N4BP1 nucleus entry defective model for researching N4BP1 cytoplasm function

Abstract

The invention provides an N4BP1 nucleus entry defective model for researching N4BP1 cytoplasm function, and relates to the technical field of biomedicine, N4BP1 as a key site for regulating inflammatory response plays an important role under physiological and pathological conditions. In an inflammatory state, N4BP1 negatively regulates expression of cell factors and is cut by caspase 8. In the virus infection process, N4BP1 can directly degrade virus RNA and is cut by MALT. Initially, N4BP1 can be observed at the nucleolus of a cell, however, recent studies find that N4BP1 is a cytoplasm protein. The application finds that N4BP1 is a nucleoplasm shuttle protein and mainly exists in insoluble components in the nucleus. When Leptomycin B is used for treating cells, N4BP1 can be accumulated in a nucleus, the mechanism is that 151-396 amino acid sequences of the N4BP1 participate in regulation, and NLS of the N4BP1 transferred from cytoplasm to the nucleus is 279-299 amino acid sequences. Therefore, an N4BP1 nucleus entry defect type cell model is constructed on the basis to research which functions of N4BP1 in cytoplasm and which mechanism of N4BP1 participating in regulation.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to an N4BP1 nuclear import defect model for studying the cytoplasmic function of N4BP1. Background Art

[0002] Biological processes such as DNA replication and protein synthesis occur in different cellular compartments: the nucleus and the cytoplasm. The surface of the eukaryotic cell nucleus has a nuclear envelope (NE), which is a protective barrier between the cytoplasm and the nuclear genome and also a medium for mechanical conduction or signal transduction. It is mainly composed of the nuclear lamina, the double membrane, and the nuclear pore complex (NPC). The nuclear pore complex is a dynamic and adaptive macromolecular complex composed of approximately 30 proteins, collectively referred to as nucleoporins (Nups). Its overall structure can be divided into the nuclear basket, the core structure of the central transport channel, and the cytoplasmic fibrils. When the molecular weight of a protein is less than 40 kDa, it can be diffusely transported through the central transport channel; when the molecular weight of a protein is greater than 40 kDa, the transport protein binds to the protein by recognizing the nuclear localization signal (NLS) for nuclear import or the nuclear export signal (NES) for nuclear export to form a transport complex for active transport. The most studied nuclear import pathway is mediated by importin α / β, which mediates the nuclear import of NLS-containing proteins in a Ran-dependent manner, while the nuclear export of a large number of NES-containing cargo proteins is mediated by the exportin chromosome maintenance protein 1 (CRM1). Many proteins, such as transcription factors and RNA-binding proteins, shuttle between the nucleus and the cytoplasm, and changes in the subcellular distribution of nucleocytoplasmic shuttle proteins are related to cancer and neurodegenerative diseases. Therefore, identifying new nucleocytoplasmic shuttle proteins is of great significance for understanding their regulation and related diseases.

[0003] N4BP1 is a binding protein of NEDD4 monoubiquitination substrates and can inhibit the function of the ubiquitin E3 ligase itch. N4BP1 has four key domains, namely the KH domain, the UBA domain, the NYN domain, and the CoCUN domain starting from the amino terminus. The KH and NYN domains of N4BP1 enable it to directly degrade mRNA substrates through the coding sequence. It has been found that N4BP1 degrades the mRNA of HIV through the NYN domain with ribonuclease activity, thereby inhibiting HIV replication; when CD4+ T cells are activated, N4BP1 is cleaved by mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) at Arg 509, resulting in the inactivation of N4BP1 and the reactivation of HIV-1. N4BP1 also has two ubiquitin-related domains: the UBA domain and the CoCUN domain. Studies have shown that the UBA domain can bind to ubiquitin (Ub) and ubiquitin-like proteins (UbLs). N4BP1 can negatively regulate NF-κB by inhibiting NEMO oligomerization through the UBA and CoCUN domains.

[0004] N4BP1 can act as a negative regulator of cytokines, being cleaved and inactivated by caspase-8. It has been found that N4BP1 is an important negative regulator of selective cytokine and chemokine responses induced by TRIF-independent TLRs (TLR2, TLR7 or TLR9), playing an important role in innate immune responses. N4BP1 also plays an important role in regulating tumor progression. For example, N4BP1 inhibits both canonical and non-canonical NF-κB pathways by binding to the deubiquitinase CEZANNE, resulting in the stabilization of TRAF3 and the degradation of NF-κB-inducing kinase NIK, leading to a decrease in MHC-1 expression, thus evading T cell immune surveillance and promoting the occurrence and development of neuroblastoma. There is also data supporting that N4BP1 can inhibit the replication of porcine reproductive and respiratory syndrome virus (PRRSV).

[0005] N4BP1 plays important roles in various life activities such as inflammatory responses, inhibiting virus replication, and regulating tumor progression. However, which mechanisms and functions each domain of N4BP1 participates in regulation are still unknown. Currently, the localization study of N4BP1 is not clear. It has been found that N4BP1 is detected in the nucleolus of mouse embryonic fibroblasts. Recent studies have found that N4BP1 is a cytoplasmic protein. The unclear localization of N4BP1 is not conducive to studying its specific functions and regulatory mechanisms. Summary of the Invention

[0006] The object of the present invention is to solve the technical problem in the prior art that the localization of N4BP1 is not clear, which is not conducive to studying the specific functions and regulatory mechanisms of N4BP1.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The nuclear localization signal of N4BP1, and the nuclear localization signal of N4BP1 is the amino acid sequence of 279-299 of N4BP1.

[0009] Preferably, the amino acid sequence of the nuclear localization signal is as shown in SEQ ID NO: 01.

[0010] The present application also provides the application of the nuclear localization signal of N4BP1 in the construction of an N4BP1 nuclear import defective model.

[0011] Preferably, the method for constructing the model is to construct an N4BP1 expression vector and knock out the nuclear localization signal of N4BP1.

[0012] Preferably, the method for constructing the model is to construct an N4BP1 expression vector and mutate the KRR amino acids of the nuclear localization signal of N4BP1 to SSS mutants or the KK amino acids to SS mutants.

[0013] Preferably, the model construction method is to directly knockout or mutate the amino acid sequence of N4BP1 at the nuclear localization signal of N4BP1 in cells by CRISPR technology.

[0014] The present application also provides an N4BP1 nuclear import defective model, which is constructed by using the above-mentioned construction method.

[0015] The present application also provides the application of the N4BP1 nuclear import defective model in studying the cytoplasmic function of N4BP1.

[0016] The present application deeply studies the localization of N4BP1 in cells, discovers that N4BP1 is a nucleocytoplasmic shuttle protein, and further studies its shuttle mechanism. During the research process, we found that the NLS for nuclear import of N4BP1 is the 279-299 amino acid sequence (ERICQKRRFSDSEERHTKKOF), and constructed an N4BP1 nuclear import defective model. This model plays an important role in our continued study of the regulatory mechanism of N4BP1 in the cytoplasm and its impact on diseases. Brief Description of the Drawings

[0017] Figure 1 Used to show the overexpression of FLAG-N4BP1 in 293T and HeLa, and immunofluorescence analysis of its localization;

[0018] Figure 2 Used to show the distribution of the stable cell line overexpressing N4BP1 in 293T by immunofluorescence analysis;

[0019] Figure 3 Used to show the overexpression of FLAG-N4BP1 in 293T and HeLa cells, treated with DMSO, Importazole (10 μM), Ivermectin (10 μM) or Leptomycin B (10 μM) for 1 hour, and immunofluorescence analysis of the localization map of FLAG-N4BP1.

[0020] Figure 4 Used to show the protein levels of FLAG-N4BP1 in the cytoplasm and nucleus detected by Western Blot.

[0021] Figure 5 Used to show the overexpression of FLAG-N4BP1 in HeLa cells, treated with Leptomycin B

[0022] (10 μM) for 24 hours, and Western Blot detection of the protein levels of FLAG-N4BP1 in the soluble and insoluble fractions of the cytoplasm and nucleus;

[0023] Figure 6 Used to show the protein levels of N4BP1 in the cytoplasmic, nuclear soluble and insoluble fractions of HeLa cells detected by Western Blot after treatment with Leptomycin B (10 μM) for 24 hours;

[0024] Figure 7 This is a schematic diagram of the truncated and point mutant plasmids of N4BP1 in one embodiment of the present invention;

[0025] Figure 8 Used to show the overexpression of full-length and truncated mutants of N4BP1 in HeLa cells, and immunofluorescence analysis of their localization in cells;

[0026] Figure 9 Used to show the overexpression of full-length and truncated mutants of N4BP1 in HeLa cells, treated with DMSO and Leptomycin B (10 μM) for 4 hours, and immunofluorescence analysis of their localization;

[0027] Figure 10 Used to show the overexpression of full-length and point mutants of N4BP1 in HeLa cells, treated with DMSO and Leptomycin B (10 μM) for 4 hours, and immunofluorescence analysis of their localization;

[0028] Figure 11 This is a schematic diagram of full-length N4BP1, NLS deletion and NLS point mutation in one embodiment of the present invention;

[0029] Figure 12 Used to show the overexpression of full-length N4BP1, NLS deletion and NLS point mutation in HeLa cells, and immunofluorescence analysis of their localization;

[0030] Figure 13 Used to show the overexpression of GFP and GFP-NLS in HeLa cells, and immunofluorescence analysis of their localization. Detailed implementation mode

[0031] The following combines specific embodiments to further elaborate on the present invention in detail.

[0032] This application provides a nuclear localization signal (NLS) of N4BP1, and the nuclear localization signal is the amino acid sequence of 279-299 of N4BP1, and the amino acid sequence of the nuclear localization signal is shown in Table 1.

[0033] Table 1 Nuclear localization signal (279-299aa) of human N4BP1

[0034] Protein Name Species Sequence Name Sequence Position Amino Acid Sequence N4BP1 Human Nuclear Localization Signal 279-299 ERICQKRRFSDSEERHTKKOF

[0035] This application also constructs a nuclear localization defective model of N4BP1 and its construction method based on the nuclear localization signal of N4BP1.

[0036] Based on the above model, this application also provides the application of the nuclear localization defective model of N4BP1 in studying the cytoplasmic function of N4BP1.

[0037] The above content is elaborated as follows with specific examples:

[0038] Example 1: Studying the localization of N4BP1 in cells

[0039] FLAG-N4BP1 was overexpressed in 293T and HeLa cells, and N4BP1 was distributed in both the cytoplasm and the nucleus ( Figure 1 ).

[0040] To further verify this view, in this example, a cell line stably expressing N4BP1 was constructed in 293T, and the same phenomenon was also observed ( Figure 2 ).

[0041] Then, to verify whether N4BP1 shuttles between the nucleus and the cytoplasm, in this example, FLAG-N4BP1 was overexpressed in 293T and HeLa cells, and treated with the nuclear export inhibitor Leptomycin B and two nuclear import inhibitors, Importazole and Ivermectin. Immunofluorescence showed that Leptomycin B inhibited the nuclear export of FLAG-N4BP1 ( Figure 3 ).

[0042] To further prove this view, in this example, the cytoplasmic protein and nuclear protein of cells were separated, but FLAG-N4BP1 was not detected in the nucleus, suggesting that FLAG-N4BP1 might be located in the insoluble fraction of the nucleus ( Figure 4 ).

[0043] On this basis, the protein of the insoluble fraction of the nucleus was extracted and treated with 10 μM Leptomycin B for 24 hours, and it was found that the amount of FLAG-N4BP1 in the insoluble fraction of the nucleus increased ( Figure 5 ). Similarly, after endogenous N4BP1 was treated with 10 μM Leptomycin B for 24 hours, its protein level also showed an upward trend in the insoluble fraction of the nucleus ( Figure 6 ).

[0044] The above experiments illustrate that N4BP1 is a nucleocytoplasmic shuttle protein and is located in the insoluble fraction in the nucleus.

[0045] Example 2: Studying the shuttle mechanism of N4BP1:

[0046] First, truncation and point mutation plasmids of N4BP1 were constructed ( Figure 7 ). Immunofluorescence showed that the KH-only domain was only present in the cytoplasm, but the addition of the 151-396 amino acid sequence enabled its localization in both the cytoplasm and the nucleus, while mutants lacking the 151-396 amino acid sequence (dKH and NYN-CoCUN) were mainly present in the cytoplasm, indicating that the 151-396 amino acid sequence is involved in regulating the nucleocytoplasmic shuttling of N4BP1 ( Figure 8 ). Similar to FL, treatment with Leptomycin B could inhibit the nuclear export of the KH-UBA mutant, but had less effect on the KH-only, dKH, and NYN-CoCUN mutants ( Figure 9 ). However, after mutating the functions of the KH domain (G71D and G93D) and the UBA domain (L350A, L379 / 380A), treatment with Leptomycin B did not affect the distribution of N4BP1 ( Figure 10 ). The above results indicate that the 151-396 amino acid sequence of N4BP1 is involved in regulating its localization in cells.

[0047] Example 3: Verification of the function of NLS

[0048] The transport of proteins from the cytoplasm to the nucleus is mediated by a nuclear localization signal (NLS). In this application, first, the potential NLS of human N4BP1 was predicted to be the 279-299 amino acid sequence (ERICQKRRFSDSEERHTKKOF) by an NLS prediction program (https: / / nls-mapper.iab.keio.ac.jp / cgi-bin / NLS_Mapper_form.cgi).

[0049] To verify the function of the predicted NLS, in this application, plasmids encoding N4BP1 NLS deletion mutants were first constructed, and point mutations were also constructed to analyze the key amino acids affecting the NLS function ( Figure 11 ). Immunofluorescence results showed that full-length N4BP1 was distributed in both the cytoplasm and the nucleus, while the NLS deletion mutant was localized in the cytoplasm, and the point mutations (KRR to SSS or KK to SS) were also localized in the cytoplasm ( Figure 12 ). These results indicate that the NLS of N4BP1 affects its nuclear import. To further verify the importance of NLS, we fused it to the C-terminus of GFP, and the results showed that wild-type GFP was mainly present in the cytoplasm, while GFP fused with NLS was mainly located in the nucleus ( Figure 13 ). The above research results indicate that 279-299 (NLS) of N4BP1 is involved in regulating its nuclear distribution.

[0050] In summary, N4BP1 is a nucleocytoplasmic shuttle protein that is mainly located in the insoluble fraction in the nucleus. Its shuttle mechanism is regulated by the 151-396 amino acid sequence of N4BP1, and Leptomycin B can cause N4BP1 to accumulate in the nucleus. Among them, 279-299 of N4BP1 is the nuclear localization signal for N4BP1 to be transported from the cytoplasm to the nucleus, which plays an important role in regulating its nuclear entry. This provides a theoretical basis for constructing a nuclear entry-deficient model of N4BP1 and continuing to study the mechanism and function of N4BP1 in the cytoplasm.

[0051] The following are some of the experiments and their steps in the above embodiments:

[0052] (1) Cell culture

[0053] 293T and HeLa cells were cultured in DMEM (containing 10% serum and 1% penicillin-streptomycin mixture), placed in an incubator at 37°C, and the carbon dioxide concentration was 5%.

[0054] (2) Transfection and construction of stable cell lines

[0055] Cells were evenly plated in six-well plates and transfected when the cell density reached 50-60%. Prepare two sets of 1.5 mL EP tubes, add 100 μL of Opti-MEM medium to all EP tubes. Add 2 μg of plasmid to each tube in the first set of EP tubes, and add 4 μL of Lipo-2000 transfection reagent to each tube in the second set of EP tubes. Add the medium containing plasmid in the first set to the corresponding medium containing Lipo-2000 respectively. After standing for 15 minutes, add the mixture to the six-well plates respectively. Change the medium 6-8 hours later, observe the fluorescence under the microscope 48 hours later, and add 1 μg / mL of Puromycin for screening. The drug concentration can be increased according to the actual situation. When all the wild-type cell groups are dead, the Puromycin concentration can be determined. Culture with complete medium containing Puromycin for about one week, and it can be verified by real-time fluorescence quantitative PCR or Western Blot.

[0056] (3) Immunofluorescence

[0057] Place round glass slides in a 24-well plate in advance, inoculate 293T or HeLa cells, and culture them in an incubator at 37°C. When the cell density reaches 70%, fix with 4% paraformaldehyde at room temperature. After 1 hour, wash 3 times with PBS. Then discard the PBS, add PBS containing 0.1% Triton X-100, and permeabilize for 10 minutes. After discarding the waste liquid, add PBS containing 1% BSA and block at room temperature for 30 minutes. Discard the waste liquid, add the primary antibody, and incubate overnight at 4°C. The next day, recover the primary antibody and wash the cells 5 times with PBS for a total of 20 minutes. Then add the fluorescent secondary antibody and incubate at room temperature for 1 hour. Discard the fluorescent secondary antibody, add Hoechst 33342, and incubate at room temperature for 1 hour. After 1 hour, discard Hoechst 33342 and wash 5 times with PBS for a total of 20 minutes. Then drop the anti-quencher on the glass slide, label the cell name and date. Take out the round glass slide, invert it on the anti-quencher, fix the round glass slide with transparent nail polish, and let it stand in the dark at room temperature for 1 hour. After the nail polish dries, the confocal microscope can be used to take images.

[0058] (4) Western Blot

[0059] Collect cells with a cell scraper, add an appropriate amount of RIPA lysis buffer and the protease inhibitor PMSF. Sonicate on ice 1-3 times for 5 seconds each time. After lysing on ice for 30 minutes, centrifuge at 12,000 rpm for 15 minutes, take the supernatant, add 5×SDS loading buffer, and boil at 100°C for 15 minutes to denature. Add the sample to an SDS-PAGE gel with the corresponding concentration and perform electrophoresis. After running at a constant voltage of 60V for 30 minutes, run at a constant voltage of 120V for 1 hour. Then transfer the proteins in the SDS-PAGE gel to the NC membrane at a constant current of 300 mA for 1 hour. Subsequently, block with 5% skim milk at room temperature for 1 hour, incubate the primary antibody overnight at 4°C. Recover the primary antibody the next day and wash 3 times with 1×TBST. Incubate the corresponding species secondary antibody at room temperature for 1 hour, wash 3 times with TBST again, and develop with a hypersensitive ECL chemiluminescence detection reagent.

[0060] In summary, this application explored the intracellular distribution of N4BP1, revealed that N4BP1 is a nucleocytoplasmic shuttle protein, and further studied its shuttle mechanism. This application found the nuclear localization signal (NLS) of N4BP1 and constructed a nuclear import defective model of N4BP1, which can be used to deeply study the specific functions and regulatory mechanisms of N4BP1 in the cytoplasm, and also provide a new theoretical basis for studying N4BP1 in disease prevention and treatment.

Claims

1. The nuclear localization signal of N4BP1 is characterized by: The nuclear localization signal of N4BP1 is the amino acid sequence 279-299 of N4BP1.

2. The nuclear localization signal of N4BP1 according to claim 1, characterized in that: The amino acid sequence of the nuclear localization signal is as shown in SEQ ID NO:

01.

3. Application of the nuclear localization signal of N4BP1 in the construction of the N4BP1 nuclear defect model.

4. The use of the nuclear localization signal of N4BP1 according to claim 3 in constructing a N4BP1 nuclear defect model, characterized in that: The model construction method is to construct an N4BP1 expression vector and knock out the nuclear localization signal NLS sequence of the N4BP1.

5. The use of the nuclear localization signal of N4BP1 according to claim 3 in constructing a N4BP1 nuclear defect model, characterized in that: The model construction method comprises constructing an N4BP1 expression vector, and mutating the KRR amino acid of the nuclear localization signal of the N4BP1 to an SSS mutant or a KK amino acid to an SS mutant.

6. The use of the nuclear localization signal of N4BP1 according to claim 3 in constructing a N4BP1 nuclear defect model, characterized in that: The model construction method is to construct a model by using CRISPR technology to directly knock out or mutate the amino acid sequence of N4BP1 in the cell according to the nuclear localization signal of N4BP1.

7. A N4BP1 nuclear defect model, characterized in that: The method according to claim 4-5 is used to construct the obtained product.

8. Application of the N4BP1 nuclear import defective model in studying the cytoplasmic function of N4BP1.

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