Oligopeptide derivative based on lysosomal membrane protein 2A and application thereof

Abstract

The invention discloses an oligopeptide derivative based on lysosomal membrane protein 2A and application of the oligopeptide derivative. The oligopeptide derivatives disclosed by the invention are L2AK104, L2AK289, L2AK132 and L2ASIM1, and the amino acid sequences of the oligopeptide derivatives are shown as SEQ ID No.6 to SEQ ID No.9 in sequence. The oligopeptide derivative is composed of cell-penetrating peptides and oligopeptides, the oligopeptides are oligopeptides based on K104, K289, K132 and SIM1 sites modified by lysosomal membrane protein 2A polypeptide SUMO, and the amino acid sequences of the oligopeptides are sequentially shown as SEQ ID No.1-4; the cell-penetrating peptide is connected to the N terminal of the short peptide, and the amino acid sequence of the cell-penetrating peptide is shown as SEQ ID No.5. The four oligopeptide derivatives in the invention do not influence the cell activity and have no cytotoxicity, and the tumor virus KSHV is used as a research model, and in-vivo experiments repeatedly confirm that the four oligopeptide derivatives have a certain inhibition effect on the infection of viruses such as the KSHV and the proliferation of tumor cells. The compound can be used for preparing related drugs for resisting KSHV virus infection and treating tumor diseases, and has important application value in prevention and targeted treatment of virus related diseases such as KSHV.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a short peptide derivative based on lysosomal membrane protein 2A and a use thereof. Background Art

[0002] According to statistics, viral infection is closely related to the occurrence and development of about 20% of tumors and is one of the main causes of their onset. Oncogenic viruses can cause tumors in humans or animals, and even induce malignant transformation of normal cells in vitro. So far, a variety of human tumor viruses have been discovered, including Kaposi's sarcoma virus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV). Viruses can promote the occurrence of tumors in many ways, including gene integration, chronic inflammation, immune escape, and regulation of host cell signaling pathways. Among them, Kaposi's sarcoma virus (Kapos's sarcoma-associated Herpesvirus, KSHV), also known as human herpesvirus-8 (Human Herpesvirus-8, HHV-8), is an enveloped virus containing double-stranded DNA. It was first identified and discovered by Chang and Moore in 1994 from tissues of Kaposi's sarcoma patients infected with human immunodeficiency virus. It belongs to the human gammaherpesvirus subfamily, the same as the Epstein-Barr virus. It is a tumor virus that can infect various types of cells, including B cells, endothelial cells and epithelial cells, and is closely related to a variety of human malignant tumors, including primary effusion lymphoma, Kaposi's sarcoma and multifocal lymphoma.

[0003] Similar to other herpes viruses, KSHV has two life cycles: latency and lytic replication, and it mainly exists in the form of latent infection in tumor tissue cells. According to statistics, the global infection rate of KSHV is about 2% to 10%, but there are large regional differences. The infection rate in South America is generally low, and the infection rate in American Indians is high. A large amount of clinical data shows that AIDS patients die not from HIV infection itself, but from complications including KSHV-induced systemic malignant lymphoma. It is estimated that about 14% of AIDS deaths are caused by cancers concurrent with KSHV-related Kaposi's sarcoma, and there are about 60,000 new cases of primary effusion lymphoma worldwide each year, but there is currently no effective targeted treatment strategy. Therefore, it is urgent to conduct in-depth research on KSHV infection and tumorigenic mechanisms in order to provide effective potential targeted intervention strategies for anti-KSHV related diseases. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems existing in the prior art and provide a short peptide derivative based on lysosomal membrane protein 2A and its use. The short peptide derivative based on lysosomal membrane protein 2A in the present invention is a polypeptide compound that can penetrate the cell membrane and enter tumor cells and is stable in vivo, and has high specificity, effectiveness and low cytotoxicity against latent infection of viruses such as KSHV, and has significant inhibitory efficiency on viral infection such as KSHV and tumor growth, proliferation and angiogenesis.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is as follows: short peptide derivatives based on lysosomal membrane protein 2A, the short peptide derivatives are L2AK104, L2AK289, L2AK132 and L2ASIM1; the amino acid sequences of L2AK104, L2AK289, L2AK132 and L2ASIM1 are shown in SEQ ID No.6-9 respectively.

[0006] Preferably, the short peptide derivative consists of a membrane-penetrating peptide and a short peptide, the short peptide is a short peptide based on the lysosomal membrane protein 2A polypeptide SUMO modification-related K104, K289, K132 and SIM1 sites, the short peptides at the K104, K289, K132 and SIM1 sites correspond to the short peptide derivatives L2AK104, L2AK289, L2AK132 and L2ASIM1, respectively, and the amino acid sequences of the short peptides at the K104, K289, K132 and SIM1 sites are shown in SEQ ID No.1-4, respectively; the membrane-penetrating peptide is connected to the N-terminus of the short peptide, and the amino acid sequence of the membrane-penetrating peptide is shown in SEQ ID No.5.

[0007] The chemical structural formula of the short peptide at the K104, K289, K132 and SIM1 sites is as follows:

[0008] Short peptide at K104 site

[0009]

[0010] Short peptide at K289

[0011]

[0012] Short peptide at K132

[0013]

[0014] Short peptide at SIM1 site

[0015]

[0016] Preferably, the short peptide derivative is prepared by solid phase synthesis.

[0017] The present invention provides a pharmaceutical preparation, which comprises one or two of the above-mentioned short peptide derivatives.

[0018] The present invention also provides the use of the short peptide derivative in preparing an antiviral infection drug, wherein the virus is Kaposi's sarcoma virus KSHV, and the short peptide derivative can inhibit the increase in the viral copy number of KSHV.

[0019] The present invention also provides the use of the short peptide derivative in preparing drugs for treating tumors, wherein the tumor is caused by Kaposi's sarcoma virus KSHV, and the short peptide derivative can inhibit the proliferation of KSHV virus-positive tumor cells, and inhibit tumor growth and angiogenesis.

[0020] Virus infection such as KSHV can induce TRIM32 to regulate SUMO modification of LAMP2A, resulting in a large accumulation of LAMP2A, which is one of the important factors for maintaining latent infection of viruses such as KSHV and inducing related diseases. LAMP2A is a key protein for heat shock protein-mediated autophagy. TRIM32 regulates SUMO modification of LAMP2A under viral infection, and the main function of its interaction is to promote high expression of LAMP2A. In the present invention, the sites where LAMP2A undergoes SUMO modification are discovered through a series of experimental tests, and polypeptides related to K104, K289, K132 and SIM1 sites are screened out. The peptide segment can competitively inhibit SUMO modification of LAMP2A and inhibit the protein expression of LAMP2A, thereby playing an anti-KSHV and other viral infections and tumors.

[0021] The present invention designs a variety of LAMP2A-derived polypeptide compounds L2AK104, L2AK289, L2AK132 and L2ASIM1, determines the peptide sequence and length through the gene sequence and specific SUMO modification site of LAMP2A; detects the effects of the polypeptide compounds on cell viability and toxicity through CCK-8 experiments; compares the effects of different polypeptide compounds on the proliferation of tumor cells associated with viruses such as KSHV through cloning; and further verifies the effects of different polypeptide compounds on the growth and angiogenesis of tumors associated with viruses such as KSHV using a mouse tumor-bearing model, thereby providing a new strategy for the treatment of diseases associated with viral infections such as KSHV.

[0022] Beneficial effects of the present invention: The present invention provides four short peptide derivatives based on lysosomal membrane protein 2A, namely L2AK104, L2AK289, L2AK132 and L2ASIM1. These four short peptide derivatives do not affect cell viability and are non-cytotoxic. In addition, the present invention uses tumor virus KSHV as a research model, and repeatedly confirms from in vivo experiments that these four short peptide derivatives have a certain inhibitory effect on viral infection such as KSHV and tumor cell proliferation, laying a foundation for the treatment of viral infection such as KSHV and tumor diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figures are the results of Example 1; A) the interaction between TRIM32 and CMA autophagy-related proteins before and after KSHV virus infection; B) the co-localization of TRIM32 and CMA autophagy-related proteins before and after KSHV virus infection; C) knocking down TRIM32 or LAMP2A in KSHV-infected cells can restore the expression level of CMA substrate MEF2D; D) the domain of interaction between TRIM32 and LAMP2A before and after simulated DNA virus infection; E) TRIM32 overexpression can increase DNA virus infection-induced SUMO2 / 3 modification of LAMP2A protein; F) RING domain deletion can inhibit TRIM32-mediated SUMO2 / 3 modification of LAMP2A.

[0024] Figure 2 The K104, 289, K132 and SIM1 sites are respectively conducive to DNA virus infection-induced SUMO2 and SUMO3 modifications of LAMP2A.

[0025] Figure 3 The results of TRIM32 regulating SUMO modification of LAMP2A to inhibit KSHV virus copy number and expression of key viral proteins in Example 3 are shown in FIG. A) TRIM32 activates and degrades RTA by modifying the K104, 289, K132, and SIM1 sites of LAMP2A; B) TRIM32 inhibits the production of sub-virus particles by modifying the K104, 289, K132, and SIM1 sites of LAMP2A.

[0026] Figure 4 The results of the effect of TRIM32-mediated SUMO modification of LAMP2A on subcutaneous tumor growth and angiogenesis in Example 3 are shown in FIG. A) is a schematic diagram of the design of the mouse tumor-bearing experiment; B) is a comparison chart of tumor size between experimental groups and a statistical chart of tumor weight; C) is a comparison of representative tumors and surrounding angiogenesis in each experimental group.

[0027] Figure 5HPLC was used to detect the purity of the synthesis of different short peptides of LAMP2A; among them, A) L2AK104; B) L2AK104R; C) L2AK289; D) L2AK289R; E) L2AK132; F) L2AK132R; G) L2ASIM1; H) L2AmSIM1.

[0028] Figure 6 CCK-8 was used to detect the effects of different short peptides on cell (iSLK-Bac16) viability.

[0029] Figure 7 Effects of different short peptide compounds of LAMP2A on the growth and angiogenesis of subcutaneous tumors infected with KSHV virus; A) Schematic diagram of mouse tumor bearing experiment design; B) Comparison of tumor size and statistical graph of tumor weight among experimental groups; C) Comparison of representative tumors and surrounding angiogenesis in each experimental group; (D) Schematic diagram of mouse weight among experimental groups.

[0030] KSHV-negative endothelial cells (including MM and iSLK) and corresponding KSHV-positive cells (including KMM and iSLK-Bac16) are disclosed in the following documents:

[0031] [1]BRULOIS KF, CHANG H, LEE A SY, et al. Construction and Manipulation of a New Kaposi's Sarcoma-Associated Herpesvirus Bacterial Artificial Chromosome Clone[J].2012,86(18):9708-20.

[0032] [2]MYOUNG J, GANEM D. Generation of a doxycycline-inducible KSHVproducer cell line of endothelial origin: Maintenance of tight latency with efficient reactivation upon induction[J]. Journal of Virological Methods, 2011,174(1):12-21.

[0033] [3]JONES T,YE F,BEDOLLA R,et al.Direct and efficient cellular transformation of primary rat mesenchymal precursor cells by KSHV[J].J ClinInvest,2012,122(3):1076-81. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0035] Example 1 Effects of viral infection on the expression and localization of TRIM32 and CMA autophagy-related protein LAMP2A and its modification level

[0036] 1. KSHV infection promotes the interaction and colocalization of TRIM32 and LAMP2A

[0037] TRIM32 is highly expressed in KS tumor tissues and is involved in the regulation of heat shock protein-mediated autophagy (CMA). To detect the association between CMA and TRIM32 before and after KSHV infection, two KSHV-negative endothelial cells (including MM and iSLK) and corresponding KSHV-positive cells (including KMM and iSLK-Bac16) were used to detect the association between CMA and TRIM32 before and after KSHV infection. [1-3] TRIM32-specific antibodies were used for co-immunoprecipitation and immunoblotting, and the specific substrate MEF2D was selected as a CMA activity marker for simultaneous detection. Figure 1 As shown in A: Compared with cells not infected with KSHV, the interaction between TRIM32 and HSC70 and LAMP2A was significantly enhanced in KSHV-positive cells, while the interaction with HSP90 was significantly weakened. It was also found that after KSHV infection, the expression levels of LAMP2A and TRIM32 were significantly increased, and the expression level of MEF2D, a CMA-specific substrate, was significantly weakened, while the levels of other proteins did not change significantly. This suggests that KSHV infection promotes the interaction between TRIM32 and HSC70 and LAMP2A, but inhibits the interaction with HSP90, thereby promoting CMA activation.

[0038] Next, immunofluorescence was used to further detect the changes in the co-localization of TRIM32 and its target proteins after KSHV infection ( Figure 1B). The results showed that compared with KSHV-negative cells, HSC70, HSP90 and LAMP2A all entered the nucleus in KSHV-positive endothelial cells, especially LAMP2A, which was significantly aggregated in the nucleus and co-localized with TRIM32 in the nucleus. This indicates that HSC70, HSP90 and LAMP2A undergo a stress response of entering the nucleus in response to KSHV infection, and co-localize with TRIM32 to varying degrees, among which the co-localization of TRIM32 and LAMP2A is the most obvious.

[0039] In summary, during viral infection, TRIM32 mainly regulates the CMA pathway by interacting with LAMP2A.

[0040] 2. TRIM32 knockdown affects LAMP2A protein expression

[0041] In order to further verify the association between TRIM32 and LAMP2A, this example uses a lentiviral packaging method to construct stable cell lines with knockdown of TRIM32 and LAMP2A. Next, the knockdown TRIM32 / LAMP2A stable cell lines constructed above were used to detect the expression of LAMP2A, TRIM32 and MEF2D by immunoblotting. Figure 1 As shown in Figure C, when the expression level of TRIM32 decreased, the expression level of LAMP2A also decreased, while the expression level of MEF2D increased; when the expression level of LAMP2A decreased, the expression level of TRIM32 had no significant effect, and the expression level of MEF2D also increased significantly. Given that downregulation of TRIM32 expression can lead to decreased LAMP2A expression and upregulation of MEF2D expression, it is suggested that TRIM32 positively regulates LAMP2A expression and acts as an upstream molecule to target and regulate LAMP2A to enhance CMA activity.

[0042] 3. TRIM32 affects the modification level of LAMP2A

[0043] In order to clarify which specific domain of TRIM32 is involved in the interaction with LAMP2A, in this example, mutants of TRIM32 with different domains deleted and tagged with EGFP were constructed, wild-type TRIM32 and its truncated form tagged with EGFP were transfected into HEK293T cells, and a control group and a simulated virus infection poly(dA:dT) treatment group were set up, and then the interaction between TRIM32 and its target protein was detected by immunoprecipitation and immunoblotting, respectively. Figure 1As shown in D, in the absence of stimulation, when the RING or CC domain of TRIM32 is missing, the interaction between LAMP2A and TRIM32 disappears or weakens, while when the NHL domain of TRIM32 is missing, the interaction between LAMP2A and TRIM32 is significantly enhanced; after poly(dA:dT) stimulation, when the CC domain is missing, TRIM32 appears as a multimer, and the interaction with the modified state of LAMP2A is significantly enhanced. This suggests that under normal physiological conditions, the interaction between LAMP2A and TRIM32 depends on the RING and CC domains of TRIM32; under poly(dA:dT) stimulation conditions, TRIM32 interacts with the modified state of LAMP2A with the help of the RING and CC domains.

[0044] In view of the effect of SUMO modification on protein localization, and the obvious co-localization of TRIM32 with LAMP2A under KSHV infection, it is speculated that TRIM32 may be involved in regulating the SUMO modification of LAMP2A. For further verification, HEK293T cells were first co-transfected with FLAG-tagged LAMP2A, HA-tagged SUMO1, SUMO2 or SUMO3, and TRIM32-myc. After 24 hours of transfection, they were stimulated with poly(dA:dT) for 24 hours, and detected by denaturing immunoprecipitation and immunoblotting experiments, such as Figure 1 As shown in E, after poly(dA:dT) stimulation, the levels of the three modifications of LAMP2A (including SUMO1, SUMO2 and SUMO3) increased, and when TRIM32 was overexpressed, the SUMO1 modification level of LAMP2A was significantly weakened, while the SUMO2 and SUMO3 modification levels were significantly increased. This shows that under poly(dA:dT) stimulation conditions, TRIM32 can induce SUMO2 and SUMO3 modifications of LAMP2A protein, while inhibiting SUMO1 modification.

[0045] In this example, LAMP2A and SUMO2 or SUMO3 were overexpressed in HEK293T cells at the same time. After 24 hours of transfection, poly(dA:dT) was used for 24 hours. The modification of LAMP2A was detected by denaturing immunoprecipitation and immunoblotting. The RING domain deletion mutant was used as a control. Figure 1 As shown in F: Overexpression of TRIM32 can significantly promote SUMO2 or SUMO3 modification of LAMP2A, while when the RING domain is missing, the SUMO2 modification level of LAMP2A is significantly reduced, while the SUMO3 modification level does not change significantly. These data indicate that TRIM32 mainly induces SUMO2 modification of LAMP2A through the RING domain, while its SUMO3 modification level is not dependent on the RING domain.

[0046] The experimental steps involved in this embodiment are as follows:

[0047] 1. Preparation of cell sample lysis:

[0048] 1) Collect an appropriate amount of cells, centrifuge at 2500 rpm for 3 min at room temperature, and discard the culture medium.

[0049] 2) Wash twice with pre-cooled PBS, centrifuge at 2500 rpm for 3 min at room temperature, and discard PBS.

[0050] 3) Add an appropriate amount of RIPA protein lysis buffer containing protease inhibitors (1% NP-40, 2mM EDTA, 150mM NaCl, 50mM Tris (pH7.6), 1mM PMSF, 1mM NEM, 1g / mL Aprotinin, 1g / mL leupeptin and 1g / mL pepstatin) and lyse on ice for 30 minutes.

[0051] 4) Oscillate once every 5 minutes to ensure complete lysis.

[0052] 5) The fully lysed cells were centrifuged at 14500 rpm for 5 min in a 4°C centrifuge.

[0053] 6) Transfer the supernatant after centrifugation to a new EP tube, which is the whole cell lysate. Measure the protein concentration using the Coomassie Brilliant Blue G-250 method.

[0054] 7) The protein samples were divided into smaller portions according to the loading amount, 6X protein loading was added, and the samples were boiled in a metal bath at 100°C for 5 min until the proteins were denatured.

[0055] 8) Protein samples were frozen at -80℃ for future use or directly used for immunoblotting experiments.

[0056] 2. Western blotting experimental steps:

[0057] 1) SDS-PAGE electrophoresis: Add the above denatured protein samples into SDS-PAGE gel of appropriate concentration for gel electrophoresis separation.

[0058] 2) Transfer: 0.45 μm NC membrane was immersed in 1X NC membrane transfer equilibrium solution and transferred using GenScript eBlot TM L1 fast wet transfer instrument standard program transfer, about 10 minutes.

[0059] 3) Blocking: Place the NC membrane in 5% skim milk powder (PBS) and incubate at room temperature for 1 h.

[0060] 4) Primary antibody incubation: Wash three times with TBST at room temperature for 5 min each time, add primary antibody diluted in appropriate ratio, and incubate at 4°C overnight.

[0061] 5) Secondary antibody incubation: Wash three times with TBST at room temperature, 5 min each time, and use fluorescent secondary antibody 800 of the corresponding species.

[0062] 6) Incubate at room temperature in the dark for 1 hour; wash three times with TBST at room temperature, 5 minutes each time.

[0063] 7) Development: Use Odyssey two-color imager for development.

[0064] 3. Construction of recombinant plasmid:

[0065] 1) Primer design: The target sequence was queried from NCBI and primers were designed. The following primer sequences are shown in SEQ ID No. 16-39.

[0066]

[0067]

[0068]

[0069] 2) PCR amplification of target fragments: reverse transcription product acquisition; existing plasmid acquisition and use Max DNAPolymerase (R045A, Takara)

[0070] PCR amplification system preparation

[0071]

[0072] PCR amplification reaction procedure

[0073]

[0074] 3) Enzyme digestion:

[0075] Preparation of enzyme digestion system

[0076]

[0077] Reaction Procedure

[0078]

[0079]

[0080] 4) Ligation sequencing: Ligation was performed using the Biotime Seamless Cloning Kit (D7010M). For specific precautions, please refer to the product manual.

[0081] 4. Construction of Short hairpin RNA (ShRNA) plasmid

[0082] 1. Primer design: Design the target gene targeting sequence according to the following method. The following primers are shown in SEQ ID No. 40-55.

[0083] (1) Review relevant literature on the target gene

[0084] (2) Merck website: https: / / www.sigmaaldrich.cn

[0085] (3) Thermo company website: https: / / rnaidesigner.thermofisher.com

[0086]

[0087]

[0088] 2. Synthesize Oligos sequence:

[0089] (1) Prepare 20 μL of synthetic oligos sequence system

[0090] Preparation of synthetic Oligos system

[0091]

[0092] (2) Annealing procedure: Place in a 100°C water bath for 5 min and then slowly cool to room temperature.

[0093] 3. Enzyme digestion: Linearize the pLKO.1 vector with restriction endonucleases AgeⅠ and EcoRⅠ

[0094] Preparation of enzyme digestion system

[0095]

[0096] 4. Connect

[0097] Use Bio-Tech Ultra-Fast T4 DNA Ligase (D7009M). Please refer to the product manual for specific precautions.

[0098] 5. Lentivirus packaging and purification:

[0099] 1) Prepare cells: Each batch of HEK293T cells should be passaged no more than 20 times, and virus packaging can begin at the third generation after recovery.

[0100] 2) Prepare plasmid:

[0101] a) Two-plasmid system: total plasmid volume 40 μg (target plasmid 15 μg, pSPA 18 μg, pMD2G 7 μg)

[0102] b) Three-plasmid system: total plasmid volume 58 μg (target plasmid 15 μg, pRSV.REV 18 μg, pMD2G 18 μg, pMDLg-pRRE 7 μg)

[0103] 3) Prepare for transfection: Use product (Polyethylenimine Linear (PEI) MW40000 (rapid lysis)) or Lipofectamine TM 3000 for transfection operation.

[0104] 4) Collect supernatant: Collect cell supernatant 24h and 48h after medium replacement into 50mL centrifuge tubes

[0105] 5) Concentrated supernatant: Centrifuge the cell supernatant at 2000 rpm for 5 minutes and filter using a 0.45 μm filter.

[0106] 6) Centrifuge at 10,000 g for 18 h at 4 °C, acceleration 5, deceleration 0.

[0107] 7) Preservation of the virus: Remove the virus tube from the centrifuge, carefully aspirate the supernatant, leaving about 1.5 mL of supernatant, mix thoroughly, divide into 3 tubes, and freeze at -80°C. Label the virus name, date, and batch.

[0108] 6. Construction of stable cell lines:

[0109] 1) Determine the optimal antibiotic screening concentration: Before lentiviral infection, first determine the optimal killing antibiotic screening concentration for the desired target cells.

[0110] 2) Cell plating: For adherent cells, add 500 μL of 0.5×10 6 The cells to be infected were plated in 24-well plates and waited for transfection the next day.

[0111] 3) Prepare the virus: Take out the virus stored in a -80°C freezer in advance and place it on ice, waiting for it to thaw.

[0112] 4) Virus infection: Take out the cells plated the day before and add the prepared virus dropwise.

[0113] 5) Supplement the culture medium: After 24h and 48h, replenish the medium and divide the wells in time.

[0114] 6) Cell screening: Cell screening was started 72 hours after the virus was inoculated, according to the optimal antibiotic screening concentration for target cell killing determined previously.

[0115] 7) Cell verification: One week after screening, if there is fluorescence, by observing the fluorescence rate reaching 80-90%, collect total cell RNA or protein to detect the virus infection effect, and prepare to freeze the cells.

[0116] 7. Immunofluorescence of adherent cells:

[0117] 1) Cell treatment: Count the cells first to determine the number of cells on the slide, gently mix with 1 mL of 4% paraformaldehyde and fix for 20 min, wash once with PBS; add 1 mL / well of Blocking Buffer to immerse the slide and block for 30 min.

[0118] 2) Primary antibody incubation: Dilute the primary antibody into Blocking Buffer according to the antibody instructions and incubate at room temperature for 3 hours.

[0119] 3) Secondary antibody incubation: (room temperature, away from light) Same as the primary antibody incubation procedure above, incubate at room temperature for 1 hour.

[0120] 4) DAPI staining (room temperature and away from light): dilute DAPI into PBS at a ratio of 1:1000 and incubate for 3 min away from light.

[0121] 5) Sealing: Use mounting medium and nail polish for sealing.

[0122] 6) Use a confocal SP8 confocal microscope to take pictures.

[0123] 8. Co-immunoprecipitation:

[0124] 1) Ordinary cell protein preparation: Same as western blot, at least 30 million cells are required.

[0125] 2) Protein A / G pretreatment: 30 μL of Protein A / G were washed with TBS, centrifuged at 4°C, 1000 g, 30 s, and repeated 3 times.

[0126] 3) Obtaining whole cell lysate: Add an appropriate volume of RIPA lysis buffer containing protease inhibitors according to the number of cells, place on ice at 4°C for 30 minutes, and shake every 5 minutes to mix.

[0127] 4) Protein supernatant after cell lysis:

[0128] a) IP group: add appropriate amount of negative control (Control IgG) and appropriate amount of washed Protein A / G premixed agarose beads, incubate at 4°C with sufficient rotation for 30min-1h to eliminate nonspecific background.

[0129] b) Input group: Take 2.5%-5% whole cell lysate as the input group, add 6X protein loading, cook at 100℃ for 10 min, and put in a -20℃ refrigerator for later use.

[0130] 5) Antibody incubation:

[0131] a) After IgG incubation, centrifuge at 4°C, 1000g for 30s and let stand at room temperature for 10min to allow the IgG antibody and its bound protein to slowly precipitate. After centrifugation, keep both the supernatant and the precipitate, and the precipitate is used as the IgG negative control group.

[0132] b) Supernatant: Transfer the supernatant to a new EP tube, add an appropriate volume of primary antibody according to the antibody instructions, and incubate at 4°C with slow rotation overnight (8-12h) to facilitate full binding of the antibody and target protein.

[0133] c) Precipitation (IgG negative control group): add 1 mL TBS to resuspend, centrifuge at 4°C, 1000 g, for 30 s, repeat this three times; add 20 μL TBS to resuspend, add 6X protein loading to boil the protein for 10 min, and store in a refrigerator at -20°C.

[0134] 6) Sample protein denaturation:

[0135] a) The next day, add 30 μL of Protein A / G premixed agarose beads to the supernatant and continue to incubate at 4°C with slow rotation for 3 h to allow the supernatant to fully bind to the beads.

[0136] b) After 3 hours, centrifuge at 4°C, 1000g for 30s and discard the supernatant. Rinse the precipitate with 1mL of pre-cooled TBS three times, centrifuge at 4°C, 1000g for 30s each time. Resuspend with 20μL of TBS, add 4μL of 6X protein loading to boil the protein, and store in a -20°C refrigerator as the IP group.

[0137] 7) Western blotting analysis: Input group, IgG group, and IP group were detected together.

[0138] 9. Denaturing immunoprecipitation:

[0139] 1) Preparation of common cell proteins: Same as the previous immunoprecipitation procedure

[0140] 2) Protein A / G pretreatment: Same as the previous immunoprecipitation procedure.

[0141] 3) Obtaining whole cell lysate:

[0142] a) Add 200 μL of a mixed solution containing protein lysis buffer RIPA (containing required protease inhibitors PMSF, NEM, aprotitin, leupeptin and pepstin) and 2% SDS to each sample according to the cell amount, and shake the cell sample to mix well.

[0143] b) Place the shaken and mixed sample in a 100°C water bath for 10 min to thermally denature the protein.

[0144] c) After 10 minutes, remove the sample and wait for it to cool to room temperature. Remember not to place it directly on ice.

[0145] d) After the sample returns to room temperature, add 1.8 mL of RIPA lysis buffer containing protease inhibitors (including the required protease inhibitors PMSF, NEM, aprotitin, leupeptin and pepstin), place on ice at 4°C for 30 min, and shake every 5 min to mix.

[0146] 4) The rest of the operation methods are consistent with co-immunoprecipitation.

[0147] Example 2 Identification of SUMO2 / 3 modification specific sites and SIM domains in LAMP2A

[0148] In this example, a series of online SUMO modification site and interaction SIM motif prediction websites (SUMOplot, SUMOsp, JASSA and GPS-SUMO2) were used to comprehensively evaluate and discover that LAMP2A has 7 potential SUMO modification sites and 2 SIM motifs, and the corresponding modification site mutants of LAMP2A were constructed. LAMP2A mutants with FLAG tags were co-expressed with HA-tagged SUMO2 or SUMO3 in human embryonic kidney KEK293T cells, and then denaturing immunoprecipitation was used to determine that the specific high-level binding sites with SUMO2 were the 104th amino acid and the 289th amino acid in LAMP2A, and the specific high-level binding sites with SUMO3 were the 132nd amino acid and the 391st to 393rd amino acid segment in LAMP2A. Subsequently, a double mutant modified by LAMP2A was constructed based on the main sites modified by SUMO2 and SUMO3. The same method as above was used to detect and analyze the changes in the modification level of LAMP2A by overexpression and poly(dA:dT) treatment. The results are as follows Figure 2 As shown: When the K104 and K289 sites of LAMP2A mutated simultaneously, its SUMO2 modification level was significantly weakened, while the SUMO3 modification level did not change significantly. Conversely, when the K132 and SIM1 sites of LMAP2A mutated simultaneously, its SUMO3 modification level was significantly weakened, while the SUMO2 modification did not change significantly.

[0149] In summary, LAMP2A is mainly modified by SUMO2 at the K104 and K289 sites, while SIM1 is conducive to its SUMO3 modification at the K132 site.

[0150] Example 3 Detection of SUMO2 / 3 specific site modification function in LAMP2A

[0151] 1. TRIM32 regulates SUMO modification of LAMP2A to inhibit KSHV viral copy number and expression of key viral proteins

[0152] In order to further verify whether TRIM32 regulates CMA by mediating SUMO modification of LAMP2A and exerts the above function, in this example, LAMP2A and its modified mutants were co-expressed with TRIM32 in the iSLK-Bac16-shLAMP2A stable transfection cell line. After 24 hours of transfection, KSHV was activated by Dox induction, and the expression of RTA and MEF2D was detected by immunoblotting ( Figure 3 A), and the viral load in the cell supernatant was detected by fluorescence quantification ( Figure 3 B), the results show that during the KSHV latent period, TRIM32 promotes the activation of CMA by mediating SUMO modification of LAMP2A at K104, K289 and K132, but the extent to which different types of modifications at different sites play a role is different; during the KSHV lytic replication period, TRIM32 also mediates SUMO modification of LAMP2A to activate CMA. In summary, under KSHV latent infection, TRIM32 mediates SUMO modification of LAMP2A to activate CMA, thereby promoting the degradation of RTA and inhibiting the release of viral particles, maintaining KSHV latent infection.

[0153] 2. Effect of TRIM32 on SUMO modification of LAMP2A and KSHV-related tumors

[0154] In order to further verify whether TRIM32 exerts tumorigenic effects by mediating SUMO modification of LAMP2A, in this example, different SUMO modification site mutants of LAMP2A were co-expressed with TRIM32 in the KMM-shLAMP2A stable cell line, and an empty vector control was set to carry out a mouse subcutaneous tumor model experiment. The results are as follows: Figure 4AC shows: Compared with overexpression of wild-type LAMP2A, when LAMP2A-K289,104R was overexpressed, the volume and weight of mouse tumors were significantly reduced, and angiogenesis was significantly reduced. When TRIM32 was co-expressed at the same time, there were no significant changes in the volume, weight and angiogenesis of mice; when LAMP2A-K132R-SIM1 was overexpressed, the volume and weight of mouse tumors were also significantly reduced, and angiogenesis was also significantly reduced. When TRIM32 was co-expressed at the same time, there were no significant changes in the volume, weight and angiogenesis of mice. This suggests that when KSHV is infected, TRIM32 can mediate SUMO modification of LAMP2A at K104, K132 and K289 sites, respectively, and then regulate LAMP2A to induce CMA activation, thereby promoting RTA degradation and tumor formation, and different SUMO modification sites have certain differences in the degree of promoting the occurrence and development of KSHV-related tumors.

[0155] The experimental steps involved in this embodiment are as follows:

[0156] 1. Quantification of viral copy number:

[0157] 1) Cultivate and process cells according to experimental requirements

[0158] 2) Concentrate the virus particles in the cell supernatant: Use a universal virus concentration kit (C2901M, Beyotime).

[0159] 3) Take out the cells cultured and treated in the incubator and collect the cell supernatant.

[0160] 4) Concentrate the collected cell supernatant using a virus concentration kit. For detailed operation methods, please refer to the universal virus concentration kit (C2901M, Beyotime).

[0161] 5) Extract virus supernatant particles: use Viral DNA / RNA Kit (ER201-01, Transgene Biotech) was used to extract the viral concentrate obtained above.

[0162] 6) Quantification of viral copy number: Quantification is performed based on the rapid detection and quantitative reagents and kits for each subtype of human herpes virus in our laboratory (patent number 20171011884609) and conventional Q-PCR methods. Q-PCR primers are shown in Table 1 below:

[0163] Table 1

[0164]

[0165] 3. BALB / c nude mouse tumor model:

[0166] 1) Prepare mice: Purchase 4-6 week old female BALB / c-nu athymic nude mice, and breed them in an SPF mouse room for 7 days. The mice are weighed and recorded one day before inoculation.

[0167] 2) Prepare cells: Culture cells to 1.5 times the required inoculation volume and collect them in centrifuge tubes on the day of inoculation; mix Matrigel with blood-free and antibiotic-free culture medium at a ratio of 1:1 according to inoculation requirements; dispense the cell mixture into EP tubes and place on ice for inoculation.

[0168] 3) Mouse inoculation: subcutaneous injection.

[0169] 4) Tumor observation: After injection, the growth of the tumor and the body weight of the mice were measured every 2 days.

[0170] 5) Dissection of mice: About 20 days after tumor formation, the mice were dissected, and the size and weight of the tumors in each group were detected and recorded.

[0171] Example 4 Synthesis of short peptides at LAMP2A SUMO modification related sites

[0172] In this embodiment, according to the short peptides based on the lysosomal membrane protein 2A polypeptide SUMO modification related K104, K289K, 132 and SIM1 sites screened out in Example 2, the corresponding short peptide derivatives L2AK104, L2AK289, L2AK132 and L2ASIM1 for treating KSHV-related tumors are designed. The short peptide derivatives are composed of the above short peptides and the leading transmembrane peptide; the amino acid sequences of the short peptides at the K104, K289, K132 and SIM1 sites are SEQ ID No.1: WIANFTKAASTYS, SEQ ID No.2: DFVFAVKNEN RFY, SEQ ID No.3: FPDAEDKGILTVD, SEQ ID No.4: LAGVL ILVLLAY. The leading transmembrane peptide is connected to the N-terminus of the above short peptide, and the amino acid sequence of the transmembrane peptide is as shown in SEQ ID No.5: YGRKKRRQRRR. The short peptides at the K104, K289, K132 and SIM1 sites correspond to the short peptide derivatives L2AK104, L2AK289, L2AK132 and L2ASIM1, respectively. Therefore, the amino acid sequences of the short peptide derivatives L2AK104, L2AK289, L2AK132 and L2ASIM1 are SEQ

[0173] ID No.6: YGRKKRRQRRRWIANFTKAASTYS, SEQ ID No.7: YGRKKRRQRRRDFVFAVKNENRFY, SEQ ID No.8: YGRKKRRQRRRFPDAEDKGI LTVD, SEQ ID No.9: YGRKKRRQRRRLAGVLILVLLAY.

[0174] In order to further verify the importance of the binding site with SUMO molecules in the polypeptide sequence, the corresponding binding site mutation short peptide derivatives L2AK104R, L2AK289R, L2AK132R and L2AmSIM1 were designed in this example, and their amino acid sequences are SEQ ID No.10: YGRKKRRQRRRWIANFTRAASTYS, SEQ ID No.11: YGRKKRRQRRRD FVFAVRNENRFY, SEQ ID No.12: YGRKKRRQRRRFPDAEDRGILTVD, SEQ ID No.13: YGRKKRRQRRRLAGAAAAAALAY. The above polypeptide sequences (short peptide derivatives and binding site mutation short peptide derivatives) were prepared by solid phase synthesis, and the purity of the polypeptides was detected by HPLC. Figure 5 As shown in Figures A-H and Tables 2A-H, the purity of the four polypeptides is greater than 95%.

[0175] Table 2A. L2AK104 HPLC test parameters

[0176]

[0177] Table 2B. L2AK104R HPLC detection parameters

[0178]

[0179] Table 2C. L2AK289 HPLC detection parameters

[0180]

[0181] Table 2D. L2AK289R HPLC detection parameters

[0182]

[0183] Table 2E. L2AK132 HPLC detection parameters

[0184]

[0185] Table 2F. L2AK132R HPLC detection parameters

[0186]

[0187]

[0188] Table 2G. L2ASIM1 HPLC detection parameters

[0189]

[0190] Table 2H. L2AmSIM1 HPLC detection parameters

[0191]

[0192] Example 5 Effects of LAMP2A-related SUMO-modified polypeptides on cell viability and toxicity

[0193] The CCK8 method was used to detect cell activity and observe the effects of peptides L2AK104, L2AK289, L2AK132 and L2ASIM1 on the activity of KSHV-positive cells. The KSHV-positive cell line iSLK-Bac16 was plated in a 96-well plate, with 20,000 cells per well. 5uM, 10uM, 20uM, and 30uM peptides were added at the same time. Untreated groups and Control peptide-treated groups were set up and returned to the cell culture incubator for culture. After treatment for 24 hours, 10uL of CCK-8 solution was added at room temperature and protected from light for 2 hours. The plate was read at 450nm with an ELISA reader, and the cell viability was calculated according to the formula. Figure 6 As shown, the results showed that these peptides had no obvious killing effect on KSHV-positive cell lines, indicating that they had low cytotoxicity.

[0194] The experimental steps involved in this embodiment are as follows:

[0195] 1. Cell proliferation-toxicity assay: CCK-8 kit (Lablead, CK001)

[0196] 1) Prepare 100 μl of cell suspension in a 96-well plate and culture (at 37° C., 5% CO 2 ) for 24 hours according to experimental requirements.

[0197] 2) Add different concentrations of the drug to be tested to stimulate the culture plate.

[0198] 3) Incubate the plate in an incubator for an appropriate period of time.

[0199] 4) Add 10 μl of CCK-8 solution to each well.

[0200] 5) Continue incubating in the cell culture incubator for 1-4 hours. The length of time depends on the experimental conditions such as cell type and cell density.

[0201] 6) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0202] 7) If the substance to be tested is oxidizing or reducing, fresh culture medium can be replaced before adding CCK8 (remove the culture medium, wash the cells twice with the culture medium, and then add new culture medium) to remove the drug effect. Of course, if the drug effect is relatively small, the culture medium can be left unchanged, and the blank absorption after adding the drug to the culture medium can be directly subtracted.

[0203] 8) Calculation of CCK-8 cell survival rate:

[0204] Cell survival rate (%) = [(AS-Ab) / (AC-Ab)] × 100%

[0205] AS: Experimental wells (containing cell culture medium, CCK-8, toxic substances)

[0206] AC: Control well (containing cell culture medium, CCK-8, no toxic substances)

[0207] Ab: Blank well (does not contain cell culture medium, toxic substances, or CCK-8)

[0208] Example 6 LAMP2A SUMO-modified site short peptide inhibits KSHV-related tumor growth

[0209] Given that synthetic short peptides may inhibit the function of LAMP2A by competitively inhibiting the SUMO modification of LAMP2A, it is unclear whether synthetic short peptides can inhibit the function of LAMP2A, thereby inhibiting the growth and proliferation of KSHV-related tumors. To further verify this hypothesis, in this example, KSHV-positive cells (KMM and iSLK-Bac16) were transfected with FLAG-tagged LAMP2A and various peptides, and a PBS treatment group and a Control peptide treatment group were set up to conduct a mouse subcutaneous tumor model experiment. The results are shown in Figure 7As shown in AD: Compared with the PBS and Control control groups, the tumor volume and weight of mice in the peptide L2AK289, L2AK289+L2AK104, L2ASIM1 and L2AK132+L2ASIM1 groups were significantly reduced, and angiogenesis was significantly reduced, while the corresponding L2AK289R, L2AK289R+L2AK104R and L2AmSIM1 groups had no significant changes in tumor volume and weight and angiogenesis, while the tumor volume and weight of mice in the L2AK132R+L2AmSIM1 group were also significantly reduced. This indicates that the peptides L2AK289 and L2ASIM1 competitively inhibit the SUMO modification of LAMP2A through the modification sites of LAMP2A, reduce the expression level of LAMP2A, and then regulate the growth, proliferation and angiogenesis of KSHV-related tumors.

[0210] The specification and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative labor, all of which are within the protection scope of the present invention.

Claims

1. A short peptide derivative based on lysosomal membrane protein 2A, characterized in that: The short peptide derivatives are L2AK104, L2AK289, L2AK132 and L2ASIM1; the amino acid sequences of L2AK104, L2AK289, L2AK132 and L2ASIM1 are shown in SEQ ID No.6-9 respectively.

2. The short peptide derivative according to claim 1, characterized in that: The short peptide derivative consists of a membrane-penetrating peptide and a short peptide, wherein the short peptide is a short peptide based on the lysosomal membrane protein 2A polypeptide SUMO-modified related K104, K289, K132 and SIM1 sites, and the short peptides at the K104, K289, K132 and SIM1 sites correspond to the short peptide derivatives L2AK104, L2AK289, L2AK132 and L2ASIM1, respectively, and the amino acid sequences of the short peptides at the K104, K289, K132 and SIM1 sites are shown in SEQ ID No. 1-4, respectively; The cell-penetrating peptide is connected to the N-terminus of the short peptide, and the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID No.

5.

3. The short peptide derivative according to claim 1, characterized in that: The short peptide derivative is prepared by solid phase synthesis.

4. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises one or two of the short peptide derivatives as claimed in claim 1.

5. Use of the short peptide derivative according to claim 1 in the preparation of an antiviral infection drug, characterized in that: The virus is Kaposi's sarcoma virus KSHV, and the short peptide derivative can inhibit the increase of the viral copy number of KSHV.

6. Use of the short peptide derivative according to claim 1 in preparing a drug for treating tumors, characterized in that: The tumor is a tumor caused by Kaposi's sarcoma virus KSHV, and the short peptide derivative can inhibit the proliferation of tumor cells positive for KSHV virus infection, and inhibit tumor growth and angiogenesis.

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