030] The design basis for the K50R mutation is: (a) In large-scale proteomics studies included in the PhosphoSitePlus proteomics database, K50 was identified as the ubiquitination modification site of NOL12. (b) The mutation of lysine to arginine is a conservative substitution, both of which are positively charged polar amino acids, which helps to maintain local charge properties and protein physicochemical properties as much as possible; (c) Arginine does not provide the lysine side chain ε-amino for ubiquitin chain linkage, so this substitution may reduce ubiquitination modification at this site and improve the stability of NOL12 under inflammatory stress conditions.

[0031] Example 2: Changes in the stability of endogenous NOL12 protein in L02 cells under in vitro sepsis-associated inflammatory pyroptosis induced by LPS combined with ATP, and an experiment involving MG132 intervention. This embodiment establishes an in vitro sepsis-related pyroptosis condition in L02 normal human hepatocytes by inducing LPS combined with the classic two-step ATP signaling pathway. The experiment, combined with intervention using the proteasome inhibitor MG132, evaluates the changes in endogenous NOL12 protein levels and the potential involvement of proteasome-related degradation processes under this condition, as detailed below: 1. Cell Culture L02 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin, and incubated at 37°C in a 5% CO2 incubator.

[0032] 2. LPS treatment and MG132 reversal experiment An in vitro sepsis-associated pyroptosis model was established using a two-step method combining LPS and ATP. Cultured L02 cells were divided into four groups for treatment: (1) Blank control group: only an equal volume of DMSO was added, without the addition of LPS, ATP and MG132; (2) LPS+ATP model group: LPS (final concentration 1 μg / mL) was first used for 7 hours, followed by ATP (final concentration 5 mmol / L) for 1 hour to establish in vitro sepsis-related pyroptosis conditions; (3) LPS+ATP+MG132 group: LPS (final concentration 1 μg / mL) was added and treated for 7 hours, followed by ATP (final concentration 5 mmol / L) and proteasome-specific inhibitor MG132 (final concentration 10 μM) and continued treatment for 1 hour. (4) LPS+ATP+chloroquine group: LPS (final concentration 1 μg / mL) was added and treated for 7 hours, followed by ATP (final concentration 5 mmol / L) and chloroquine (final concentration 10 μM), an inhibitor of autophagy-lysosomal pathway, and continued treatment for 1 hour.

[0033] After each group was treated, cell lysates were collected, and Western blot was used to detect the NOL12 protein level in each group, with γ-Tubulin as the internal reference protein.

[0034] 3. Experimental Results The results are as follows Figure 1 As shown, the in vitro sepsis-associated pyroptosis induced by LPS combined with ATP can lead to a decrease in the level of endogenous NOL12 protein in L02 cells. The decrease in NOL12 protein was significantly reduced after MG132 intervention. The NOL12 protein level in the chloroquine intervention group was not significantly different from that in the LPS+ATP model group, suggesting that the change in NOL12 protein stability under this condition may be related to the proteasome-related degradation process.

[0035] Example 3: Comparison of protein stability between HA-NOL12 WT and HA-NOL12 K50R under LPS- and ATP-induced in vitro sepsis-associated pyroptosis conditions. This embodiment uses plasmid overexpression experiments with L02 normal human hepatocytes as the experimental system to directly compare the protein stability of HA-NOL12 wild-type (WT) and HA-NOL12 K50R mutant under in vitro sepsis-related pyroptosis conditions induced by LPS combined with ATP two-step signaling. The effect of K50R mutation on NOL12 protein stability is evaluated as follows: 1. Plasmid construction and transfection (1) Using pCMV Using HA as the basic vector, the NOL12 gene and the NOL12(K50R) gene were inserted respectively to construct the expression plasmid pCMV. HA NOL12 WT and pCMV HA NOL12 K50R expression plasmid.

[0036] (2) Express plasmid pCMV HA NOL12 WT and pCMV HA NOL12 K50R expression plasmid was transfected into L02 cells (liposome method). 24 hours after transfection, the cells were replaced with RPMI containing 10% FBS. Wild-type cells HA-NOL12-WT and mutant cells HA-NOL12-K50R were obtained from 1640 medium and cultured for another 24 hours before being treated with LPS+ATP.

[0037] 2. LPS+ATP treatment and protein detection An in vitro sepsis-associated pyroptosis model was established using a two-step method combining LPS and ATP. Specifically, wild-type HA-NOL12-WT cells and mutant HA-NOL12-K50R cells were treated with LPS at a final concentration of 1 μg / mL, with treatment groups at 0, 4, 8, and 12 hours. The 0-hour group served as the untreated control. For the 4, 8, and 12-hour groups, ATP was added to a final concentration of 5 mmol / L one hour before the end of the treatment period to provide an activation signal. Lysis buffer was collected after treatment. Western blot analysis was performed using an HA-tagged monoclonal antibody as a probe, with γ-Tubulin as an internal control protein.

[0038] 3. Experimental Results The results are as follows Figure 2 As shown, under the in vitro sepsis-associated pyroptosis induced by two-step signaling of LPS and ATP, the NOL12 protein level in the HA-NOL12-WT group decreased rapidly with LPS treatment time, dropping to less than 10% of the initial level after 12 hours of LPS treatment; while the NOL12-K50R protein level in the HA-NOL12-K50R group decreased significantly and slowly, remaining above 50% of the initial level after 12 hours of LPS treatment. The difference between the two groups was statistically significant (p<0.05).

[0039] The above results indicate that under LPS- and ATP-induced in vitro sepsis-associated pyroptosis conditions, the HA-NOL12-WT protein level decreased with treatment time, while the decrease in HA-NOL12-K50R protein level was significantly delayed. This suggests that the K50R mutation can significantly improve the protein stability of NOL12 under sepsis-associated pyroptosis conditions, and the K50 site is involved in the stability regulation of NOL12 in this pathological stress environment.

[0040] Example 4: Sequence design of the NOL12 mutant fusion protein 1. Design of NOL12 mutant fusion protein structure At the N-terminus of the NOL12(K50R) mutant, the HIV TAT membrane-penetrating peptide (YGRKKRRQRRR, SEQ ID NO: 3) and the flexible linker (GGGGSGGGGS, SEQ ID NO: 4) were sequentially linked to construct the NOL12 mutant fusion protein TAT-NOL12(K50R).

[0041] NOL12 carries its own nuclear localization signal (NLS) and nucleolar localization signal (NoLS), eliminating the need for an external nuclear localization sequence. The N-terminal to C-terminal structure of the fusion protein is as follows: initiation methionine-glycine-TAT transmembrane peptide-GGGGSGGGGS-NOL12(K50R); the C-terminal His tag is provided by the pET28a(+) vector.

[0042] 2. Codon optimization JCat (Java Codon Adaptation Tool) was used to optimize the codon preference of the full-length fusion protein coding sequence for Escherichia coli. After optimization, the CAI increased from 0.208 to 1.0, rare codons were completely eliminated, and the GC content was homogenized to 51.69%.

[0043] 3. Enzyme cleavage site design An NcoI restriction site is introduced at the 5' end of the coding sequence, and an XhoI restriction site is introduced at the 3' end. The ATG in the NcoI recognition sequence (CCATGG) serves as the start codon for the fusion protein, followed by GGC which encodes glycine (Gly), making the N-terminus of the fusion protein actually a Met-Gly-TAT transmembrane peptide sequence; this glycine residue is used to ensure the correct open reading frame.

[0044] The amino acid sequence of the NOL12 mutant fusion protein is shown in SEQ ID NO: 5, and the codon-optimized nucleotide sequence is shown in SEQ ID NO: 6.

[0045] Example 5 Construction of fusion protein expression vector and preparation of NOL12 mutant fusion protein 1. Gene sequence for synthesizing the NOL12 mutant fusion protein.

[0046] 2. The synthesized gene sequence and pET28a(+) vector were double-digested with NcoI and XhoI, respectively, and purified and recovered by 1% agarose gel electrophoresis.

[0047] 3. The digested gene fragment and vector fragment were ligated with T4 DNA ligase, transformed into Escherichia coli DH5α competent cells, plated on LB agar plates containing kanamycin (50 μg / mL), and incubated at 37°C for 12-16 hours.

[0048] 4. The grown single-clone colonies were screened by colony PCR and verified by Sanger sequencing. The positive recombinant plasmid pET28a(+)-TAT-NOL12(K50R) was obtained, and its plasmid map is shown below. Figure 3 As shown, its nucleotide sequence is shown in SEQ ID NO: 7.

[0049] 5. Transform the recombinant plasmid into Escherichia coli BL21(DE3) competent cells, culture at 37°C until OD600≈0.6, add 0.5 mM IPTG, and induce at 18°C ​​for 16 hours.

[0050] 6. Collect the bacterial cells by centrifugation, resuspend the bacterial cells in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0), sonicate to disrupt, and collect the supernatant by centrifugation at 13000 g.

[0051] 7. Perform Ni-NTA affinity chromatography on the collected supernatant: wash with lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 20 / 50 mM imidazole, pH 8.0), elute with elution buffer (20 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, pH 8.0), replace the protein with PBS (pH 7.4) on a PD-10 desalting column, and store at -80°C.

[0052] The purified NOL12 mutant fusion protein was obtained and subjected to Western blot analysis. The results are as follows: Figure 4 As shown, the purified NOL12 mutant fusion protein exhibits a single band, and its molecular weight is consistent with the theoretical value.

[0053] Example 6TAT Verification of cell penetration and nuclear localization of recombinant NOL12(K50R) protein This embodiment uses L02 cells (human normal liver cell line) as the experimental subject. Immunofluorescence assays were used to evaluate the ability of the NOL12 mutant fusion protein TAT-NOL12(K50R) to enter cells and localize to the cell nucleus without the aid of any transfection reagents. The details are as follows: 1. Experimental Grouping L02 cells were plated using standard methods and then divided into two groups for treatment the following day (without using any transfection reagents): (1) Wild-type control group (NOL12-WT): Only an equal mass of wild-type protein NOL12 was added; (2) Recombinant proteome (NOL12-K50R): Add NOL12 mutant fusion protein TAT-NOL12(K50R) (final concentration 50 μg / mL).

[0054] 2. Immunofluorescence detection After adding the corresponding substances and culturing for 12 hours, the cells were washed twice with PBS (to fully remove extracellular residual proteins), fixed with paraformaldehyde, blocked with BSA, and then subjected to immunofluorescence staining: using anti-His-tagged monoclonal antibody (1:200) as the primary antibody, Alexa Fluor 594-labeled secondary antibody (red) to display protein signals, and DAPI (blue) for nuclear staining; the cells were observed and images were acquired using a laser confocal microscope.

[0055] 3. Experimental Results The results are as follows Figure 5As shown, the wild-type control group cells showed negative His tag signal and a clean background; the recombinant protein group cells showed significant overlap between the red fluorescence signal of the His tag and the blue nuclear staining signal of DAPI, indicating that the NOL12 mutant fusion protein TAT-NOL12(K50R) can enter cells via TAT transmembrane peptides and partially localize to the nucleus under conditions without transfection reagents. These results support the ability of the NOL12 mutant fusion protein TAT-NOL12(K50R) to penetrate cells and localize to the nucleus.

[0056] Example 7: The mutant NOL12 fusion protein TAT-NOL12 (K50R) alleviates LPS- and ATP-induced in vitro sepsis-related hepatocyte pyroptosis signaling. In this embodiment, an in vitro sepsis-related hepatocyte pyroptosis model was established by inducing L02 normal human hepatocytes with LPS combined with the classic two-step ATP signaling, and the effects of the NOL12 mutant fusion protein TAT-NOL12(K50R) on pyroptosis-related indicators such as NLRP3 protein expression, Caspase1 p20 production, and GSDMD cleavage were evaluated.

[0057] 1. Cell Culture L02 cells were cultured in RPMI-1640 medium containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C and 5% CO2.

[0058] 2. Experimental Grouping The cultured L02 cells were divided into 5 groups and treated separately, with 3 replicate wells for each group: (1) Blank control group: only an equal volume of PBS was added, without adding LPS, ATP or any protein; (2) TAT-NOL12-K50R treatment group: NOL12 mutant fusion protein TAT-NOL12(K50R) (final concentration of 50 μg / mL) was added, and culture was continued without adding LPS or ATP; (3) LPS+ATP model group: LPS (final concentration of 1 μg / mL) was used for 7 hours (Priming), followed by ATP (final concentration of 5 mmol / L) for 1 hour (Activation). (4) TAT membrane-penetrating peptide + LPS + ATP control group: pure TAT membrane-penetrating peptide (YGRKKRRQRRR, final concentration of 50 μg / mL) was added 12 h before LPS treatment, and then treated according to the protocol of group (3); (5) LPS+ATP+TAT-NOL12-K50R treatment group: NOL12 mutant fusion protein TAT-NOL12(K50R) (final concentration of 50 μg / mL) was added 12 h before LPS treatment, and then treated according to the protocol of group (3).

[0059] 3. Western blot detection (1) Collect cells from each group after treatment, and perform RIPA lysis on ice, BCA quantification, and take an equal amount of total protein (20 μg) from each group for SDS-PAGE electrophoresis, PVDF membrane transfer, and block with 5% skim milk powder for 30 minutes.

[0060] (2) Primary antibodies were incubated (overnight at 4°C) for NLRP3 antibody (1:1000), Caspase 1 antibody (1:1000, for simultaneous detection of full length and p20), GSDMD antibody (1:1000, for simultaneous detection of full length GSDMD and GSDMD-NT), and β-actin antibody (1:5000).

[0061] (3) Then HRP secondary antibody incubation (1:5000) was performed, and finally ECL color development and ImageJ grayscale quantification were performed.

[0062] 4. Experimental Results (1) NLRP3 protein detection results ( Figure 6 A, Figure 6 B): The baseline NLRP3 expression level was low in the blank control group; the NLRP3 protein level in the TAT-NOL12-K50R-only treatment group was not statistically different from that in the blank control group (p>0.05), suggesting that the mutant fusion protein had little effect on the baseline NLRP3 protein expression under non-inflammatory conditions; the NLRP3 protein expression in the LPS+ATP model group was significantly increased (p<0.05); the NLRP3 protein level in the TAT transmembrane peptide + LPS+ATP control group was not statistically different from that in the model group (p>0.05); the NLRP3 protein expression in the TAT-NOL12-K50R + LPS+ATP treatment group was significantly lower than that in the model group (p<0.05), close to the blank control level.

[0063] (2) Detection results of full-length Caspase 1 and p20 ( Figure 6 A, Figure 6C): No obvious Caspase 1 p20 band was observed in the blank control group and the TAT-NOL12-K50R alone treatment group; obvious Caspase 1 p20 band was observed in the LPS+ATP model group; the Caspase 1 p20 band in the TAT transmembrane peptide + LPS+ATP control group was similar to that in the model group; the Caspase 1 p20 band was significantly weakened in the TAT-NOL12-K50R + LPS+ATP treatment group.

[0064] (3) GSDMD test results ( Figure 6 A, Figure 6 D): In the blank control group and the TAT-NOL12-K50R-only treatment group, the GSDMD-FL band was normal, and there was no obvious GSDMD-NT band; in the LPS+ATP model group, the GSDMD-FL band was significantly lighter, and at the same time, an obvious GSDMD-NT band appeared; the TAT transmembrane peptide + LPS+ATP control group was similar to the model group; in the TAT-NOL12-K50R + LPS+ATP treatment group, the GSDMD-FL band recovered to a level close to that of the blank control group, and the GSDMD-NT band was significantly weakened or basically undetectable.

[0065] 5. Conclusion The results are as follows Figure 6 As shown, the NOL12 mutant fusion protein TAT-NOL12(K50R), when directly added to cells without transfection reagents, reduced NLRP3 protein expression, decreased Caspase 1 p20 fragment production, and reduced GSDMD-NT fragment levels in an in vitro sepsis-associated hepatocyte pyroptosis model induced by LPS and ATP in L02 hepatocytes, while relatively restoring the full-length GSDMD protein level. These results suggest that the NOL12 mutant fusion protein TAT-NOL12(K50R) can alleviate NLRP3 / Caspase 1 / GSDMD-related pyroptosis signaling in sepsis-associated hepatocytes.

[0066] Example 8: Drugs for treating sepsis-related hepatocellular pyroptosis-related liver injury The drug in this embodiment includes the NOL12 mutant fusion protein TAT-NOL12(K50R) and pharmaceutically acceptable excipients. The concentration of the NOL12 mutant fusion protein TAT-NOL12(K50R) in the drug is 10~100 μg / mL.

[0067] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A NOL12 mutant, characterized in that: Compared with the amino acid sequence of wild-type NOL12 shown in SEQ ID NO: 1, the NOL12 mutant has a lysine at position 50 mutated to arginine.

2. The NOL12 mutant according to claim 1, characterized in that: The amino acid sequence of the NOL12 mutant is shown in SEQ ID NO:

2.

3. A NOL12 mutant fusion protein, characterized in that: The NOL12 mutant fusion protein includes a TAT membrane-penetrating peptide, a flexible linker, and the NOL12 mutant as described in claim 1 or 2; The amino acid sequence of the TAT membrane-penetrating peptide is shown in SEQ ID NO:

3.

4. The NOL12 mutant fusion protein according to claim 3, characterized in that: The amino acid sequence of the flexible linker is shown in SEQ ID NO:

4.

5. The NOL12 mutant fusion protein according to claim 4, characterized in that: The amino acid sequence of the NOL12 mutant fusion protein is shown in SEQ ID NO:

5.

6. A gene encoding a NOL12 mutant fusion protein, characterized in that: The gene nucleotide sequence of the NOL12 mutant fusion protein is shown in SEQ ID NO:

6.

7. A method for preparing the NOL12 mutant fusion protein according to any one of claims 3 to 5, characterized in that: Includes the following steps: (1) The gene sequence of the NOL12 mutant fusion protein was synthesized and double-digested with NcoI and XhoI to obtain the gene fragment; (2) The pET28a(+) vector was double-digested with NcoI and XhoI to obtain the vector fragment; (3) The gene fragment and the vector fragment are ligated to obtain a recombinant plasmid; (4) The recombinant plasmid was transferred into Escherichia coli BL21(DE3) competent cells, and protein expression was performed by shaking culture. Then, the protein was subjected to sonication and Ni-NTA affinity chromatography to obtain the NOL12 mutant fusion protein.

8. The use of the NOL12 mutant fusion protein according to any one of claims 3 to 5 in the preparation of a medicament for sepsis-associated hepatocellular pyroptosis-related liver injury.

9. A drug for sepsis-related hepatocellular pyroptosis-related liver injury, characterized in that: The drug comprises the NOL12 mutant fusion protein according to any one of claims 3 to 5, wherein the concentration of the NOL12 mutant fusion protein in the drug is 10 to 100 μg / mL.

10. The medicament according to claim 9, characterized in that: The concentration of the NOL12 mutant fusion protein in the drug is 50 μg / mL.

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