Targeted molecular chaperonin TRIC / CCT polypeptide and application thereof

Abstract

The invention relates to the field of biotechnology and medicine, and discloses a polypeptide targeting molecular chaperonin TRIC / CCT and application thereof. The sequence of the polypeptide is consistent with or truncated from the C-terminal sequence of a CCT subunit, and the polypeptide can be competitively combined with CRL4-DCAF12 ubiquitin ligase, so that the ubiquitination of the CCT subunit is reduced, and the formation of a TRIC / CCT compound is inhibited; the polypeptide inhibits the formation of a TRIC / CCT compound and blocks the activation of key metastasis promoting signal channels such as YAP, STAT3 and mTOR, thereby inhibiting the metastasis of lung cancer. According to systematic in-vivo and in-vitro experiments, the compound has excellent performance in the aspect of inhibiting lung cancer cell migration and metastasis, has the characteristics of clear target spot, clear action mechanism and the like, and provides a new treatment strategy for developing a new generation of lung cancer metastasis resisting medicines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and medicine, and particularly relates to polypeptides targeting chaperone protein TRiC / CCT and applications thereof. BACKGROUND

[0002] Lung cancer is the most burdensome malignant tumor in the world, and its high mortality rate is mainly due to treatment failure and poor prognosis caused by tumor metastasis. Although targeted therapy (such as EGFR-TKI and ALK inhibitors) and immunotherapy (such as PD-1 / PD-L1 antibodies) have made breakthrough progress in recent years, effective treatment methods for metastatic lung cancer are still very limited, and new targeted therapy strategies need to be developed. Chaperone protein TRiC / CCT (TCP-1 ring complex / chaperone containing TCP-1) is an important protein folding auxiliary complex in eukaryotic cells, and its unique ring structure is composed of 8 subunits. This complex not only participates in the regulation of cytoskeletal proteins (such as Actin and Tubulin) folding, but also participates in the function maintenance of various oncogenic proteins and key molecules of signaling pathways (such as STAT3, mTOR pathway regulatory proteins Raptor and mLST8). A large number of studies have shown that the TRiC / CCT complex presents an abnormal expression pattern in various malignant tumors including lung cancer, and promotes tumor progression and metastasis by regulating key biological processes such as cell cycle progression, migration and invasion ability, and epithelial-mesenchymal transition (EMT). These important findings make the TRiC / CCT complex a new target for anti-tumor drug development.

[0003] Currently, inhibitors targeting TRiC / CCT mainly include two types of small molecule compounds (such as HSF1A) and polypeptide molecules. However, the existing inhibitors have obvious limitations. Polypeptide inhibitors (such as CT20p) competitively block substrate protein recognition and loading by specifically binding to the top domain of CCT2 subunit, induce misfolded protein accumulation and activate the endoplasmic reticulum stress apoptosis pathway. However, it has problems such as poor in vivo stability and potential immunogenicity; small molecule inhibitors (such as HSF1A) mainly interfere with the protein folding function of TRiC / CCT by binding to the CCT1 subunit, but have insufficient targeting and off-target risks. Based on this, developing new TRiC / CCT targeting polypeptides with high efficiency and specificity has important clinical translation value. SUMMARY

[0004] The present application aims to provide a polypeptide targeting chaperone protein TRiC / CCT and its application, which prevents ubiquitination of CCT subunits by competitively binding CRL4-DCAF12 ubiquitin ligase, destroys TRiC / CCT complex assembly or formation, inhibits correct folding of cytoskeletal proteins, and blocks activation of key metastasis-promoting signaling pathways such as YAP, STAT3 and mTOR, thereby inhibiting lung cancer metastasis.

[0005] To achieve the above-mentioned purpose, the present application provides a polypeptide targeting chaperone protein TRiC / CCT, which comprises an amino acid sequence of a CCT5-targeting polypeptide as shown in SEQ ID NO: 1-3, an amino acid sequence of a CCT7-targeting polypeptide as shown in SEQ ID NO: 4-6, and an amino acid sequence of a CCT3-targeting polypeptide as shown in SEQ ID NO: 7.

[0006] Preferably, the CCT5-targeting polypeptide comprises CCT5-2W-1 as shown in SEQ ID NO: 1, CCT5-2W-2 as shown in SEQ ID NO: 2, and CCT5-2W-3 as shown in SEQ ID NO: 3; the CCT5-2W-2 is obtained by deleting 10 amino acids from the C-terminal of CCT5-2W-1, and the CCT5-2W-3 is obtained by deleting 20 amino acids from the C-terminal of CCT5-2W-1; the CCT7-targeting polypeptide comprises CCT7-2W-1 as shown in SEQ ID NO: 4, CCT7-2W-2 as shown in SEQ ID NO: 5, and CCT7-2W-3 as shown in SEQ ID NO: 6; the CCT7-2W-2 is obtained by deleting 10 amino acids from the C-terminal of CCT5-2W-1, and the CCT7-2W-3 is obtained by deleting 20 amino acids from the C-terminal of CCT5-2W-1; and the CCT3-targeting polypeptide comprises CCT3-2W as shown in SEQ ID NO: 7.

[0007] Preferably, the amino acid sequence of CCT5-2W-1 is the same as the C-terminal 50 amino acid sequence of CCT5 subunit; the amino acid sequence of CCT7-2W-1 is the same as the C-terminal 50 amino acid sequence of CCT7 subunit; and the amino acid sequence of CCT3-2W is the same as the C-terminal 19 amino acid sequence of CCT3 subunit.

[0008] Preferably, the CCT5 polypeptide inhibits ubiquitination of the CCT5 subunit by targeting the CCT5 subunit of the TRiC / CCT complex, thereby inhibiting assembly of the TRiC / CCT complex; the CCT7 polypeptide inhibits ubiquitination of the CCT7 subunit by targeting the CCT7 subunit of the TRiC / CCT complex, thereby inhibiting assembly of the TRiC / CCT complex; and the CCT3 polypeptide inhibits ubiquitination of the CCT3 subunit by targeting the CCT3 subunit of the TRiC / CCT complex, thereby inhibiting assembly of the TRiC / CCT complex.

[0009] The present application provides a polynucleotide comprising a nucleotide sequence encoding the above-mentioned CCT5-targeting polypeptide as shown in SEQ ID NO: 8-10, a nucleotide sequence encoding the above-mentioned CCT7-targeting polypeptide as shown in SEQ ID NO: 11-13, and a nucleotide sequence encoding the above-mentioned CCT3-targeting polypeptide as shown in SEQ ID NO: 14.

[0010] The present application provides a recombinant vector comprising the above-mentioned polynucleotide and a Lenti-teton-EGFP-zeocin vector as shown in SEQ ID NO: 8.

[0011] The present application provides a recombinant lentivirus comprising the above-mentioned polynucleotide or the above-mentioned recombinant vector.

[0012] The present application also provides use of the above-mentioned polypeptide targeting the chaperone protein TRiC / CCT, mainly for use in preparation of an anti-lung cancer metastasis drug.

[0013] Preferably, the lung cancer is non-small cell lung cancer.

[0014] The present application also provides a pharmaceutical composition comprising the above-mentioned polypeptide targeting the chaperone protein TRiC / CCT and a pharmaceutically acceptable carrier.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] (1) The C-terminal of the CCT5 subunit, the CCT7 subunit and the CCT3 subunit is an important position for binding of the CRL4-DCAF12 ubiquitin ligase, and the present application designs a CCT5-targeting polypeptide, a CCT7-targeting polypeptide and a CCT3-targeting polypeptide by using the C-terminal amino acid sequence of the CCT5 subunit, the CCT7 subunit and the CCT3 subunit, respectively, wherein the polypeptide sequence is identical to or truncated from the C-terminal sequence of the CCT subunit, the polypeptide can competitively bind to the CRL4-DCAF12 ubiquitin ligase, thereby reducing ubiquitination of the CCT subunit and further inhibiting formation of the TRiC / CCT complex.

[0017] (2) The polypeptide of the present application inhibits the activation of key metastasis-promoting signaling pathways such as YAP, STAT3 and mTOR by inhibiting the formation of TRiC / CCT complex, thereby inhibiting the metastasis of lung cancer. The polypeptide realizes subunit selective binding through domain matching, and has a high specificity molecular basis. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Western blot for detecting interaction of CCT5 subunit and its truncated type;

[0019] Figure 2 Western blot for detecting interaction of CCT7 subunit and its truncated type;

[0020] Figure 3 Docking diagram of DCAF12 and CCT3 polypeptide molecular interaction;

[0021] Figure 4 Western blot for detecting inhibitory effect of CCT5 polypeptide and its truncated type on ubiquitination of CCT5 subunit;

[0022] Figure 5 Western blot for detecting inhibitory effect of CCT7 polypeptide and its truncated type on ubiquitination of CCT7 subunit;

[0023] Figure 6 Western blot for detecting inhibitory effect of CCT3 polypeptide on ubiquitination of CCT3 subunit;

[0024] Figure 7 Western blot for detecting content change of CCT complex Short exposure;

[0025] Figure 8 Western blot for detecting content change of CCT complex Long exposure;

[0026] Figure 9 Western blot for detecting content of each subunit of CCT;

[0027] Figure 10 Western blot for detecting expression amount of YAP signaling pathway related proteins of H1299 stable cell line; Figure 11 Western blot for detecting expression amount of STAT3 and mTOR signaling pathway related proteins of H1299 stable cell line;

[0028] Figure 12 Transwell result diagram of H1299 cells stably expressing CCT5-2W-2 and CCT7-2W-1;

[0029] Figure 13 Statistical chart of Transwell results of H1299 cells stably expressing CCT5-2W-2 and CCT7-2W-1;

[0030] Figure 14 Chart of Transwell results of H1299 cells stably expressing CCT3-2W;

[0031] Figure 15 Statistical chart of Transwell results of H1299 cells stably expressing CCT3-2W;

[0032] Figure 16 Chart of Transwell results of A549 cells stably expressing CCT5-2W-2 and CCT7-2W-1;

[0033] Figure 17 Statistical chart of Transwell results of A549 cells stably expressing CCT5-2W-2 and CCT7-2W-1;

[0034] Figure 18 Chart of Transwell results of A549 cells stably expressing CCT3-2W;

[0035] Figure 19 Statistical chart of Transwell results of A549 cells stably expressing CCT3-2W;

[0036] Figure 20 Fluorescence chart of lung of lung metastasis mice of H1299 cells stably expressing CCT5-2W-2. DETAILED DESCRIPTION

[0037] The following examples are only used to more clearly illustrate the technical solutions of the present application, and cannot be used to limit the protection scope of the present application.

[0038] The compounds used in the examples are all commercially available, and are not subjected to any further purification treatment.

[0039] Human embryonic kidney cells HEK293T and human non-small cell lung cancer cells (H1299 and A549) are purchased from the Cell Bank of Culture Collection, Chinese Academy of Sciences; high-metastatic mouse lung adenocarcinoma cells 889DTC are donated by Winslow Laboratory of Stanford University, USA.

[0040] All cells are cultured in a constant temperature incubator at 37℃ and 5% CO2, and the specific culture medium is as follows:

[0041] ①HEK293T cells: cultured in DMEM medium containing 10% fetal bovine serum (FBS);

[0042] ②H1299, A549 and 889DTC cells: cultured in RPMI-1640 medium containing 10% FBS.

[0043] Example 1

[0044] CCT subunit and CRL4-DCAF12 ubiquitin ligase binding site determination, including the following steps:

[0045] NCBI gene database (http: / / www.ncbi.nlm.nih.gov / ) to obtain the complete coding sequence of CCT subunit, using DNAMAN 9.0 software design specific primers. The primer design parameters are: length 25-40 bp, Tm value 60-65℃, GC content 40%-60%. After specific verification of all primer sequences by NCBI BLAST tool, Nanjing Kings River Biotechnology Co., Ltd. was entrusted to synthesize, purified by HPLC (purity≥98%), and stored at-20℃ for standby. The genomic DNA of HEK293T cells was used as a template to amplify the genes of CCT5 and CCT7 subunits and their C-terminal truncated genes, which were cloned into pcDNA3HA3 vector to obtain pcDNA3HA3-CCT5, pcDNA3HA3-CCT5Δ50, pcDNA3HA3-CCT5Δ100, pcDNA3HA3-CCT5Δ150, pcDNA3HA3-CCT7, pcDNA3HA3-CCT7Δ50, pcDNA3HA3-CCT7Δ100, pcDNA3HA3-CCT7Δ150. The constructed C-terminal truncated mutants were co-transfected with FLAG-tagged DCAF12 into HEK293T cells, and the cells were collected 48 hours after transfection and total protein was extracted using NP40 lysis buffer. Then, anti-HA magnetic beads were used to specifically enrich CCT proteins and their interaction complexes, and finally Western blot was used to detect the binding of FLAG-tagged DCAF12 to each truncated body. The experiment was set up twice independently, and the wild-type full-length protein was used as a positive control, and the empty vector transfection group was used as a negative control. Western blot detection of CCT5 subunit and its truncated type interaction as shown in Figure 1 Western blot detection of CCT7 subunit and its truncated type interaction as shown in Figure 2

[0046] ​Experimental data showed that the C-terminal 50 amino acids of CCT5 and CCT7 were critical for their binding to DCAF12. For CCT5, the wild-type (1-541) showed strong interaction signal, while the mutant lacking 50 amino acids (1-491) had significantly reduced binding ability, and further truncation to 100 or 150 amino acids (1-441 / 1-391) resulted in almost complete loss of binding. The wild-type of CCT7 (1-543) could effectively bind to DCAF12, but after deletion of 50 amino acids (1-493), the binding ability decreased, and further deletion (Δ100 / Δ150) completely destroyed the interaction. These results indicated that the C-terminal protein domain of the CCT subunit played a key role in its interaction with DCAF12.

[0047] Example 2

[0048] The design of targeting CCT5, CCT7 and CCT3 polypeptides, respectively, included the following steps:

[0049] Based on the sequence characteristics of the C-terminal domains of CCT5 and CCT7 subunits (50 amino acids each), a rational design strategy of "full sequence coverage + gradient truncation" was used to systematically construct three classes of inhibitory polypeptide variants. The three-dimensional structure of CCT3 protein was downloaded from the Uniprot database (https: / / www.uniprot.org / ) in PDB format. The original PDB file was edited using Notepad2 to remove water molecules (HOH), cofactors, ligand molecules, and other non-protein components, leaving only the atomic coordinate information of the target protein to ensure the accuracy of subsequent docking calculations. The processed protein structure was imported into AutoDockTools 1.5.6 (ADT) for protein structure optimization, including supplementing missing hydrogen atoms, calculating Gasteiger-Marsili charge distribution, and correcting atomic types to meet the force field requirements of molecular docking. The optimized protein structure was saved in PDBQT format as the receptor file for molecular docking. AutoDockVina was used for molecular docking, with the following specific steps: ① Docking box setting: Based on the active site or key residues of the target protein, define the docking search space (grid box) to ensure sufficient coverage of the ligand binding region. ② Docking parameter optimization: Adjust the exhaustiveness (search depth) parameter of Vina (usually set to 8-20) to improve the reliability of the docking results. ③ Docking run: Perform docking calculations to generate multiple possible binding conformations and sort them by binding free energy (ΔG, kcal / mol). After docking is complete, use PyMOL 2.6.1 for structure analysis and visualization: ① Binding mode analysis: Observe the key interactions between the ligand and the protein (such as hydrogen bonds, hydrophobic interactions, π-π stacking, etc.). ② Structure superimposition: Superimpose the docking conformation with the experimental structure (such as the crystal complex) to evaluate the accuracy of the docking. ③ Image rendering: Use PyMOL's high-quality rendering function to generate publication-level molecular interaction diagrams, highlighting key binding sites and interacting residues.

[0050] The interaction mode between DCAF12 and CCT3 was systematically analyzed by molecular docking technology, and the following key molecular characteristics were found in the interface between the two: (1) hydrogen bond network: ARG-228, ASN-235, and LYS-234 of CCT3 form four stable hydrogen bonds with GLN-139, THR-123, ILE-122, and HIS-85 of DCAF12, respectively; (2) hydrophobic interaction: HIS-302 of CCT3 forms a significant hydrophobic interaction with PRO-133 of DCAF12. Based on the above structural characteristics, we designed a 19-amino acid polypeptide CCT3-2W targeting the 222-240 key functional region of CCT3 (including the three key residues ARG-228, ASN-235, and LYS-234).

[0051] The sequences of CCT5, CCT7, and CCT3 peptides are shown in Table 1, and the docking diagram of the CCT3 peptide is shown in Table 1. Figure 3 As shown.

[0052] Table 1: Amino acid sequence listing of peptides targeting CCT5, CCT7, and CCT3

[0053]

[0054] Example 3

[0055] The validation of the inhibitory effects of targeting CCT5, CCT7, and CCT3 peptides on CCT subunit ubiquitination included the following steps:

[0056] Based on the amino acid sequences targeting CCT5, CCT7, and CCT3 peptides respectively, the corresponding nucleotide sequences were designed and synthesized (as shown in Table 2 below) and constructed into the lentiviral expression vector Lenti-teton-EGFP-zeocin (sequence shown in SEQ ID NO: 15). EGFP-P2A-CCT5, EGFP-P2A-CCT7, or EGFP-P2A-CCT3 peptides and their truncated expression vectors were co-transfected into HEK293T cells with ubiquitin ligase vectors and 6×His-Ub vectors at a ratio of 1:1.5:1. Simultaneously, an empty vector was used to replace the ubiquitin ligase expression vector as a negative control. The culture medium was changed regularly to ensure good cell growth. Cells were collected 48 hours after transfection, and total protein was extracted using NP40 lysis buffer. The ubiquitin-protein complex was specifically enriched using anti-Ni magnetic beads. The ubiquitination of the CCT subunits was detected by Western blot. The experiment was performed in two independent replicates, with a wild-type full-length protein positive control and an empty vector negative control. The inhibitory effects of EGFP-P2A-CCT5 peptide and its truncated form on CCT5 subunit ubiquitination are as follows: Figure 4 As shown, the inhibitory effects of the EGFP-P2A-CCT7 peptide and its truncated form on CCT7 subunit ubiquitination are as follows: Figure 5 As shown, the inhibitory effect of EGFP-P2A-CCT3 peptide on CCT3 subunit ubiquitination is as follows: Figure 6 As shown.

[0057] Table 2. Nucleotide sequence list of peptides targeting CCT5, CCT7, and CCT3

[0058]

[0059]

[0060] like Figure 4As shown, CCT5-2W-1 and CCT5-2W-2 could significantly reduce the ubiquitination modification of CCT5 mediated by DCAF12, while CCT5-2W-3 only showed partial inhibition, which was consistent with the Co-IP results. This phenomenon further supported that CCT5-2W-1 / 2 could effectively prevent the binding of DCAF12 to CCT5, thereby inhibiting its ubiquitination regulation function. As shown in Figure 6B, the ubiquitination level of CCT5 in the presence of DCAF12 was significantly reduced in the presence of CCT5-2W-1 / 2, but not in the presence of CCT5-2W-3. This result further confirmed that CCT5-2W-1 / 2 could effectively prevent the binding of DCAF12 to CCT5, thereby inhibiting its ubiquitination regulation function. As shown in Figure 6C, the ubiquitination level of CCT5 in the presence of DCAF12 was significantly reduced in the presence of CCT5-2W-1 / 2, but not in the presence of CCT5-2W-3. This result further confirmed that CCT5-2W-1 / 2 could effectively prevent the binding of DCAF12 to CCT5, thereby inhibiting its ubiquitination regulation function. Figure 5 As shown, CCT7-2W-1, CCT7-2W-2 and CCT7-2W-3 could all significantly inhibit the ubiquitination modification of CCT7 by DCAF12, further confirming that these polypeptides could prevent DCAF12 from binding to the C-terminal domain of CCT7, thereby interfering with the subsequent ubiquitination process. As shown in Figure 8B, the ubiquitination level of CCT7 in the presence of DCAF12 was significantly reduced in the presence of CCT7-2W-1 / 2 / 3, but not in the presence of CCT7-2W-4. This result further confirmed that CCT7-2W-1 / 2 / 3 could effectively prevent the binding of DCAF12 to the C-terminal domain of CCT7, thereby inhibiting its ubiquitination regulation function. As shown in Figure 8C, the ubiquitination level of CCT7 in the presence of DCAF12 was significantly reduced in the presence of CCT7-2W-1 / 2 / 3, but not in the presence of CCT7-2W-4. This result further confirmed that CCT7-2W-1 / 2 / 3 could effectively prevent the binding of DCAF12 to the C-terminal domain of CCT7, thereby inhibiting its ubiquitination regulation function. Figure 6 As shown, CCT3-2W showed significant inhibition of CCT3 ubiquitination, verifying that the 19-peptide could effectively target the DCAF12-CCT3 interaction interface. Molecular docking analysis showed that its inhibitory effect may be due to specific blocking of the key hydrogen bond network of DCAF12-CCT3 (ARG-228 / ASN-235 / LYS-234).

[0061] Example 4

[0062] Construction and screening of stably transfected cell lines targeting CCT5, CCT7 and CCT3 polypeptides, including the following steps:

[0063] HEK293T cells were used as lentivirus packaging cell line, which were seeded in 6-well plates. The seeding density was precisely controlled to ensure that the cell confluence reached 70-80% at the time of transfection. Cell culture was performed in DMEM complete medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator overnight. Plasmid transfection operation: ① Plasmid preparation: prepare the transfection mixture according to the optimized ratio, the total plasmid amount is 2.5 μg, the specific ratio is as follows: lentivirus vector plasmid 1 μg, packaging plasmid psPAX2 1 μg, envelope plasmid pMD2.G 0.5 μg; ② Transfection process: use Lipofectamine 2000 transfection reagent (Invitrogen) for plasmid DNA transfection, strictly follow the instructions. ③ Post-transfection treatment: the transfection time is 6 hours, and the fresh complete medium must be replaced. Virus collection process: ① First collection: the first medium replacement was performed at 24 hours after transfection, and 2 mL of fresh medium was added. ② Subsequent collection: starting from 36 hours after transfection, virus supernatant was collected every 12 hours (i.e. 36, 48, 60, 72 hours) and fresh medium was replaced. The supernatant was filtered through a 0.45 μm filter to remove cell debris, which can be directly used for cell infection (or aliquoted and stored at -80°C for long-term storage). A549 and H1299 cells in logarithmic growth phase were seeded in 12-well culture plates at an appropriate density, and when the cell confluence reached 40-60%, virus infection was performed. The original culture medium was discarded, and the mixed solution (total volume 1 mL / well) was added according to the ratio of virus liquid: fresh medium = 1:2, and incubated in a 37°C, 5% CO2 incubator for 16-18 hours. The next day, the virus mixed solution was removed, and 2 mL of fresh complete medium was added, and cultured for 24 hours. Subculture to 6-well plates. Add complete medium containing 400 μg / mL bleomycin for screening, wild-type cells not infected with virus as negative control group, and blank wells containing only medium as blank control group; replace the fresh medium containing bleomycin every 48 hours, and closely observe the cell state. When the negative control group cells died completely, the screening was terminated. The surviving experimental group cells were transferred to normal culture medium for expansion, which were stable cell lines.

[0064] Example 5

[0065] The inhibitory effect of polypeptides targeting CCT5, CCT7 and CCT3, respectively, on the assembly of TRiC / CCT complex includes the following steps:

[0066] H1299 stable expression cell lines were treated with non-denaturing lysis method after 1 μg / mL Dox induction for 72 hours to maintain the natural state of protein complex, and then the TRiC / CCT complex with a molecular weight of about 1 MDa was separated and detected by non-denaturing gel (BN-PAGE) electrophoresis technology, and the content change of CCT complex was detected by Western blot, and after short exposure, as shown in Figure 7 , and after long exposure, as shown in Figure 8 ; the change of the content of each subunit of CCT was detected by denaturing gel, as shown in Figure 9 .

[0067] According to Figure 7 , it can be known that the polypeptides targeting CCT5, CCT7 and CCT3 respectively can significantly reduce the formation of TRiC / CCT complex; according to Figure 8 , it can be known that the polypeptides targeting CCT5, CCT7 and CCT3 respectively can increase the amount of CCT3 subunit, indicating that more CCT3 subunits do not participate in the formation of TRiC / CCT complex, resulting in the decrease of TRiC / CCT complex content; according to Figure 9 , it can be known that the total amount of each subunit of CCT remains unchanged after CCT5 / CCT7 / CCT3 polypeptide treatment, indicating that CCT5 / CCT7 / CCT3 polypeptide does not inhibit the synthesis of each subunit of CCT, but inhibits the ubiquitination of the subunit, and reduces the formation of TRiC / CCT complex.

[0068] Example 6

[0069] The regulatory effect of polypeptides targeting CCT5, CCT7 and CCT3 respectively on tumor metastasis related signal pathways includes the following steps:

[0070] H1299 stable cell lines stably expressing EGFP, CCT3-2W, CCT5-2W-2 and CCT7-2W-1 were taken respectively, and the cells stably expressing EGFP were used as the control group. After 72 hours of Dox induction, the cells were collected, and the activity changes of multiple key signal pathways were detected by Western blot technology, and the results are shown in Figure 10 and Figure 11 .

[0071] The results show that the three polypeptide treatments can significantly up-regulate p-YAP(S127) levels, while reducing the expression of p-STAT3(Y705); in the mTOR signaling pathway, the phosphorylation levels of p-4E-BP1(T37 / 46) and p-S6K1(T389) are significantly decreased. These data show that the CCT5 / CCT7 / CCT3 polypeptides can interfere with the function of the TRiC / CCT complex, and synergistically inhibit the activation of YAP, STAT3 and mTOR, and other multiple metastasis signaling pathways.

[0072] Example 7

[0073] Functional verification of polypeptides targeting CCT5, CCT7 and CCT3 respectively to inhibit the migration activity of lung cancer cells in vitro, including the following steps:

[0074] 600 μL of serum-free medium was added to the lower chamber of a 24-well plate, and 100 μL of serum-free medium was added to the upper chamber of a Transwell (Corning, pore size 8 μm). Place in a 37°C, 5% CO2 incubator for 2h to eliminate the influence of membrane surface tension on cell migration. After 72h of Dox induction, the A549 and H1299 cells stably expressing EGFP, CCT3-2W, CCT5-2W-2 and CCT7-2W-1 were digested with 0.05% trypsin, centrifuged at 800g for 5 minutes to collect the cells; washed twice with PBS to completely remove serum residues; resuspended the cells with serum-free medium, and adjusted the cell concentration to 4×10 5 4 cells / mL. Take 100 μL of cell suspension and inoculate it into the upper chamber of the Transwell; add 600 μL of medium containing 15% fetal bovine serum to the lower chamber of the 24-well plate as a chemoattractant; be careful not to generate bubbles between the upper and lower chambers during operation to ensure consistency of the migration direction. H1299 cells were cultured at 37°C for 7h, and A549 cells were cultured at 37°C for 24h; remove the Transwell chamber, remove the culture medium, and gently wipe the Matrigel and cells in the chamber with a PBS-wetted cotton swab or cotton. Add 600 μL of 4% paraformaldehyde fixing solution to the clean wells of the 24-well plate, and place the chamber in the fixing solution for 30 minutes. Discard the fixing solution and wash the chamber inside and outside once with PBS. Add 600 uL of crystal violet staining solution to the clean wells of the 24-well plate, and place the chamber in the staining solution for 10 minutes. Remove the chamber, wash the chamber inside and outside 3 times with PBS. After appropriate air-drying, observe under a microscope for qualitative research; take 3-5 fields and take photos, then use ImageJ to count and take the average value for quantitative research. The Transwell results of H1299 cells stably expressing CCT5-2W-2 and CCT7-2W-1 are shown in Figure 12 Figure 13 ​As shown; Transwell results of H1299 cells that stably express CCT3-2W are as follows. Figure 14 As shown, the relative migration statistics of cells are as follows: Figure 15 As shown; Transwell results of A549 cells that stably express CCT5-2W-2 and CCT7-2W-1 are as follows. Figure 16 As shown, the relative migration statistics of cells are as follows: Figure 17 As shown; Transwell results of A549 cells that stably express CCT3-2W are as follows. Figure 18 As shown, the relative migration statistics of cells are as follows: Figure 19 As shown.

[0075] Quantitative analysis showed that, compared with the control group, all three CCT-targeting peptides exhibited significant migration inhibition effects, with inhibition efficiencies exceeding 70% (P<0.0001). These data fully demonstrate that CCT-targeting peptides can effectively block the migration ability of lung cancer cells in in vitro experiments.

[0076] Example 8

[0077] In vivo pharmacodynamic evaluation of CCT5-targeted peptides in inhibiting lung cancer metastasis, including the following steps:

[0078] Six- to eight-week-old male BALB / c nude mice (n=6 / group) were selected and housed in a standardized SPF-grade animal facility with strictly controlled environmental parameters such as temperature and humidity. H1299 lung cancer cell lines stably expressing the EGFP-P2A-CCT5-2W-2 fusion protein were expanded and cultured. A control group expressing only EGFP was also established. All cells were labeled with TurboRFP fluorescence for monitoring. Cells were inoculated via tail vein injection with 1×10⁻⁶ cells / mL. 6 Fluorescently labeled tumor cells were inoculated into mice, and the mice were closely monitored for 30 minutes after inoculation to rule out the risk of acute embolism. To precisely regulate peptide expression, an optimized dual induction protocol was used: intraperitoneal injection of 200 μL of 0.53 mMDox (single dose) combined with continuous drinking of a solution containing 1 μg / ml Dox (containing 2.5% sucrose), with freshly prepared medicated drinking water provided every 3 days. After 6 weeks of experimental observation, mice were euthanized using CO2 asphyxiation combined with cervical dislocation, and lung tissue was removed for fluorescence microscopy observation. The results are as follows: Figure 20 As shown.

[0079] like Figure 20 As shown, analysis using a fluorescence microscopy system revealed that, compared to the control group, the number of metastatic lesions in the lung tissue of mice expressing CCT5-2W-2 was significantly reduced, thus confirming the anti-metastatic activity of CCT5-2W-2 in vivo and providing important reference data for subsequent preclinical studies.

[0080] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. A polypeptide targeting the molecular chaperone protein TRiC / CCT, characterized in that, The polypeptide comprises an amino acid sequence of a targeting CCT5 polypeptide as shown in SEQ ID NO: 1-3, an amino acid sequence of a targeting CCT7 polypeptide as shown in SEQ ID NO: 4-6, and an amino acid sequence of a targeting CCT3 polypeptide as shown in SEQ ID NO:

7.

2. A polynucleotide, comprising, The polynucleotide comprises a nucleotide sequence encoding the targeting CCT5 polypeptide as claimed in claim 1 as shown in SEQ ID NO: 8-10, a nucleotide sequence encoding the targeting CCT7 polypeptide as claimed in claim 1 as shown in SEQ ID NO: 11-13, and a nucleotide sequence encoding the targeting CCT3 polypeptide as claimed in claim 1 as shown in SEQ ID NO:

14.

3. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide as claimed in claim 2 and a Lenti-teton-EGFP-zeocin vector as shown in SEQ ID NO:

15.

4. A recombinant lentivirus, characterized in that, The recombinant lentivirus comprises the polynucleotide as claimed in claim 2 or the recombinant vector as claimed in claim 3.

5. Use of a polypeptide targeting the chaperone protein TRiC / CCT, characterized in that, The application is the use of the polypeptide in the preparation of a drug for resisting lung cancer metastasis.

6. Use of a polypeptide targeting the chaperone protein TRiC / CCT according to claim 5, characterized in that, The lung cancer is non-small cell lung cancer.

7. A pharmaceutical composition, characterized by, The application also provides a pharmaceutical composition comprising the polypeptide targeting the chaperonin TRiC / CCT as claimed in claim 1 and a pharmaceutically acceptable carrier.

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