Non-phosphorylated and non-ubiquitinated CREPT proteins and their applications

Abstract

This invention relates to a non-phosphorylated and non-ubiquitinated CREPT protein and its applications. Specifically, this invention relates to a protein obtained by modifying CREPT or its homologous proteins, wherein the modification prevents degradation of the modified protein when it is located in eukaryotic cells in the G1 terminal phase or G1 / S transition phase, resulting in the inability of the dual MCM hexamer to separate, thereby arresting the cell cycle, generating a genomic stress response, and ultimately leading to cell death. This invention also relates to methods for screening non-phosphorylated and non-ubiquitinated CREPT modifiers, methods for identifying whether a substance is a phosphorylation inhibitor of CREPT, methods for using CREPT to identify eukaryotic cells in the G1 terminal phase or G1 / S transition phase, and methods for treating cancer by inducing apoptosis in cancer cells based on non-phosphorylated and non-ubiquitinated CREPT.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to a CREPT protein capable of maintaining a non-phosphorylated and non-ubiquitinated state and its applications. Background Technology

[0002] DNA replicates during the S phase of the cell cycle. Erroneous DNA replication accumulates in stem cells and is closely associated with cell death and aging. Therefore, DNA replication, which determines cell fate, needs to be strictly controlled. The DNA replication mechanism in eukaryotic cells is highly complex. The origin of DNA replication is located in the G1 phase of the cell cycle, and pre-RC (pre-replication complex) proteins are loaded at all potential origin sites in the genome. First, the ORC complex (origin recognition complex, ORC1-6) with ATPase activity is recruited to the origin of replication. Further, the hexamer complex of CDC6, CDT1 (CDC10-dependent transcript 1), and MCM2-7 (mini-chromosome maintenance) is loaded onto the OCR complex to form the MCM helicase complex. This step is called origin licensing. Activation of replication initiation involves the formation of the pre-IC (pre-initiation complex) complex and the activation of the MCM helicase complex. Pre-IC assembly is triggered by DDK (DBF4-dependent kinase) and CDK (Cyclin-dependent kinase) during the G1 / S transition. DDK and CDK phosphorylate several proteins involved in DNA replication, such as MCM10, CDC45, RECQL4 (ATP-dependent DNA helicase Q4), treslin, GINS, and TOPBP1 (DNA topoisomerase 2-binding protein 1). Furthermore, DDK and CDK can also phosphorylate several bases in the MCM2-7 complex, leading to helicase activation and unwinding of the DNA. During helicase activation, the MCM2-7 dihexamer loaded on the DNA splits into two separate hexamers, which continue to unwind at the two replication forks originating from the site of replication.

[0003] The cell cycle is precisely regulated by the ubiquitin-proteasome system (UPS)-mediated degradation of cyclin-associated proteins and CDK inhibitors. UPS achieves efficient protein degradation by adding ubiquitin to the lysine (K) residues of substrate proteins for proteasome recognition. There are three main types of enzyme-controlled UPS: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). Ligase E3 is the most diverse enzyme, providing target specificity. During the G1 / S phase, the SCF complex of the RING-finger subfamily of E3 ligases, composed of SKP1, CUL1, and F-box proteins (FBP), recognizes the substrate to be degraded. FBP primarily determines the target specificity of the SCF complex. The SCF complex preferentially binds to phosphorylated degradation determinants, which are phosphorylated substrate motifs that produce surfaces for E3 ligase recognition. SKP2 (S-phase kinase-associated protein 2), a member of the FBP family, is one of the E3 ligases regulating the G1 / S transition. The SKP2-CKS1-p27 complex mediates the proteasome degradation of the CDK inhibitor p27 and releases Cyclin E / CDK2 to promote G1 / S switching.

[0004] Cell cycle regulation is closely related to tumorigenesis and development. In previous studies, our laboratory discovered and cloned a gene, CREPT (Cell-cycle-Related and Expression-elevated Protein in Tumor, Chinese Patent No. 200510135513.4, the corresponding protein sequence of which is associated with cell cycle regulation and tumor formation), in human cells. Furthermore, we found that this gene has highly conserved homologs in various eukaryotes, such as yeast, mouse, dog, cat, chicken, toad, zebrafish, fruit fly, nematode, and Arabidopsis thaliana cells. It is known that the CREPT protein promotes cell proliferation by regulating the expression of Cyclin D1 and B1.

[0005] However, the specific mechanism by which CREPT regulates the cell cycle remains unclear. Summary of the Invention

[0006] Through extensive research, the inventors discovered that the CREPT protein in human cells promotes the cell cycle into the S1 phase by undergoing degradation during the G1 / S transition, thereby playing a role in cell cycle regulation.

[0007] Specifically, the inventors discovered that in multiple cells within the same culture, CREPT expression was very low in some cells and remained high in others. This low expression was found to be due to CREPT degradation during the G1 / S transition, which is crucial for cell transition from G1 to S phase. CREPT degradation is mediated by ubiquitination, which depends on phosphorylation at two sites on CREPT, S134 and S166. The inventors further discovered that if sites S134 and S166 of the CREPT protein are kept non-phosphorylated during the G1 / S transition and the protein itself remains non-ubiquitinated, CREPT cannot be degraded. This non-degradable CREPT cannot separate from the MCM complex, leading to cell death.

[0008] Furthermore, the inventors discovered that homologous proteins in non-human eukaryotic cells have the same or similar cell cycle regulatory effects and mechanisms as human CREPT, and that this cell cycle regulatory effect still exists when human CREPT protein is expressed in non-human eukaryotic cells.

[0009] Based on the above findings, the inventors completed this invention.

[0010] In a first aspect, the present invention provides a protein obtained by phosphorylation inactivation modification of sites 134 and 166 of SEQ ID No:4, wherein the phosphorylation inactivation modification causes sites 134 and 166 to remain in a non-phosphorylated state and the protein to remain in a non-ubiquitinated state when the protein is located in a eukaryotic cell in the G1 terminal phase or the G1 / S transition phase, thereby preventing the protein from being degraded in the eukaryotic cell.

[0011] In the first aspect, the eukaryotic cells may be cells of humans, yeast, mice, dogs, cats, chickens, toads, zebrafish, fruit flies, nematodes, or Arabidopsis thaliana, preferably human cells, and more preferably human cancer cells.

[0012] In the first aspect, the phosphorylation inactivation modification at positions 134 and 166 may be an amino acid mutation and / or a chemical modification.

[0013] In the first aspect, the phosphorylation inactivation modification at sites 134 and 166 may be a mutation of serine (S) to alanine (A).

[0014] In a first aspect, the present invention also provides a protein having (i) more than 90% sequence identity and (ii) the same phosphorylation inactivation modifications at sites 134 and 166 as each of the proteins described above. In a second aspect, the present invention provides a protein having a tag sequence or guide sequence attached to the N-terminus and / or C-terminus of the protein of the first aspect.

[0015] In a third aspect, the present invention provides nucleic acids encoding the proteins described in the first and second aspects.

[0016] In a fourth aspect, the present invention provides a vector comprising the nucleic acid described in the third aspect.

[0017] In a fifth aspect, the present invention provides cells comprising the carrier described in the fourth aspect.

[0018] In a sixth aspect, the present invention provides the use of the proteins, nucleic acids or carriers described in the first to fifth aspects in the preparation of reagents for inhibiting eukaryotic cell proliferation, inhibiting eukaryotic cell DNA replication, regulating the cell cycle of eukaryotic cells or killing eukaryotic cells.

[0019] In a seventh aspect, the present invention provides the use of the proteins, nucleic acids or carriers described in the first to fifth aspects in the preparation of anticancer drugs.

[0020] In an eighth aspect, the present invention provides a method for treating cancer, the method comprising administering to a subject an effective amount of the protein, nucleic acid, or vector described in the first to fifth aspects; or, the method comprising editing the CRPET gene in the genome of a subject's cancer cells using CRISPR / Cas9-based gene editing technology to cause the cancer cells to express the protein of SEQ ID NO:2. The subject may be a mammal, preferably a human. The expression of wild-type CREPT in the subject's cancer cells may be reduced or eliminated before, during, or after administering an effective amount of the protein, nucleic acid, or vector to the subject.

[0021] In an eighth aspect, the present invention provides a method for screening non-phosphorylated and non-ubiquitinated modifiers for CREPT protein, wherein the modifier maintains the S134 and S166 sites of the CREPT protein in a continuously non-phosphorylated state, thereby ensuring that the CREPT protein remains non-ubiquitinated in the cell and does not degrade; the amino acid sequence of the CREPT protein is SEQ ID No:4, and the method comprises:

[0022] i) A candidate modifier simulating a non-phosphorylated state was added to eukaryotic cells expressing CREPT protein that were synchronized to the G1 phase, and then the eukaryotic cells were released and cultured. The phosphorylation levels of the S134 and S166 sites of the CREPT protein were examined while the eukaryotic cells were still alive.

[0023] or

[0024] ii) In vitro, candidate modifiers simulating the non-phosphorylated state were incubated with the CREPT protein, and the phosphorylation levels at S134 and S166 sites of the CREPT protein were examined under Cyclin E / CDK2 kinase catalysis.

[0025] If the phosphorylation levels at sites S134 and S166 of the CREPT protein treated in step i) or ii) decrease relative to the untreated control, for example, by more than 10%, more than 20%, more than 30%, or more than 40%, then the candidate modifier is screened as a non-phosphorylated, non-ubiquitinated modifier for the CREPT protein.

[0026] This invention also provides a method for identifying whether a substance is a phosphorylation inhibitor of CREPT protein, wherein the inhibitor maintains the S134 and S166 sites of CREPT protein in a continuously non-phosphorylated state, thereby keeping the CREPT protein in a non-ubiquitinated state in the cell and preventing its degradation; the amino acid sequence of the CREPT protein is SEQ ID No:4, and the method includes:

[0027] i) The substance to be identified is added to eukaryotic cells expressing CREPT protein that have been synchronized to the G1 phase, then the eukaryotic cells are released and cultured, and the phosphorylation levels of the CREPT protein at sites S134 and S166 are examined while the eukaryotic cells are still viable; or

[0028] ii) The substance to be identified was incubated with CREPT protein in vitro, and the phosphorylation levels of the S134 and S166 sites of the CREPT protein were examined under the catalytic conditions of Cyclin E / CDK2 kinase.

[0029] If the phosphorylation levels at sites S134 and S166 of the CREPT protein treated in step i) or ii) decrease relative to the untreated control, for example, by more than 10%, 20%, 30%, or 40%, then the substance is identified as a phosphorylation inhibitor of the CREPT protein; otherwise, the substance is not a phosphorylation inhibitor of the CREPT protein.

[0030] In the above method, mass spectrometry or immunoprecipitation can be used to examine the phosphorylation levels of the S134 and S166 sites of the CREPT protein; preferably, the immunoprecipitation method may include performing immunoprecipitation using antiphosphorylation antibodies that recognize phosphorylation at the S134 and S166 sites of the CREPT protein.

[0031] The present invention also provides a method for identifying whether a substance is a phosphorylation inhibitor of CREPT protein, wherein the inhibitor maintains the S134 and S166 sites of CREPT protein in a persistent non-phosphorylated state, thereby keeping the CREPT protein in a non-ubiquitinated state in the cell and preventing degradation; the amino acid sequence of the CREPT protein is SEQ ID No:4; the method includes: i) adding the substance to be identified to eukaryotic cells expressing CREPT protein and culturing the eukaryotic cells; and ii) examining the ubiquitination level of CREPT protein in the eukaryotic cells using immunoprecipitation; if the ubiquitination level of CREPT protein in the treated cells decreases, for example, by more than 10%, more than 20%, more than 30%, or more than 40%, compared with the ubiquitination level of CREPT protein in control cells not treated with the substance, then the substance is identified as a phosphorylation inhibitor of CREPT protein; otherwise, the substance is not a phosphorylation inhibitor of CREPT protein. Prior to step i), the method may further include: designing the substance to be identified using the prediction tools SwissTargetPrediction and SEA targeting CREPT. Furthermore, step ii) may include quantifying the ubiquitination level of the CREPT protein using an anti-CREPT antibody that recognizes the CREPT protein and a ubiquitin antibody that recognizes ubiquitin.

[0032] In a ninth aspect, the present invention provides a homologous protein of human CREPT derived from a non-human eukaryote, having a phosphorylation-inactivating modification at homologous sites corresponding to sites 134 and 166 of SEQ ID No:4, wherein the phosphorylation-inactivating modification causes the homologous protein to remain non-phosphorylated at homologous sites 134 and 166 of SEQ ID No:4 and to remain non-ubiquitinated when the homologous protein is located in G1-telophase or G1 / S-transition cells of the eukaryote, thereby preventing the homologous protein from being degraded in the cells, thereby leading to cell cycle arrest and apoptosis.

[0033] In the ninth aspect, the eukaryote may be yeast, mouse, dog, cat, chicken, toad, zebrafish, fruit fly, nematode, or Arabidopsis thaliana.

[0034] In the ninth aspect, the phosphorylation inactivation modification at the homology sites corresponding to sites 134 and 166 of SEQ ID No:4 can be an amino acid mutation and / or a chemical modification.

[0035] In the ninth aspect, the phosphorylation inactivation modification at the homology sites corresponding to sites 134 and 166 of SEQ ID No:4 can be a mutation of serine to alanine.

[0036] In the ninth aspect, the amino acid sequence of the homologous protein may be SEQ ID No:6.

[0037] In a tenth aspect, the present invention provides a protein selected from:

[0038] i) A protein having a tag sequence or guide sequence attached to its N-terminus and / or C-terminus as described in the ninth aspect; or

[0039] ii) A protein that has more than 90% sequence identity with the protein described in the ninth aspect and has the same modification at the homology site.

[0040] In the eleventh aspect, the present invention provides nucleic acids encoding the proteins described in the ninth and tenth aspects.

[0041] In a twelfth aspect, the present invention provides a vector comprising the nucleic acid described in the eleventh aspect.

[0042] In a thirteenth aspect, the present invention provides cells comprising the carrier described in the twelfth aspect.

[0043] In a fourteenth aspect, the present invention provides a method for identifying eukaryotic cells in the late G1 phase or the G1 / S transition phase, the method comprising:

[0044] 1) Prepare eukaryotic cells capable of endogenously expressing human CREPT protein with a detectable label or its homologous protein in non-human eukaryotes, and culture the eukaryotic cells under conditions that allow cell cycle progression;

[0045] 2) Observe or measure the expression level of the human CREPT protein or its homologous protein in the eukaryotic cells using the detectable marker;

[0046] 3) Identify the cells with the lowest expression levels of the human CREPT protein or its homologous proteins as cells in the G1 phase or G1 / S transition phase;

[0047] The sequence of the human CREPT protein is SEQ ID No:4.

[0048] In the fourteenth aspect, the detectable marker may be an isotope marker, a fluorescent marker, or a quantum dot marker, or a marker that can be further combined with an isotope marker, a fluorescent marker, or a quantum dot marker, preferably GFP.

[0049] In the fourteenth aspect, the eukaryotic cells may be cells of humans, yeast, mice, dogs, cats, chickens, toads, zebrafish, fruit flies, nematodes, or Arabidopsis thaliana.

[0050] In a fifteenth aspect, the present invention provides a method for inhibiting the degradation of CREPT protein in eukaryotic cells, the method comprising:

[0051] 1) To introduce an inhibitor selected from SKP2 inhibitors, CUL1 inhibitors, neddylation inhibitors, and CDK2 inhibitors into eukaryotic cells expressing CREPT; and / or

[0052] 2) Phosphorylation inactivation modification is performed on sites 134 and 166 of the CREPT protein, so that when the modified protein is located in eukaryotic cells in the G1 terminal phase or G1 / S transition phase, sites 134 and 166 remain in a non-phosphorylated state and the modified protein remains in a non-ubiquitinated state, thereby preventing the modified protein from being degraded.

[0053] In this method, the eukaryotic cells can be cells of humans, yeast, mice, dogs, cats, chickens, toads, zebrafish, fruit flies, nematodes, or Arabidopsis thaliana, preferably human cells, and more preferably human cancer cells.

[0054] In this method, the phosphorylation inactivation modification at sites 134 and 166 can be an amino acid mutation and / or chemical modification. Preferably, the phosphorylation inactivation modification at sites 134 and 166 is to mutate serine (S) to alanine (A).

[0055] In this method, the SKP2 inhibitor can be a double-stranded siRNA targeting SKP2 with the sequence AAUCUAAGCCUGGAAGGCCUGdTdT; the CUL1 inhibitor can be a double-stranded siRNA targeting CUL1 with the sequence UAGACAUUGGGUUCGCCGUdTdT; and the neddylation inhibitor can be MLN4924.

[0056] In a sixteenth aspect, the present invention also provides a protein selected from: 1) a protein obtained by mutating serine at position 166 of SEQ ID No:4 to alanine; 2) a protein obtained by mutating serine at position 136 of SEQ ID No:8 to alanine; 3) a protein obtained by mutating serine at position 174 of SEQ ID No:8 to alanine; and 4) a protein having more than 90% sequence identity with any one of proteins 1) to 3) and having the same alanine mutation. The present invention also provides a nucleic acid encoding said protein and a vector comprising said nucleic acid. Attached Figure Description

[0057] Specific embodiments of the present invention will now be described with reference to the accompanying drawings, but neither the drawings nor the following detailed description should be construed as limiting the scope of the invention. In the drawings:

[0058] Figures 1A-1E The expression level of CREPT was shown to oscillate during the cell cycle; Figure 1A A representative fluorescence image of CREPT in tumor cells. White circles indicate cells without CREPT expression. Scale bar, 10 μm. Figure 1B Time-lapse microscopic images of live cells with GFP-CREPT knocked in. Scale bar, 10 μm. Figure 1C CREPT expression during the cell cycle; DLD1 cells were synchronized to the G1 / S phase and released using 2 mM diathymidine arrest (DTB). Cell lysates were collected at the indicated time points and analyzed by Western blotting. Cyclin A / B1 / E and SKP2 were detected to confirm cell cycle progression. CREPT protein levels reached their lowest point during the G1 / S transition. Figure 1D Representative fluorescence image of CREPT in DLD1 Fucci cells synchronized with DTB. Scale bar, 10 μm. Figure 1E Fluorescence image ( Figure 1D Quantitative results of the expression levels of CREPT, EdU, and CDT1 in ). Statistical significance (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001); P>0.05, not significant [ns], generated by t-test.

[0059] Figures 2A-2K This shows that the degradation of CREPT during the G1 / S transition depends on ubiquitin modification; Figure 2A CREPT expression in cells treated with actinomycin (CHX). CREPT degradation began 10 hours after CHX treatment. Figure 2B CREPT expression levels remained stable after MG132 treatment. DLD1 cells were treated with or without 25 μg / ml MG132 for 4 hours prior to the second thymidine release. Figure 2C ) Figure 2B Quantitative immunoblotting results of CREPT in China. Figure 2D CREPT can be ubiquitinated. In vivo ubiquitination assays were performed in 293T cells transfected with the specified plasmid. Figure 2E K11-polyubiquitination-mediated CREPT degradation. In vivo ubiquitination assays were performed in 293T cells transfected with the specified construct. Figure 2F K11 ubiquitination accelerates CREPT degradation. HeLa cells were transfected with the HA-Ub-K11 plasmid and treated with CHX. Figure 2G ) Figure 2F The quantitative results of CREPT immunoblotting in the sample. Figure 2H K48 ubiquitination had no effect on CREPT degradation. HeLa cells were treated with CHX and transfected with the HA-Ub-K48 plasmid. Figure 2I ) Figure 2HThe quantitative results of CREPT immunoblotting in the sample. Figure 2J K63 ubiquitination had no effect on CREPT degradation. HeLa cells were treated with CHX and transfected with the HA-Ub-K63 plasmid. Figure 2K ) Figure 2J The quantitative results of CREPT immunoblotting were used. Statistical significance was calculated using t-tests (*P<0.05; **P<0.01; ****P<0.0001).

[0060] Figures 3A-3M Display CRL1 SKP2 CREPT is directly ubiquitinated during the G1 / S transition; Figure 3A SKP2 exhibits the highest binding affinity to CREPT. CREPT pull-down proteins in synchronized cells were analyzed by mass spectrometry. These proteins were ranked according to their unique peptide composition and log10 coverage. The top 10 proteins with known E3 ligase activity are shown. The area of ​​the dots represents the relative coverage value. Figure 3B The interaction between exogenous CREPT and exogenous SKP2 was investigated. Cell extracts from HEK293T cells expressing HA-CREPT and Flag-SKP2 were immunoprecipitated with anti-HA beads, and Western blot analysis was performed using the antibodies indicated. Figure 3C The interaction between endogenous CREPT and endogenous SKP2 was investigated. Cell extracts from DLD1 cells were immunoprecipitated with anti-CREPT beads, and Western blot analysis was performed using the indicated antibodies. Figure 3D Purified prokaryotic expression of SKP2 and CREPT was used for in vitro co-IP assay, followed by immunoblotting analysis. Figure 3E SKP2 mediates CREPT ubiquitination. In vivo ubiquitination assays were performed in 293T cells transfected with the indicated plasmid, with or without MG132 treatment. Figure 3F )and( Figure 3G The effect of SKP2 overexpression on CREPT degradation. Figure 3H )and( Figure 3I The effect of SKP2 knockdown on CREPT degradation. Figure 3J )and( Figure 3K The levels of SKP2 and CREPT were measured after synchronizing cells to the G1 / S phase and releasing them. Figure 3L )and( Figure 3M Synchronize cells to M phase and release SKP2 and CREPT levels.

[0061] Figures 4A-4G This shows that the S134A and S166A mutations in CREPT cannot interact with Ub; Figure 4ACREPT is ubiquitinated in the CID domain. In vivo ubiquitination was measured in 293T cells transfected with the specified plasmid and treated with MG132. Figure 4B CREPT modification was identified by mass spectrometry. Modified mass spectrometry analysis was performed in 293T cells synchronized to G1 / S phase, and phosphorylation levels at serine (S) sites on the CREPT protein were determined by #PSMs. Figure 4C A schematic diagram of the amino acids in CREPT and RTT103 within the CREPT 135-170 region. Figure 4D CREPT S134A / S166A mutants cannot be ubiquitinated. Immunoassay of ubiquitinated CREPT and CREPT mutants overexpressed in 293T cells. Figure 4E SKP2 recognizes the phosphorylated form of CREPT. 293T cells were transfected with the specified plasmid and harvested for co-IP assay, followed by Western blot analysis. Figure 4F The interaction between endogenous CREPT and endogenous CDK2 / Cyclin E was investigated. Cell extracts from DLD1 cells were immunoprecipitated with anti-CREPT beads, and Western blot analysis was performed using the indicated antibodies. Figure 4G CREPT is phosphorylated by CDK2 / Cyclin E1 at S134 / S166. Flag-CDK2 and Flag-Cyclin E1 were purified by Co-IP using an anti-Flag antibody from HEK293T cells transfected with expression vectors of Flag-CDK2 and Flag-Cyclin E1. GST, GST-CREPT, and GST-CREPT (S134A / S166A) were purified from the prokaryotic expression system using GST beads. Phosphorylation of GST or GST-labeled CREPT and its mutants was detected by a universal antiphosphorylation antibody (top). CREPT(SA): CREPT(S134A / S166A).

[0062] Figures 5A-5G The results showed that the CREPT (S134A / S166A) mutation led to apoptosis; Figure 5A The CREPT (S134A / S166A) mutant caused cell death. HeLa wild-type (Mock) and CREPT knockout (KO) cells were transfected with the plasmids shown for 48 hours, followed by Annexin V and PI staining for FACS analysis. Figure 5B ) Figure 5A Statistical analysis of flow cytometry data. Figure 5C Cell death is triggered by apoptosis. HeLa wild-type (Mock) and CREPT knockout (KO) cells were transfected with the plasmids shown for 48 hours, followed by Western blotting. Figure 5DCell growth inhibition was caused by CREPT (S134A / S166A) overexpression. Cell viability was determined by CCK-8 assay 48 hours after transfection of HeLa wild-type (Mock) and CREPT knockout (KO) cells with the plasmids shown. Figure 5E CREPT (S134A / S166A) inhibits tumor growth. 1×10 6 Three B16 cells overexpressing the plasmid were injected into C57BL / 6 mice (n=3). Mice were sacrificed on day 10 and tumor size was measured. Figure 5F The CREPT (S134A / S166A) mutant is lethal to *Saccharomyces cerevisiae*. The growth sensitivity to temperature was compared between WT, RTT103KO (Rtt103Δ), and human CREPT WT or mutant plasmids in By4741 RTT103-KO cells. Figure 5G The Rtt103(S136A / S174A) mutant is lethal to *Saccharomyces cerevisiae*. The growth sensitivity to temperature was compared between WT, Rtt103Δ, and yeast RTT103 WT or mutant plasmids in By4741RTT103-KO cells. CREPT(SA) represents the CREPT(S134A / S166A) mutant, and CREPT(SE) represents the CREPT(S134E / S166E) mutant.

[0063] Figures 6A-6G Undegraded mutant CREPT showed that it prevented cells from entering S phase; Figure 6A CREPT did not bind to the MCM complex during the G1 / S transition. ChIP-MS analysis was performed in DLD1 cells, and proteins were sequenced according to #PSMs. Cell cycle synchronization was performed using DTB. Figure 6B The interaction between exogenous CREPT and exogenous MCM5 was investigated. Cell extracts from HEK293T cells expressing Myc-CREPT and Flag-MCM5 were immunoprecipitated with anti-Myc beads and analyzed by Western blot with specified antibodies. Figure 6C Interaction between endogenous CREPT and endogenous MCM5. Cell extracts from DLD1 cells were cross-linked with 1% paraformaldehyde for 10 minutes. Immunoprecipitation was then performed with anti-CREPT beads, followed by Western blot analysis with the specified antibody. Figure 6D The interaction between CREPT and MCM5 during the cell cycle. DLD1 cells were synchronized to the G2 / M phase. Cell lysates were collected at the indicated time points for endogenous co-IP assays. Figure 6E The CREPT(S134A / S166A) mutant pulled down more MCM protein. 293T cells were transfected with the specified plasmid and harvested for co-IP. Figure 6F)-( Figure 6G Undegraded CREPT mutants prevent cells from entering S phase. Representative fluorescence images of MCM5 and EdU in HeLa cells, cells overexpressed with CREPT(S134A / S166A) mutants for 16 hours and then stained using a pre-extraction method. Scale bar, 10 μm. CREPT(WT) represents the wild-type CREPT plasmid, CREPT(SA) represents the CREPT(S134A / S166A) mutant, and CREPT(SE) represents the CREPT(S134E / S166E) mutant.

[0064] Figures 7A-7D The presence of undegraded CREPT caused the replication fork to abort; Figure 7A Representative fluorescence image of CREPT in HeLa cells. Cells were transfected with the specified plasmid for 12 hours. Immunofluorescence assay results for RPA2 quantification (right figure). Scale bar, 10 μm. Figure 7B Representative fluorescence image of CREPT in HeLa cells. Quantitative results of p-RPA2 by immunofluorescence assay (right panel). Scale bar, 10 μm. Figure 7C DNA replication rates in WT and S134A / S166A mutants treated with HU. First, asynchronous HeLa CREPT KO cells were treated with 4 mM HU for 4 h. Then, CIdU was added to the culture for 0.5 h, and cells were harvested for DNA fiber analysis to measure the length and distribution of CIdU fibers. Scale bar, 10 μm. Figure 7D The percentage of terminated replication forks in WT and S134A / S166A mutants during HU blockade and release. DNA fibrillation assays were performed in HeLa CREPT KO cells to measure terminated replication forks. Statistical significance (*P<0.05; **P<0.01; ****P<0.0001) was calculated by t-test. CREPT(WT) represents the CREPT wild-type plasmid, and CREPT(SA) represents the CREPT(S134A / S166A) mutant.

[0065] Figure 8 A schematic diagram illustrating the mechanism by which CREPT regulates the cell cycle is shown.

[0066] Figures 9A-9G The expression level of CREPT was shown to oscillate during the cell cycle, where, ( Figure 9A A schematic diagram of the GFP-CREPT fusion protein knocked into the HeLa cell line. GFP is fused after the ATG sequence in the EXON1 of CREPT. Figure 9B Western blotting confirmed that GFP-CREPT expression was knocked into cells. Figure 9CAt the release time points shown, synchronous DLD1 cells were analyzed by FACS using PI staining. Figure 9D CREPT expression during the cell cycle. HeLa cells are synchronized to the G1 / S phase and released via DTB. Figure 9E Real-time quantitative PCR analysis of CREPT mRNA expression was performed at the indicated release time points after DTB treatment. Figure 9F Representative fluorescence image of CREPT in DLD1 Fucci cells synchronized with DTB. Scale bar: 10 μm. Figure 9G Fluorescence image ( Figure 9F The quantitative results of CREPT and CDT1 expression levels in ) were obtained. Statistically significant (P<0.0001); P>0.05, not significant [ns], were calculated using t-tests.

[0067] Figures 10A-10D This shows that CREPT degrades via the ubiquitin pathway during the G1 / S transition; Figure 10A ) Figure 2A Quantitative immunoblotting results of CREPT in China. Figure 10B FACS analysis of DLD1 cells synchronized with or without MG132 treatment. Figure 10C CREPT degradation is not mediated by autophagy. DLD1 cells were treated with the lysosomal inhibitor chloroquine or leucine 6 hours before the second thymidine release. Figure 10D Six hours before the second thymidine release, HeLa cells were treated with the lysosomal inhibitor chloroquine or leucopeptide.

[0068] Figures 11A-11I Display CRL1 SKP2 CREPT is directly ubiquitinated during the G1 / S transition. Figure 11A GST-CERPPT expression was verified. Gels containing purified GST-CERPPT protein were stained with Coomassie blue. Figure 11B The expression of His-SKP1 and His-SKP2 was verified. Gels containing purified His-SKP1 and His-SKP2 proteins were stained with Coomassie blue. Figure 11C The expression of His-SKP2 was verified. The purified SKP2 protein was subjected to immunoassay at different purification steps. Figure 11D At the release time points shown, synchronous DLD1 cells were analyzed by FACS using PI staining. Figure 11EActivation of cullin ligase is crucial for CREPT accumulation. Immunoassay was performed on DLD1 cells treated with the 1 μM neddylation inhibitor MLN4924 for 0–8 hours. siCtrl cells were control cells, and siSKP2 cells were cells with SKP2-lowered using siRNA targeting SKP2. Figure 11F CUL1 interacts with CREPT. 293T cells were transfected with the plasmid shown and harvested for co-immunoprecipitation (co-IP) assays, followed by Western blot analysis. Figure 11G CUL1 mediates CREPT ubiquitination. In vivo ubiquitination assays were performed in 293T cells transfected with the plasmid shown, with or without MG132 treatment. Figure 11H Overexpression of CUL1 accelerates CREPT degradation. HeLa cells were treated with CHX at the indicated time points, with or without MG132 treatment. Figure 11I Knockdown of CUL1 prolongs the half-life of CREPT. HeLa cells were treated with CHX to knock down CUL1 or N / C via siRNA.

[0069] Figures 12A-12E This shows that the S134A and S166A mutations in CREPT cannot interact with Ub; Figure 12A CREPT ubiquitination is independent of a single lysine (K) mutation in the CID domain. In vivo ubiquitination assays were performed in 293T cells transfected with the plasmid shown. Figure 12B CREPT ubiquitination is independent of a single K mutation in the linker region. In vivo ubiquitination assays were performed in 293T cells transfected with the plasmid shown. Figure 12C CREPT ubiquitination is K-dependent. In vivo ubiquitination assays were performed in 293T cells transfected with the indicated plasmid and harvested for co-IP assays. Figure 12D CREPT ubiquitination is independent of threonine (T) or cysteine ​​(C). In vivo ubiquitination assays were performed in 293T cells transfected with the plasmid shown. Figure 12E CREPT is phosphorylated in eukaryotic cells. Purified GST-CREPT in prokaryotic (E. coli) and eukaryotic (mammalian) cells was examined by Western blotting using a universal antiphosphorylation antibody. p-CREPT(S / T / Y) represents the panphosphorylation antibody.

[0070] Figures 13A-13F The results showed that the CREPT (S134A / S166A) mutation led to apoptosis; Figure 13ACell growth inhibition and cell death caused by undegraded CREPT mutant protein. HeLa wild-type (Mock) and CREPT knockout (KO) cells were transfected with the plasmid shown for 48 hours. Figure 13B Overexpression of CREPT (S134A / S166A) led to cell growth inhibition. Cell viability was determined by CCK-8 assay 48 hours after transfection of 293T and NCM460 cells with the indicated plasmids. Figure 13C )and( Figure 13D The CREPT (S134A / S166A) mutation resulted in reduced clonogenicity. HeLa wild-type and CREPT knockout cells were transfected with the indicated plasmids for 6 hours, and then 1000 cells were counted for clonogenic assay. Figure 13E CREPT (S134A / S166A) inhibits tumor growth. 1×10 6 Three B16 cells overexpressing the plasmid were injected into C57BL / 6 mice (n=3). Mice were sacrificed on day 10 and tumor size was measured. Figure 13F Compare the temperature-dependent growth sensitivity of By4741 RTT103 WT cells with WT, RTT103KO (Rtt103Δ) and yeast RTT103 WT or mutant plasmids. CREPT(SA): CREPT (S134A / S166A) mutation; CREPT(SE): CREPT (S134E / S166E) mutation.

[0071] Figures 14A-14G Undegraded CREPT showed that it prevented cells from entering S phase; Figure 14A CREPT interacts with MCM2. 293T cells were transfected with the plasmid shown and harvested for co-IP. Figure 14B CREPT interacts with MCM7. 293T cells were transfected with the plasmid shown. Figure 14C In the cross-linked state, CREPT binds to endogenous MCM5. Endogenous co-IP was measured in DLD1 cells after cell fixation with 1% paraformaldehyde. Figure 14D CREPT does not bind directly to chromatin. Representative fluorescence images of MCM5 and EdU in HeLa cells, stained using direct fixation and pre-extraction methods. Scale bar, 10 μm. Figure 14E FACS analysis of CREPT KO HeLa cells transfected with the indicated plasmid for 12 hours was performed. Figure 14F ) Figure 6G Quantitative results of MCM5 by immunofluorescence assay. Figure 14G ) Figure 6G The diameter of the cell nucleus. WT: CREPT(WT); SA: CREPT(S134A / S166A).

[0072] Figures 15A-15D The presence of undegraded CREPT caused the replication fork to abort; Figure 15A Representative fluorescence images of TUNEL signaling in HeLa cells. TUNEL staining of HeLa wild-type (Mock) and CREPT knockout (KO) cells overexpressing the indicated plasmid. CREPT(SA): CREPT (S134A / S166A) mutation; CREPT(SE): CREPT (S134E / S166E) mutation. Scale bar, 10 μm. Figure 15B Representative fluorescence images of γH2AX in HeLa cells. HeLa CREPT knockout cells 12 hours after overexpression of the indicated plasmid, CREPT(SA): CREPT(S134A / S166A) mutation, CREPT(SE): CREPT(S134E / S166E) mutation. Scale bar, 10 μm. Figure 15C The quantitative value of γH2AX intden in cells stained with immunofluorescence. Figure 15D The number of γH2AX focal points in cells stained with immunofluorescence. Statistical significance (P < 0.0001) was calculated using a t-test.

[0073] Figure 16 This is the screening result for small molecule inhibitors of CREPT phosphorylation and ubiquitination. The numbers #1 to #5 at the top indicate cells treated with candidate compounds #1 to #5, and the numbers in the middle (0.1 to 1.1) represent the relative ubiquitination levels of each sample. SA: CREPT (S134A / S166A).

[0074] Figure 17 The effects of candidate compounds #1 to #5 on the proliferation of DLD1 cells (A) and MGC803 cells (B) are shown. Detailed Implementation

[0075] definition

[0076] The term “human CREPT protein” or “CREPT protein” as used in this article refers to human wild-type CREPT protein unless otherwise specified.

[0077] As used in this article, the terms "CREPT protein variant" and "modified CREPT protein" refer to protein variants obtained by amino acid mutations and / or chemical modifications based on wild-type CREPT protein.

[0078] The term "homogeneous protein" as used in this article refers to proteins whose amino acid sequences are homologous and which perform the same or similar functions in different organisms.

[0079] The term “phosphorylation inactivation modification” as used in this article refers to modified amino acid residues in a protein that can maintain or mimic a non-phosphorylated state in eukaryotic cells, and residues modified by phosphorylation inactivation cannot be phosphorylated by kinases in eukaryotic cells.

[0080] The following will describe in detail the specific embodiments of the present invention.

[0081] Through extensive research, the inventors discovered the mechanism of action of the CREPT protein in regulating the cell cycle, and thus completed this invention.

[0082] First, the inventors discovered that the expression level of the CREPT protein oscillates during the cell cycle. Figure 1A This is due to the degradation of CREPT protein during the G1 / S transition phase. This degradation leads to its separation from the MCM complex, thereby driving the cell into S phase and initiating DNA replication. Therefore, CREPT protein expression is at its lowest level during the G1 / S transition phase, and gradually recovers as the cell enters S phase. Figure 1B and Figure 1D ).

[0083] Therefore, in one aspect of the present invention, a method is provided for identifying eukaryotic cells in the late G1 phase or the G1 / S transition phase, the method comprising:

[0084] 1) Prepare eukaryotic cells capable of endogenously expressing human CREPT protein with a detectable label or its homologous protein in non-human eukaryotes, and culture the eukaryotic cells under conditions that allow cell cycle progression;

[0085] 2) Observe or measure the expression level of the human CREPT protein or its homologous protein in the eukaryotic cells using the detectable marker;

[0086] 3) Identify the cells with the lowest expression levels of the human CREPT protein or its homologous proteins as cells in the G1 phase or G1 / S transition phase;

[0087] The sequence of the human CREPT protein is SEQ ID No:4.

[0088] In one embodiment, the detectable marker is an isotope marker, a fluorescent marker, or a quantum dot marker, or a marker that can be further combined with an isotope marker, a fluorescent marker, or a quantum dot marker, preferably GFP.

[0089] In one embodiment, the eukaryotic cells are cells of humans, yeast, mice, dogs, cats, chickens, toads, zebrafish, fruit flies, nematodes, or Arabidopsis thaliana, preferably human cells or yeast cells.

[0090] The inventors further discovered that this degradation of the CREPT protein during the G1 / S transition phase of the cell depends on ubiquitination-mediated proteasome degradation, which is mediated by the E3 ligase CRL1. SKP2 Catalysis, and the recognition and catalytic activity of SKP2 depend on the phosphorylation of CREPT protein at sites S134 and S166. The inventors simulated the phosphorylation and non-phosphorylation states of CREPT at sites S134 and S166 by mutating these two sites. The results showed that CREPT protein remaining non-phosphorylated at these two sites remained non-ubiquitinated. The non-phosphorylated and non-ubiquitinated S134A / S166A double mutant protein did not degrade at G1 telophase or G1 / S transition. This non-degradable CREPT protein variant did not separate from the MCM complex, leading to cell arrest at G1 telophase or G1 / S transition when MCM complex expression levels are high. This prevented the cell cycle from entering S phase and halted the DNA replication fork, ultimately leading to cell death. Figures 5A-5G Individual S134A or S166A mutant proteins can affect ubiquitination to some extent. Figure 4D On the other hand, the S134E / S166E double mutant protein, mimicking the phosphorylation state, had no effect on cell ubiquitination or survival. Figure 4D , Figures 5A-5B This indicates that, like the wild-type CREPT protein, it degrades normally during the G1 / S transition. This demonstrates that the CREPT protein variant that maintains non-phosphorylation at positions 134 and 166 and remains non-ubiquitinated cannot degrade at the end of G1 or during the G1 / S transition, thus leading to cell death.

[0091] It is known in the art that ligases responsible for protein ubiquitination recognize substrates based on phosphorylation at specific sites on the substrate (typically serine, threonine, or tyrosine). Phosphorylation essentially involves a change in the protein's charge. Therefore, by mimicking the phosphorylation state at the corresponding sites (i.e., carrying a negative charge), it is often possible to induce a phosphorylated state in the substrate protein. In this case, the phosphorylated protein can be recognized by the ubiquitination ligase, thereby promoting protein degradation. The mimicry of phosphorylation / non-phosphorylation is typically achieved through mutation or chemical modification. For example, persistent activating mutations (i.e., mutations mimicking phosphorylation) include mutating residues to aspartic acid (D) or glutamic acid (E), as these two amino acids are the only two negatively charged amino acids; while persistent repressive mutations (i.e., mutations mimicking non-phosphorylation) most commonly mutate serine to alanine (A), because alanine carries a positive charge and can persistently inhibit the activity of that residue site. On the other hand, activating chemical modifiers (i.e., chemical modifiers mimicking phosphorylation) can include phosphate donors such as acetyl phosphate, phosphoramide salts, carbamoyl phosphate, and sodium pyrophosphate, as well as beryllium trifluoride. In addition, some CDK4 / 6 specific small molecule inhibitors, such as palbociclib, ribociclib, or abemaciclib, can also achieve the effect of keeping proteins non-phosphorylated (Maianiet et al., 2021; Simoneschi et al., 2021).

[0092] Based on the above findings of the inventors, those skilled in the art can reasonably infer that as long as residues 134 and 166 of CREPT can remain in a non-phosphorylated state during the G1 phase or the G1 / S transition phase, the protein will not be ubiquitinated, thus preventing degradation and leading to cell death.

[0093] Therefore, the present invention provides a non-phosphorylated and non-ubiquitinated CREPT protein variant, wherein when the CREPT protein is located in eukaryotic cells in the G1 phase or G1 / S transition phase, positions 134 and 166 of the CREPT protein are kept non-phosphorylated and the protein is kept non-ubiquitinated, thereby preventing the protein variant from degrading during this phase.

[0094] Furthermore, it is known that the absence of SKP2 ligase or CDK2 kinase can inhibit cell growth but not cell death (Lin et al., 2010; Zhu, 2010; Berthet et al., 2003; Tadesse et al., 2019), indicating that CREPT plays a crucial role in regulating the cell cycle. The S residues at positions 134 and 166 of CREPT are located in the linker region of CREPT, constituting a phosphorylation degradation determinant recognized by SKP2 ligase, and are highly conserved among homologous proteins. Figure 4CThe above demonstrates that the phosphorylation state of residues 134 and 166 plays a crucial role in regulating the cell cycle.

[0095] The inventors also discovered that the S134A / S166A mutant form of human CREPT protein induces apoptosis in other eukaryotes. The inventors exogenously expressed human CREPT S134A / S166A in *Saccharomyces cerevisiae*, and the results showed that this protein variant impaired yeast survival at different temperatures. Figure 13F To eliminate the influence of endogenous Rtt103 (a homolog of CREPT) in yeast, human CREPT S134A / S166A was also exogenously expressed in Rtt103-deficient yeast strains, which significantly inhibited yeast growth. Figure 5F This demonstrates that the cell cycle regulation function and mechanism of CREPT protein and its homologs are universally applicable in eukaryotes, and also shows that the exogenous introduction of CREPT mutants can control the cell cycle of eukaryotic cells, select tumor cells, and induce apoptosis.

[0096] Therefore, another aspect of the present invention provides a protein obtained by phosphorylation inactivation modification of residues 134 and 166 in the CREPT protein sequence (SEQ ID NO:4), wherein the phosphorylation inactivation modification causes the 134 and 166 sites to remain in a non-phosphorylated state and the protein to remain in a non-ubiquitinated state when the protein is located in a eukaryotic cell in the G1 terminal phase or the G1 / S transition phase, thereby preventing the protein from being degraded in the eukaryotic cell.

[0097] In one embodiment, the eukaryotic cells are cells of humans, yeast, mice, dogs, cats, chickens, toads, zebrafish, fruit flies, nematodes, or Arabidopsis thaliana, preferably human cells, and more preferably human cancer cells.

[0098] In one embodiment, the phosphorylation inactivation modification at sites 134 and 166 is an amino acid mutation and / or chemical modification.

[0099] In one embodiment, the phosphorylation inactivation modification at positions 134 and 166 involves mutating both serine (S) to alanine (A), at which point the amino acid sequence of the protein is SEQ ID No:2, i.e., the S134A / S166A double mutant form of CREPT.

[0100] In one embodiment, the N-terminus and / or C-terminus of the protein may be linked with a tag sequence or a guide sequence. In one embodiment, the link is covalent. In one embodiment, the protein with the tag sequence or guide sequence is a fusion protein. In one embodiment, the protein with the tag sequence or guide sequence is a conjugated protein. In one embodiment, the tag sequence may be, for example, a purification tag, a fluorescent tag, a solubilization tag, an affinity tag, or an antigenic epitope tag. In one embodiment, the guide sequence may be a polypeptide sequence that guides the protein across the cell membrane into the cell, including, for example, cell-penetrating peptides not based on endocytosis, and peptide or protein sequences that are readily endocytosed into the cell.

[0101] The present invention also provides a protein having the same phosphorylation inactivation modification at sites 134 and 166 as the above-described protein and having sequence identity of 90% or more, 95% or more, preferably 98% or more, or 99% or more.

[0102] In another aspect, the present invention provides a nucleic acid encoding the above-mentioned protein, a vector containing the nucleic acid, and a cell containing the vector.

[0103] Methods for introducing a target protein (e.g., CREPT S134A / S166A of the present invention) into target cells (e.g., cancer cells) may include introducing a vector expressing the target protein into the target cells via transfection, infection, or other means, or may employ chemically modified mRNA (modRNA) to achieve expression of the target protein in the target cells. Furthermore, the target protein may be directly introduced into cells using, for example, the guide sequence described above. However, the present invention is not limited thereto. For example, precise gene editing technologies (e.g., prime editors) may be used to directly mutate target sites in the tumor genome (Anzalone, et al., 2019), for example, by mutating the corresponding bases of the CREPT genome to induce cell expression of CREPT S134A / S166A.

[0104] In another aspect, the present invention provides the use of the above-mentioned proteins, nucleic acids or carriers in the preparation of reagents that inhibit eukaryotic cell proliferation, inhibit eukaryotic cell DNA replication, regulate eukaryotic cell cycle or kill eukaryotic cells.

[0105] In one embodiment, the eukaryotic cells are cells of humans, mice, dogs, cats, chickens, toads, zebrafish, fruit flies, nematodes, yeast, or Arabidopsis thaliana.

[0106] Since CREPT is highly expressed in most cancers (Li et al., 2021; Lu et al., 2012), in another respect, the present invention provides the use of the above-mentioned protein, nucleic acid or carrier in the preparation of anticancer drugs.

[0107] In another aspect, the present invention provides a method for treating cancer, the method comprising administering an effective amount of the aforementioned protein, nucleic acid, or vector to a human subject; or, the method comprising editing the CRPET gene in the genome of a subject's cancer cells using CRISPR / Cas9-based gene editing technology to cause the cancer cells to express the protein of SEQ ID NO:2. In one embodiment, the cancer is liver cancer, kidney cancer, gastric cancer, or colorectal cancer. In one embodiment, the method comprises introducing the aforementioned nucleic acid into tumor cells. In one embodiment, before, during, or after administering an effective amount of the protein, nucleic acid, or vector to the subject, the expression of wild-type CREPT in the subject's cancer cells is reduced or eliminated. In another aspect, the present invention provides a pharmaceutical composition comprising: the aforementioned protein, nucleic acid, or vector, and a pharmaceutically acceptable carrier, excipient, or medium. In one embodiment, the pharmaceutical composition is used to treat cancer, such as liver cancer, kidney cancer, gastric cancer, or colorectal cancer.

[0108] Since the phosphorylation state of residues 134 and 166 of the CREPT protein plays a crucial role in regulating the cell cycle, another aspect of the present invention provides a method for screening non-phosphorylated, non-ubiquitinated modifiers of the CREPT protein or a method for identifying whether a substance is a phosphorylation inhibitor of the CREPT protein. The modifier or inhibitor maintains the S134 and S166 sites of the CREPT protein in a persistently non-phosphorylated state, thereby ensuring that the CREPT protein remains non-ubiquitinated in the cell and does not degrade. Specifically, when the CREPT protein is located in eukaryotic cells at the G1 end-phase or G1 / S transition phase, the S134 and S166 sites can maintain a persistently non-phosphorylated state, and the protein can remain non-ubiquitinated, thus preventing degradation. The amino acid sequence of the CREPT protein is SEQ ID No:4.

[0109] In one embodiment, the method may be performed as follows: adding a candidate modifier or substance to be identified in a simulated non-phosphorylated state to eukaryotic cells expressing CREPT protein that have been synchronized to the G1 phase, then releasing and culturing the eukaryotic cells, and examining the phosphorylation levels of the S134 and S166 sites of the CREPT protein while the eukaryotic cells are still viable. In another embodiment, the method may be performed as follows: incubating the candidate modifier or substance to be identified in a simulated non-phosphorylated state with the CREPT protein in vitro, and examining the phosphorylation levels of the S134 and S166 sites of the CREPT protein under Cyclin E / CDK2 kinase catalysis. In the above method, if the phosphorylation levels of the S134 and S166 sites of the CREPT protein treated in step i) or ii) decrease relative to their respective untreated controls, for example, by more than 10%, 20%, 30%, or 40%, preferably by more than 50%, 60%, 70%, 80%, or 90%, then the candidate modifier is screened as a non-phosphorylation, non-ubiquitination modifier of the CREPT protein, or the substance is identified as a phosphorylation inhibitor of the CREPT protein; otherwise, the candidate modifier is not a non-phosphorylation, non-ubiquitination modifier of the CREPT protein, and the substance is not a phosphorylation inhibitor of the CREPT protein. In the above method, mass spectrometry or immunoprecipitation can be used to examine the phosphorylation status of the S134 and S166 sites of the CREPT protein. The immunoprecipitation method may include: performing immunoprecipitation using an antiphosphorylation antibody that recognizes phosphorylation at sites S134 and S166 of the CREPT protein, thereby quantifying the phosphorylation level at site S134 of the CREPT protein. Specifically, the CREPT protein can be precipitated using an anti-CREPT antibody, and the total amount of CREPT protein can be measured as a background level. For the protein precipitated with the anti-CREPT antibody, the phosphorylated protein can be detected and quantified using an antiphosphorylation antibody. In this case, the phosphorylation level can be a relative value of the amount of phosphorylated protein relative to the background amount of CREPT protein, and can be normalized for a control.

[0110] This invention also relates to the following compounds:

[0111]

[0112] The compound is also described in its use in the preparation of phosphorylation inhibitors at S134 and S166 sites of the CREPT protein or ubiquitination inhibitors of the CREPT protein, and in the preparation of medicaments for treating cancer. In one embodiment, the cancer is melanoma, liver cancer, kidney cancer, gastric cancer, or colorectal cancer.

[0113] Since phosphorylation at S134 and S166 sites of the CREPT protein is a necessary condition for its ubiquitination, the level of ubiquitination can be used to identify phosphorylation-ubiquitination inhibitors of the CREPT protein. Therefore, the present invention also provides a method for identifying whether a substance is a phosphorylation inhibitor of CREPT protein, wherein the inhibitor maintains the S134 and S166 sites of CREPT protein in a persistent non-phosphorylated state, thereby keeping the CREPT protein in a non-ubiquitinated state in the cell and preventing degradation; the amino acid sequence of the CREPT protein is SEQ ID No:4, and the method includes: i) adding the substance to be identified to eukaryotic cells expressing CREPT protein and culturing the eukaryotic cells, and ii) examining the ubiquitination level of the CREPT protein in the eukaryotic cells using immunoprecipitation; if the ubiquitination level of the CREPT protein in the treated cells decreases, for example, by more than 10%, more than 20%, more than 30%, or more than 40%, compared with the ubiquitination level of the CREPT protein in control cells not treated with the substance, then the substance is identified as a phosphorylation inhibitor of CREPT protein; otherwise, the substance is not a phosphorylation inhibitor of CREPT protein. Prior to step i), the method may further include: designing the substance to be identified using the prediction tools SwissTargetPrediction and SEA targeting CREPT. Furthermore, step ii) may include quantifying the ubiquitination level of CREPT protein using an anti-CREPT antibody that recognizes CREPT protein and a ubiquitin antibody that recognizes ubiquitin. The ubiquitination level may be a relative value of the amount of ubiquitinated protein to the total amount of CREPT protein and may be normalized against controls.

[0114] Furthermore, S134 and S166 in CREPT correspond to S136 and S174 in yeast Rtt103 (see...). Figure 4C The inventors discovered that overexpression of the Rtt103 S136A / S174A double mutant protein led to a lethal phenotype in Rtt103-deficient yeast. Figure 5G These results indicate that human CREPT protein has the same or similar mechanisms of action in regulating the cell cycle as its homologous proteins in other eukaryotic organisms.

[0115] Therefore, in another aspect, the present invention provides a homologous protein of human CREPT derived from non-human eukaryotes, which has phosphorylation inactivation modifications at homologous sites corresponding to sites 134 and 166 of the human CREPT protein. These phosphorylation inactivation modifications ensure that when the homologous protein is located in G1-terminal or G1 / S-transition cells of the eukaryote, the homologous sites remain non-phosphorylated and the homologous protein remains non-ubiquitinated, thereby preventing the homologous protein from being degraded in the cells, leading to cell cycle arrest and apoptosis.

[0116] In one embodiment, the eukaryote is yeast, mouse, dog, cat, chicken, toad, zebrafish, fruit fly, nematode, or Arabidopsis thaliana.

[0117] In one embodiment, the phosphorylation inactivation modification at the homology sites corresponding to positions 134 and 166 of the human CREPT protein is an amino acid mutation and / or chemical modification. In one embodiment, the phosphorylation inactivation modification at the homology sites corresponding to positions 134 and 166 of the human CREPT protein is a mutation of serine to alanine. In one embodiment, the homologous protein is a double mutant protein of *Saccharomyces cerevisiae* Rtt103S136A / S174A, whose amino acid sequence is SEQ ID No:6.

[0118] In one embodiment, the N-terminus and / or C-terminus of the homologous protein may be linked with a tag sequence or a guide sequence to form, for example, a fusion protein or a conjugated protein.

[0119] In one embodiment, a protein is also provided that has the same phosphorylation inactivation modification at the homology site as the above-described protein and has a sequence identity of 90% or more, 95% or more, preferably 98% or more, or 99% or more.

[0120] In one embodiment, the present invention provides a nucleic acid encoding the above-mentioned homologous protein, a vector containing the nucleic acid, and a cell containing the vector.

[0121] In one embodiment, the present invention provides the use of the above-mentioned homologous proteins, nucleic acids or vectors in the preparation of reagents that inhibit eukaryotic cell proliferation, inhibit eukaryotic cell DNA replication, regulate eukaryotic cell cycle or kill eukaryotic cells.

[0122] In one embodiment, the eukaryotic cells are cells of humans, yeast, mice, dogs, cats, chickens, toads, zebrafish, fruit flies, nematodes, or Arabidopsis thaliana.

[0123] In addition to phosphorylation inactivation at positions 134 and 166 of the CREPT protein, the inventors also discovered that even without modification of CREPT, knocking down or depleting SKP2 or CUL1 using siRNAs targeting them significantly inhibited the degradation of wild-type CREPT protein in cells (see [link to article]). Figure 3H and Figure 3I , Figure 11E and Figure 11I ).

[0124] Therefore, in another aspect, the present invention provides a method for inhibiting the degradation of CREPT protein in eukaryotic cells, the method comprising: 1) introducing an inhibitor selected from SKP2 inhibitors, CUL1 inhibitors, neddylation inhibitors, and CDK2 inhibitors into eukaryotic cells expressing CREPT; and / or 2) phosphorylating sites 134 and 166 of the CREPT protein to inactivate it, such that when the modified protein is located in eukaryotic cells in the G1 terminal phase or G1 / S transition phase, sites 134 and 166 remain in a non-phosphorylated state and the modified protein remains in a non-ubiquitinated state, thereby preventing the modified protein from being degraded. In one embodiment, the eukaryotic cells may be cells of humans, yeast, mice, dogs, cats, chickens, toads, zebrafish, fruit flies, nematodes, or Arabidopsis thaliana, preferably human cells, more preferably human cancer cells. In one embodiment, the phosphorylation inactivation modification at sites 134 and 166 may be an amino acid mutation and / or chemical modification, preferably, a mutation of serine (S) to alanine (A). In one embodiment, the SKP2 inhibitor can be a double-stranded siRNA targeting SKP2, with a sequence of AAUCUAAGCCUGGAAGGCCUGdTdT; the CUL1 inhibitor can be a double-stranded siRNA targeting CUL1, with a sequence of UAGACAUUGGGUUCGCCGUdTdT; and the neddylation inhibitor can be MLN4924.

[0125] In addition, such as Figure 4D As shown, the single mutant proteins S134A or S166A of CREPT can inhibit ubiquitination to some extent. Therefore, it can be understood that these single mutant proteins and similar single mutants of homologous proteins in other eukaryotic cells have the function of inhibiting CREPT degradation.

[0126] Therefore, in another aspect, the present invention also provides the following proteins: 1) a protein obtained by mutating serine at position 166 of SEQ ID No:4 to alanine; 2) a protein obtained by mutating serine at position 136 of SEQ ID No:8 to alanine; 3) a protein obtained by mutating serine at position 174 of SEQ ID No:8 to alanine; and 4) a protein having more than 90% sequence identity with any one of proteins 1) to 3) and having the same alanine mutation. Furthermore, the present invention also provides a nucleic acid encoding said proteins and a vector comprising said nucleic acid.

[0127] Sequence description:

[0128] SEQ ID NO:1 encodes the nucleic acid sequence of the CREPT S134A / S166A double mutant protein;

[0129] The amino acid sequence of the S134A / S166A double mutant protein; SEQ ID NO:2CREPT

[0130] The coding nucleic acid sequence of SEQ ID NO:3 wild-type CREPT;

[0131] The amino acid sequence of wild-type CREPT (SEQ ID NO:4);

[0132] SEQ ID NO:5 encodes the nucleic acid sequence of the Rtt103 S136A / S174A double mutant protein;

[0133] The amino acid sequence of the S136A / S174A double mutant protein, SEQ ID NO:6Rtt103;

[0134] The coding nucleic acid sequence of wild-type Rtt103 in SEQ ID NO:7;

[0135] The amino acid sequence of wild-type Rtt103 in SEQ ID NO:8.

[0136] Example

[0137] Example 1. CREPT degrades during the G1 / S transition and recovers during the S phase.

[0138] Immunofluorescence (IF) staining was performed on DLD1 and HeLa cells to determine the expression pattern of CREPT in tumor cells. The results showed that most tumor cells expressed abundant CREPT, but a minority of tumor cells were CREPT-negative. Figure 1A (See the dotted circle). Furthermore, CREPT-negative tumor cells have slightly larger nuclei and show uniform DAPI staining ( Figure 1A (DAPI staining). Therefore, CREPT-negative tumor cells may be due to the disappearance of CREPT during a specific cell cycle phase. To verify this hypothesis, the inventors used CRISPR-Cas9 to generate HeLa cells with GFP-CREPT knocked in. Figure 9A , Figure 9B Live-cell imaging analysis showed that GFP-CREPT remained in the cells for a period of time, then disappeared for nearly 30 minutes, and then reappeared. Figure 1B This indicates that the level of the CREPT protein oscillates during the tumor cell cycle.

[0139] To further confirm the role of CREPT protein oscillations in the cell cycle, DLD1 and HeLa cells were synchronized to the G1 / S transition phase using diathymidine arrest (DTB) and released to different cell cycle time points. Fluorescence-activated cell sorting (FACS) analysis showed that after DTB treatment, over 90% of cells were synchronized to the G1 phase (…). Figure 9C Western blot analysis showed that CREPT protein was almost undetectable during the G1 / S transition, but increased during the S phase. Figure 1C and Figure 9D Lanes 1, 0 hours, and 1 hour). This change in CREPT protein is accompanied by the opposite trend of Cyclin E and SKP2, but similar to the expression patterns of Cyclin A and Cyclin B1. Figure 1C and Figure 9D Meanwhile, the mRNA level of CREPT remained unchanged from the G1 / S phase transition to the S phase. Figure 9E These results indicate that the CREPT protein is degraded during the G1 / S transition phase of the tumor cell cycle.

[0140] To directly visualize specific time points of CREPT disappearance, a fluorescently ubiquitinated cell cycle indicator (Fucci) cell line was established in DLD1 cells. Endogenous IF staining was performed in Fucci cells embedded with 5-ethynyl-2'-deoxyuridine (EdU). Figure 1D , Figure 1E and Figure 9F , Figure 9G The results showed that CREPT disappeared after synchronization, accompanied by negative EdU staining (a marker of S phase), but positive GEMININ and CDT1 staining (a marker of G1 / S phase). Figure 1D 0 hours). As tumor cells enter the S phase, CREPT protein gradually increases ( Figure 1D Quantitative analysis showed that CREPT and EdU were at their lowest levels during the G1 / S transition phase, but recovered after tumor cells were released into the S phase. Figure 1E These results indicate that the CREPT protein is at its lowest level during the G1 / S transition phase of tumor cells.

[0141] Example 2. GREPT degrades via ubiquitination of SKP2 during the G1 / S transition.

[0142] To elucidate the degradation mechanism of CREPT during the G1 / S transition, the inventors first examined the protein stability of CREPT by blocking protein synthesis with actinomycete ketone (CHX). The results showed that CHX treatment reduced CREPT protein levels (…). Figure 2A , Figure 10A The time to 10-12 hours indicates CREPT protein degradation. Furthermore, when MG132 (an inhibitor of proteasome-induced degradation) is added during cell cycle synchronization, CREPT protein remains at a relatively high level during the G1 / S transition. Figure 2B Lane 7 vs 1 Figure 2C , Figure 10B These results indicate that the decrease in CREPT expression is due to proteasome-induced degradation.

[0143] To confirm whether CREPT degradation is ubiquitin-dependent, the inventors performed a co-immunoprecipitation (Co-IP) experiment to detect the interaction between CREPT and ubiquitin. The results showed that the antibody against Myc precipitated Myc-CREPT and HA-ubiquitin, while MG132 increased the level of precipitated HA-ubiquitin. Figure 2D This indicates that CREPT can be modified by ubiquitin. Further ubiquitin linkage type characterization experiments revealed that CREPT is primarily ubiquitinated via K11 linkages, rather than K48 linkages. Figure 2E It is worth noting that K63 ubiquitin can also moderately induce CREPT ubiquitination (see...). Figure 2E (lanes 6 and 12). This result was unexpected, as K48 ubiquitin has been widely reported to mediate protein degradation. The inventors further overexpressed different types of ubiquitin along with CREPT in the presence of CHX. The results showed that, in the presence of K11 instead of K48 or K63 ubiquitin, CREPT reduced ( Figures 2F-2K Furthermore, the inventors synchronized DLD1 and HeLa cells to the G1 / S phase using the autophagy inhibitors CQ and LEU. The results showed that CREPT protein remained degraded even 0 hours after DTR under CQ or LEU treatment. Figures 10C-10D These results indicate that the degradation of CREPT during the G1 / S transition depends on K11-linked ubiquitination.

[0144] To identify E3 ligases targeting CREPT, the inventors synchronized cells to the G1 / S phase under MG132 treatment to precipitate CREPT-interacting proteins. Mass spectrometry analysis revealed the presence of several E3 ligases in the precipitated complex. Among the top 10 E3 ligases potentially interacting with CREPT, SKP2 showed the highest probability of binding to CREPT. Figure 3A To determine whether SKP2 is an E3 ligase used for CREPT degradation, the inventors verified their interactions under different conditions. IP experiments showed that the antibody against HA precipitated HA-CREPT and Flag-SKP2. Figure 3B This indicates that HA-CREPT interacts with Flag-SKP2. Importantly, antibodies against CREPT were observed to precipitate endogenous SKP2 in DLD1 cells. Figure 3CThis indicates that CREPT and SKP2 interact in intact cells. To verify the physical interaction, the inventors purified GST-CREPT and His-SKP2 in E. coli according to previously reported strategies (Chan et al., 2013; Schulman et al., 2000). GST sedimentation assays showed that the antibody against GST simultaneously precipitated GST-CREPT and His-SKP2, indicating that SKP2 directly binds to CREPT. Figure 3D These results indicate that CREPT and SKP2 interact directly both in vivo and in vitro.

[0145] To demonstrate the role of SKP2 in CREPT ubiquitination, the inventors examined the levels of ubiquitinated CREPT. In vivo ubiquitination assays showed that overexpression of SKP2 enhanced polyubiquitination of Myc-CREPT. Figure 3E To determine whether SKP2 mediates CREPT degradation, the inventors overexpressed SKP2 in HeLa cells under CHX treatment. Western blot analysis showed that CREPT protein levels decreased in SKP2-overexpressing cells 8 hours after CHX treatment, while they decreased in control cells at 10 hours, indicating that SKP2 overexpression accelerated CREPT degradation by 2 hours. Figures 3F-3G (10-12 hours). It is worth noting that adding MG132 will weaken the degradation of CREPT ( Figure 3F The results (at 12 hours after MG132) indicate that SKP2-induced CREPT degradation is proteasome-dependent. To confirm the role of SKP2 in CREPT degradation in vivo, endogenous SKP2 was depleted in HeLa cells using siRNA. The results showed that in SKP2-depleted cells, CREPT protein levels remained stable at different time points after CHX treatment. Figures 3H-3I These findings indicate that CREPT ubiquitination is mediated by the F-box family E3 ligase SKP2.

[0146] To investigate the role of SKP2 in the degradation of CREPT during the tumor cell cycle, the inventors synchronized cells to the G1 / S phase and precipitated CREPT complexes at different release time points. The results showed that SKP2 expression transitioned from the G1 / S transition phase to the S phase during the cell cycle, while CREPT expression was lowest in the G1 / S phase and then gradually recovered. Figure 3J (lysates). Notably, the antibody against CREPT caused strong precipitation of SKP2 from 0 to 4 hours after release, but no precipitation at 10 hours, indicating that the interaction between SKP2 and CREPT occurs specifically in the G1 / S phase rather than the G2 / M phase. Figures 3J-3K (0-8 hours).

[0147] To further determine the timing of CREPT / SKP2 interaction in the tumor cell cycle, cells were synchronized to the G2 / M phase. Co-IP experiments showed that SKP2 appeared 10 hours post-release, but CREPT expression was at its lowest level. Figure 3J The interaction between CREPT and SKP2 was also observed to occur at 10 hours post-cell release and persist until 18 hours. Figure 3L-Figure 3M Flow cytometry analysis showed that the cells were still in the G1 / S phase 10 hours after release. Figure 11D These results indicate that SKP2 interacts with CREPT during the G1 / S transition and induces its degradation.

[0148] Since SKP2 belongs to the F-box family of SCF complex proteins, to determine whether the Cullin-RING E3 ubiquitin ligase (CRL1) complex is involved in CREPT degradation, the inventors used the neddylation inhibitor MLN4924, which inhibits the activation of all Cullin-RING ligase complexes. The results showed that CREPT protein levels increased in control cells (si Ctrl), but not in MLN4924-treated SKP2-depleted cells (si SKP2). Figure 11E ). Figure 11E The results indicate that in control cells, Cullin-RING ligase was gradually inactivated by MLN4924 over time (0-6 hours). Although SKP2 expression remained significant, CREPT ubiquitination gradually decreased and degradation was inhibited, with its level gradually increasing over time. In contrast, in cells where SKP2 was depleted with siRNA, CREPT ubiquitination and degradation were consistently inhibited, thus remaining at a significant level. These results demonstrate that SKP2 deficiency can significantly inhibit CREPT ubiquitination and degradation.

[0149] Correspondingly, immunoprecipitation assays revealed an interaction between Flag-Cul1 and HA-CREPT. Figure 11F Furthermore, overexpression of Cul1 increased CREPT ubiquitination levels with or without MG132 treatment. Figure 11G (See HA band). Consistent with this, the over-surface degradation of Cul1 accelerated the degradation of CREPT by 2 hours (see HA band). Figure 11H However, the deletion of Cul1 induced by siRNA protected CREPT from degradation. Figure 11I These results indicate that CRL1 SKP2 It is an E3 ligase that recognizes CREPT to be degraded, and the inactivation of this ligase can significantly inhibit the ubiquitination and degradation of CREPT.

[0150] Example 3. CREPT ubiquitination depends on phosphorylation of S134 and S166.

[0151] To identify the ubiquitination sites on the CREPT protein, the inventors expressed the Myc-tagged CID domain (Myc-CREPT-CID) and CCT domain (Myc-CREPT-CCT) in 293T cells using HA-Ub in the presence of MG132. Western blot analysis showed that Myc-CREPT-CID was ubiquitinated, while Myc-CREPT-CCT was not. Figure 4A This indicates that ubiquitination occurs within the CID domain. The inventors then mutated all individual lysine (K) residues in the CID domain to arginine (R) to map ubiquitination sites, but Western blot analysis showed that none of the mutations impaired ubiquitination levels. Figure 12A and 12B The K56R mutation increases ubiquitination levels. Figure 12A Lane 9 indicates that K56 is a repressive residue for CREPT ubiquitination. Even with mutations in all K residues, Myc-CREPT-CID remains at a high ubiquitination level. Figure 12C (last lane), however, when all K residues are mutated but K56 is not mutated, ubiquitination is at baseline ( Figure 12C (lane 4). These results indicate that ubiquitination may occur on other residues, while K56 is an inhibitory residue.

[0152] In search of other possible ubiquitin-linking residues, the inventors also mutated threonine (T) and cysteine ​​(C) residues in the CID domain, but the level of CREPT ubiquitination remained unchanged in these mutations. Figure 12D These negative results prompted the inventors to verify whether CREPT was a substrate with phosphorylation degradation determinants, since SCF complexes tend to bind to phosphorylation degradation determinants for ubiquitination. To this end, the inventors analyzed the phosphorylation modification of CREPT by mass spectrometry and found that serine 134 (S134) and serine 166 (S166) sites were highly phosphorylated. Figure 4B It is noteworthy that these two S residues are located in the linker region of CREPT and are highly conserved between CREPT and its ortholog in Saccharomyces cerevisiae, Rtt103. Figure 4CTo verify whether phosphorylation of S134 and S166 regulates CREPT ubiquitination, the inventors created different mutants, including S134A, S166A, and the S134A / S166A double mutant to simulate a loss-of-function non-phosphorylated state, and S134E, S166E, and the S134E / S166E double mutant to simulate sequential phosphorylation. Western blot analysis showed that the S134A, S166A, and S134A / S166A mutations impaired ubiquitination, but the other mutations had no effect on ubiquitination. Figure 4D This result indicates that phosphorylation of S134 and S166 is essential for the ubiquitination of CREPT. To confirm whether these two S residues are crucial for SKP2 recognition, the inventors performed an IP experiment, which showed that the Myc antibody precipitated the Myc-CREPT(S134E / S166E) / Flag-SKP2 complex, but not the Myc-CREPT(S134A / S166A) / Flag-SKP2 complex. Figure 4E The results indicate that CREPT (S134A / S166A) failed to interact with SKP2. These results suggest that S134 and S166 are involved in the formation of phosphorylation degradation determinants of CREPT.

[0153] Furthermore, amino acid sequence analysis of CREPT showed that S134 and S166 are both potential recognition sites for the Cyclin E-CDK2 complex with sp sequences. Interactions between CREPT and Cyclin E1 and CDK2 were also observed in IP experiments. Figure 4F In vitro kinase assays showed that GST-CREPT was phosphorylated by CDK2 and Cyclin E1, but GST-CREPT (S134A / S166A) was not phosphorylated. Figure 4G GST-CREPT was also observed to be phosphorylated in mammalian cells, but not in E. coli. Figure 12E These results indicate that the degradation of CREPT during the G1 / S phase depends on the degradation by CRL at sites S134 and S166. SKP2 Identified phosphorylation degradation determinants.

[0154] Example 4. Undegraded CREPT protein variants induce apoptosis.

[0155] The inventors discovered that the mutant protein CREPT (S134A / S166A) always induces cell death. Figure 13A(See the image above). Specifically, the inventors overexpressed wild-type CREPT and mutant CREPT in CREPT-deficient cells. The results showed that overexpression of wild-type (WT) CREPT and CREPT (S134E / S166E) rescued cell proliferation; however, overexpression of CREPT (S134A / S166A) led to a significant increase in cell death. Figure 13A (See figure below). FACS analysis showed that expression of CREPT (S134A / S166A) led to a significant increase in cell death in both wild-type and CREPT-deficient cells, while wild-type CREPT and CREPT (S134E / S166E) had no effect. Figures 5A-5B Furthermore, the mortality rate of CREPT-deficient cells induced by CREPT (S134A / S166A) was significantly higher than that induced by it in wild-type cells. Figures 5A-5B Correspondingly, when CREPT (S134A / S166A) is expressed in wild-type cells or CREPT-deficient cells, the cell growth rate ( Figure 5D ) and settlement formation ability ( Figure 5D and Figure 13C Significant impairment was observed. Western blot analysis revealed that overexpression of CREPT (S134A / S166A) induced the expression of cleaved caspase 7, a typical apoptosis actuator. Figure 5C The above results indicate that undegraded CREPT variants induce apoptosis.

[0156] To confirm whether the apoptosis-inducing effect of the S134A / S166A mutation exists in other species, the inventors exogenously expressed the CREPT mutant protein in *Saccharomyces cerevisiae*. The results showed that CREPT (S134A / S166A) significantly inhibited yeast growth at different temperatures. Figure 13F Exogenous expression of human p15RS had no effect on yeast survival. Figure 13F To eliminate the influence of endogenous Rtt103 (a homolog of CREPT) in yeast, human CREPT and its mutant proteins were exogenously expressed in Rtt103-deficient yeast strains. The results showed that CREPT (S134A / S166A) significantly blocked yeast growth (…). Figure 5F This indicates that CREPT (S134A / S166A) is also lethal in yeast. Furthermore, p15RS also appears to inhibit the growth of Rtt103-deficient yeast, echoing the inhibitory effect of p15RS in mammalian cells. These results suggest that the double mutation of S134A and S166A in CREPT can induce cell death in both mammalian and yeast cells.

[0157] Since S134 and S166 in CREPT correspond to S136 and S174 in yeast Rtt103 (see...) Figure 4C The inventors created the corresponding yeast mutant protein and observed that overexpression of Rtt103 (S136A / S174A) led to a lethal phenotype in Rtt103-deficient yeast. Figure 5G These results indicate that disruption of phosphorylation degradation determinants in CREPT and RTT103 leads to cell death.

[0158] Example 5. Undegraded CREPT variants prevent cells from entering S phase

[0159] Previous results have indicated that the phosphorylation degradation determinant of CREPT is crucial for the G1 / S transition. To investigate the mechanism by which CREPT phosphorylation affects the G1 / S transition, the inventors identified proteins that do not interact with CREPT during the G1 / S phase. Specifically, DLD1 cell lines were synchronized to the G1 / S phase using thymidine arrest, and proteins interacting with CREPT were analyzed using chromatin immunoprecipitation mass spectrometry (ChIP-MS). Comparison of the differences in CREPT-precipitated proteins between asynchronous and synchronous DLD1 cells revealed that the MCM hexamer proteins MCM5 and MCM7 appeared not to interact with CREPT during the G1 / S phase, but maintained interaction with CREPT in other phases. Figure 6A This result indicates that CREPT may separate from the MCM hexamer during the G1 / S transition, possibly due to the degradation of CREPT.

[0160] The MCM hexamer contains six MCMs, including MCM2 through MCM7. To verify the interaction between CREPT and the MCM hexamer, Myc-CREPT and Flag-MCM5 were overexpressed in 293T cells. IP experiments showed that Myc-CREPT and Flag-MCM5 interacted strongly in asynchronous 293T cells. Figure 6B The interaction between Myc-CREPT and Flag-MCM7 was also observed. Figure 14A Myc-CREPT interacts with Flag-MCM2. Figure 14B This indicates that CREPT binds to the MCM hexamer. To confirm the endogenous interaction, the inventors performed co-IP experiments using an antibody against CREPT under different cross-linking conditions. Figure 14C The results showed that CREPT and MCM5 interacted after 10 minutes of crosslinking. Figure 6C It was also observed that CREPT and MCM5 interacted strongly when synchronized cells were released 0 to 8 hours after release, but their interaction appeared to weaken 10 hours after release. Figure 6DNotably, CREPT remained at its lowest level 10 hours after cell release, corresponding to the emergence of SKP2 and Cyclin E1. Figure 6D These observations indicate that the CREPT / MCM interaction is impaired at the G1 / S phase due to CREPT degradation. The inventors further performed IP experiments using undegraded mutant CREPT, and the results showed that the interaction between Myc-CREPT (S134A / S166A) and Flag-MCM5 was stronger than that between WT protein and Myc-CREPT (S133E / S166E) and Flag-MCM5. Figure 6E This result indicates that the degradation of CREPT leads to its dissociation from the MCM hexamer.

[0161] To decipher the CREPT / MCM interaction and dissociation, the inventors examined the occupancy of the MCM hexamer on chromatin DNA. For this purpose, different fixation strategies were used to stain MCM5 and CREPT proteins. The results showed that direct fixation with 1% paraformaldehyde resulted in uniform staining of MCM5 within the cells. Figure 14D (See above image), but when pre-extracted with Triton X100 before fixation, it became negative in some cells ( Figure 14D (See figure below). CREPT did not stain in the pre-extracted cells, indicating that CREPT does not directly bind to DNA. To investigate whether CREPT degradation regulates the occupancy of MCM hexamers on chromatin DNA, the undegraded CREPT mutant protein Myc-CREPT (S134A / S166A) was overexpressed, and MCM5 was stained under pre-extraction conditions. The results showed that when Myc-CREPT (S134A / S166A) was overexpressed, MCM5 was strongly stained (…). Figure 6F and Figure 6G This means that the dual MCM hexamer is loaded onto chromatin DNA, and the cell cycle arrests at the end of G1 or the G1 / S phase. FACS analysis confirmed that the expression of CREPT (S134A / S166A) led to a significant reduction in S-phase cells. Figure 14E Notably, the nuclei of all MCM5-positive cells were enlarged, and EdU-negative staining was observed in cells overexpressing Myc-CREPT (S134A / S166A). Figure 6G (See EdU staining). These results indicate that all MCM5 strongly positive cells with Myc-CREPT (S134A / S166A) overexpression arrested the cell cycle before entering S phase. In summary, all results suggest that CREPT degradation leads to the dissociation of the MCM hexamer, and the failure of this dissociation arrests the cell cycle before entering S phase.

[0162] Example 6. Undegraded CREPT variants halt DNA replication forks.

[0163] Undegraded CREPT mutants cause cell death and cell cycle arrest by binding to the MCM hexamer; therefore, the inventors verified whether the CREPT-MCM complex induces genomic stress during the G1 / S transition. Since genomic stress leads to DNA damage, the inventors performed TUNEL staining to examine DNA strand breaks. The results showed that overexpression of CREPT (S134A / S166A) resulted in significant TUNEL signaling in both WT and CREPT-deficient cells, while overexpression of WT protein and CREPT (S134E / S166E) showed negative signals. Figure 15A Notably, overexpression of CREPT (S134A / S166A) produced a significantly stronger TUNEL signal in CREPT-deficient cells than in WT cells. Figure 15A (Comparison of KO and Mock). Correspondingly, γH2AX focal points were also observed in cells overexpressing CREPT (S134A / S166A). Figure 15B These results indicate that undegraded CREPT variants cause DNA damage.

[0164] To confirm whether the DNA damage was due to genomic stress caused by undegraded CREPT variants, the inventors examined foci formed by replication protein A2 (RPA2), which binds to single-stranded DNA (ssDNA) and phosphorylates in response to replication stress. IF staining with an antibody against total RPA2 showed that overexpression of CREPT (S134A / S166A) in HeLa cells led to an increase in RPA2 foci and protein levels. Figure 7A (See ctrl). When cells are treated with HU (a dNTP synthesis inhibitor that blocks DNA replication), the RPR2 focus increases significantly. Figure 7A (Comparing HU and ctrl). Further examination of phosphorylated RPA2 (p-RPA2) revealed positive focal points in cells overexpressing CREPT (S134A / S166A), and this was significantly increased under HU treatment. Figure 7B These results indicate that undegraded CREPT protein variants contribute to genomic stress.

[0165] Since MCM-DNA dissociation failed in cells expressing CREPT (S134A / S166A), we investigated whether cell death induced by undegraded CREPT variants was caused by genomic stress from DNA replication disruption. Newly synthesized DNA fibers were examined for this purpose. Cells were released after HU blockade, allowing CIdU incorporation. IF staining results showed that the length of CIdU-tagged DNA fiber trails was shortened when CREPT (S134A / S166A) expression was 12 hours prior. Figure 7C To further confirm this result, dual IdU and CIdU labeling experiments were performed. The results showed that, compared to WT cells, the length of IdU-labeled fibers was reduced in CREPT (S134A / S166A) cells. Figure 7D Consistent with this, CIdU-labeled fibers, representing the replication rate under normal conditions, are shortened in CREPT (S134A / S166A) cells. Figure 7D Quantitative analysis showed that, compared with WT cells, cells overexpressing CREPT (S134A / S166A) induced more replication-arrested fibrosis (CIdU). + IdU-trajectory)( Figure 7D These results suggest that undegraded CREPT variants may cause DNA synthesis defects by affecting stalled replication forks.

[0166] Materials and methods

[0167] The materials and methods used in the above embodiments are described below.

[0168] plasmids and siRNA

[0169] The plasmids HA-CREPT, Flag-CREPT, Myc-CREPT, Myc-CREPT-CID, Myc-CREPT-CCT, GSTCRET, GFP-P15RS, and Myc-P15RS were constructed by the inventor's laboratory. The Flag-MCM2 plasmid was a gift from Dr. Daochun Kong (School of Life Sciences, Peking University). pRK5-HA-UBI (#17608), pRK5-HA-UBI-K11 (#22901), pRK5-HA-UBI-K48 (#17605), pRK5-HA-UBI-K63 (#17606), and pSpCas9(BB)-2AGFP (PX458, #48138) were purchased from Addgene. Flag-SKP1, Flag-SKP2, Flag-CUL1, Flag-MCM5, and Flag-MCM7 were generated from cDNA. CREPT-mutated plasmids were subjected to site-directed mutagenesis. TMThe SKP2 and CUL1 siRNA duplexes were generated by Lipofectamine RNAi MAX (Invitrogen) transfection, with oligonucleotide sequences of AAUCUAAGCCUGGAAGGCCUGdTdT and UAGACAUUGGGUUCGCCGUdTdT, respectively.

[0170] CRISPR-Cas9 knock-in cell lines

[0171] HeLa cells were used to construct a CRISPR-Cas9 knock-in cell line. Short guide RNA (sgRNA) oligonucleotides were designed and optimized according to the lab instructions of Feng Zhang ( / / crispr.mit.edu / ). The sgRNA sequence was CTCCTTCTCTGAGTCGGCGC. Annealed sgRNA and a BBSI-digested Px458 vector were ligated using Solution I (Takara) to construct a Cas9 DNA splicing plasmid. Conversely, the coding sequence for GFP was cloned and ligated into a PCDNA 3.1-HA vector to construct a GFP transcription plasmid. HeLa cells were co-transfected with the Cas9 DNA splicing and GFP transcription plasmids. GFP-positive HeLa cells were sorted by flow cytometry, and single cells were then seeded in 96-well plates to select DNA recombinant clones. One week later, cells were screened using GFP expression with genomic insertion.

[0172] Cell cycle synchronization

[0173] Cells were synchronized to the G1 / S phase via thymidine arrest (DTB). Cells were treated with 2 mM thymidine for at least 18 hours, released into fresh medium for 8 hours, and then treated with 2 mM thymidine again for at least 16 hours. Cells were synchronized to the G2 / M phase via thymidine-nocodazole arrest. Cells were treated with 2 mM thymidine for at least 24 hours, released for 3 hours, and then treated with 340 nM nocodazole for at least 16 hours. Cells were harvested at the indicated time points. The cell cycle stage of the harvested cells was verified by flow cytometry analysis. For MG132 treatment, MG132 was added to DLD1 cells 4 hours prior to harvest.

[0174] In vitro protein assay

[0175] Because the SKP2 protein is unstable in prokaryotic expression systems, in vitro protein-protein interaction assays were performed using pET22b-SKP1, pET30A-SKP2, and GST-CREPT proteins. Immunoprecipitation assays validated the interaction between GST-CREPT and pET30A-SKP2.

[0176] In vitro kinase assays were performed using purified GST, GST-CREPT (wild-type), or GST-CREPT (S134A / S166A). The protein was incubated with Myc-Cyclin E / Myc-CDK2 protein for 30 minutes at 30°C in kinase buffer (10 mM HEPES (pH 7.5), 50 mM NaCl, 2 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, and 0.1 mM ATP). The reaction was stopped using SDS loading buffer. Phosphorylation of CREPT was detected by Western blotting.

[0177] DNA fiber analysis

[0178] DNA fiber experiments were performed as previously described (Genois et al., 2021). In short, DLD1 cells were first labeled with 50 mM CldU, washed twice with PBS, and then labeled with 250 mM IdU. Cells were harvested and resuspended in cold PBS to a concentration of 1–1.5 × 10⁻⁶. 6 Cells / ml were collected, and 3 μL of cell culture was mixed with 7 μL of spreading buffer (0.5% SDS, 200 mM Tris-HCl pH 7.4, 50 mM EDTA) and spread onto silanized slides. The slides were tilted at 30–60° to spread the fibers and incubated at room temperature for 15 minutes. DNA fibers were fixed in methanol:acetic acid (3:1) for 20 minutes. After drying, the slides were stored at 4°C overnight. DNA fibers were denatured in 2.5 M HCl for 30 minutes and blocked with 3% BSA for 60 minutes. CldU and IdU were detected with rat anti-BrdU and mouse anti-BrdU at room temperature for 2 hours, followed by binding with Alexa488 anti-mouse and Cy3 anti-rat secondary antibodies at room temperature for 1 hour. The slides were fixed with Prolong Gold Antifade Reagent. The fibers were imaged using a 60X objective on an Olympus FV3000 confocal microscope.

[0179] Immunofluorescence assay

[0180] In the pre-extraction method, to extract soluble proteins, live cells were first treated on ice with 1 mL of permeation buffer (containing 0.2% Triton X-100, 20 mM HEPES pH 7.4, 100 mM NaCl, and 300 mM sucrose) for 5 minutes. After removing the permeation buffer, the cells were fixed with 2% paraformaldehyde at room temperature for 10 minutes. In the direct fixation method, cells were first fixed with 2% paraformaldehyde at room temperature for 10 minutes. Then, the cells were permeated with 0.3% Triton X-100 in PBS for 15 minutes and blocked with 10% BSA in PBST. The fixed cells were incubated with the specified primary antibody at room temperature for 2 hours or overnight at 4°C. After incubation with fluorescent secondary antibody and mounting in Prolong Gold, the cells were imaged on an Olympus FV3000 confocal microscope using a 60X objective.

[0181] Example 7. Screening of CREPT phosphorylation ubiquitination inhibitors

[0182] 7.1 Prediction of small molecule compounds as potential inhibitors of CREPT phosphorylation and ubiquitination

[0183] We used the prediction tools SwissTargetPrediction (http: / / www.swisstargetprediction.ch / ) and SEA (Similarity ensemble approach; https: / / sea.bkslab.org / ) to jointly predict small molecule phosphorylation inhibitors of CREPT, and obtained and synthesized five candidate small molecule compounds #1 to #5. Since phosphorylation of CREPT is a prerequisite for its ubiquitination, these five candidate small molecule compounds #1 to #5 are potential inhibitors of CREPT phosphorylation and ubiquitination.

[0184] 7.2 Effects of candidate small molecule compounds on CREPT ubiquitination

[0185] 1) Divide 293T cells into 6cm culture dishes and incubate at 37°C for 24 hours. Transfect. Figure 16 Insert the specified plasmids (HA-Ub and / or Myc-CREPT) into the cells, and replace with fresh culture medium 5 hours later.

[0186] 2) 24 hours after transfection, collect cells using 1 ml RIPA lysis buffer and lyse at 4°C for 1 hour. Centrifuge at 13000 rpm for 10 minutes at 4°C. Take 800 μl of supernatant, add 50 μl of protein plus beads and 5 μl of anti-myc antibody, label as IP sample, and incubate overnight at 4°C. Take 50 μl of supernatant, add an equal volume of 2x loading buffer, and label as lysis buffer sample.

[0187] 3) The IP samples were eluted by centrifugation four times with cell lysis buffer, 10 minutes each time. After elution, 50 μl of 2x loading buffer was added to the IP samples. The IP samples and lysis buffer were boiled at 100°C for 10 minutes. The samples were then analyzed by SDS-PAGE.

[0188] The results are as follows Figure 16 As shown, the wild-type CREPT protein, untreated with candidate inhibitors, can be ubiquitinated by ubiquitin (Ub). Figure 16 In the third band (the negative control CREPT mutant SA control group), ubiquitination was undetectable. Among the bands with added candidate compounds #1 to #5, ubiquitination of #4 was significantly reduced (approximately 20% lower ubiquitination levels compared to the untreated wild-type protein).

[0189] The above results demonstrate that compound #4 is an effective inhibitor of CREPT phosphorylation and ubiquitination. The structural formula of compound #4 is as follows:

[0190]

[0191] 7.3 Effects of candidate small molecule compounds on cell proliferation

[0192] 1) Take DLD1 (human colorectal adenocarcinoma epithelial cells) or MGC803 (human gastric cancer cells) in logarithmic growth phase, digest them with 0.25% trypsin and gently pipette to make them into single cells. Count the viable cells and adjust the cell density to 1×10⁻⁶ cells using DMEM culture medium containing 10% fetal bovine serum. 4 Cells / L.

[0193] 2) Mix 10 mL of culture medium and 10 mL of cell diluent at a 1:1 ratio, then add 0.2 mL of the mixture to each well of a 96-well plate, for a total of 3 replicate wells. Incubate at 37°C in a 5% CO2 incubator for 12 hours.

[0194] 3) Dissolve the above five candidate small molecule compounds #1 to #5 in DMSO. The initial screening concentration for each compound is 10 μM (DLD1 cells) or 5 μM (MGC803 cells). Perform three replicates for each compound; incubate each compound at a concentration of 10 μM (DLD1 cells) or 5 μM (MGC803 cells) for 3 days, then use CCK to measure cell proliferation. Before measurement, replace each well with 10 μl of well-mixed CCK-8 solution and 90 μl of complete culture medium (wells with the corresponding amounts of CCK-8 solution and cell culture medium serve as blank controls). Incubate at 37°C for 3 hours. Measure the absorbance at 450 nm. Calculate and statistically analyze the results and plot them as shown below. Figure 17 The cells A (DLD1 cells) and B (MGC803 cells) are shown in the figure.

[0195] It can be seen that compound #4, as an inhibitor of CREPT phosphorylation and ubiquitination, significantly inhibited cell proliferation. This indicates that the tested concentration of compound #4 inhibited CREPT degradation to some extent, leading to apoptosis in some cells. This result is consistent with the results of Examples 2-4.

[0196] The technical concept and specific embodiments of the present invention have been described above. However, it should be understood that the above specific embodiments do not limit the scope of the present invention in any way. Those skilled in the art will understand that various modifications and / or changes can be made to the invention shown in the specific embodiments without departing from the spirit of the present invention, and the modified and / or changed embodiments are also covered within the scope of the present invention. Therefore, the embodiments of the present invention are merely illustrative and not restrictive.

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Claims

1. A protein having the amino acid sequence SEQ ID NO:

2.

2. The protein of claim 1, wherein, When the protein is located in a eukaryotic cell in the G1 terminal phase or the G1 / S transition phase, its 134 and 166 sites remain unphosphorylated and the protein remains unubiquitinated, thereby preventing the protein from being degraded in the eukaryotic cell, which in turn leads to cell cycle arrest and apoptosis. The eukaryotic cell is a human cancer cell or a yeast cell.

3. A protein having a tag sequence or guide sequence attached to the N-terminus and / or C-terminus of the protein described in claim 1 or 2.

4. The nucleic acid encoding the protein of claim 1.

5. A vector comprising the nucleic acid of claim 4.

6. Cells comprising the carrier of claim 5.

7. The use of the protein according to any one of claims 1 to 3 in the preparation of reagents for inhibiting eukaryotic cell proliferation, inhibiting eukaryotic cell DNA replication, regulating the cell cycle of eukaryotic cells, or killing eukaryotic cells, wherein, The eukaryotic cells are human cancer cells or yeast cells.

8. The use of the protein according to any one of claims 1 to 3 in the preparation of anticancer drugs.

9. A protein having the amino acid sequence SEQ ID NO:

6.

10. The protein of claim 9, wherein, When the protein is located in a eukaryotic cell in the G1 phase or G1 / S transition phase, its 136 and 174 sites remain unphosphorylated and the protein remains unubiquitinated, thereby preventing the protein from being degraded in the eukaryotic cell, which in turn leads to cell cycle arrest and apoptosis. The eukaryotic cell is a yeast cell.

11. A protein having a tag sequence or guide sequence attached to the N-terminus and / or C-terminus of the protein of claim 9 or 10.

12. A nucleic acid encoding the protein of claim 9.

13. A vector comprising the nucleic acid of claim 12.

14. Cells comprising the carrier of claim 13.

15. A method for screening non-phosphorylated, non-ubiquitinated modifiers for CREPT protein, wherein, The modifier maintains the S134 and S166 sites of the CREPT protein in a persistently non-phosphorylated state, thereby ensuring that the CREPT protein remains non-ubiquitinated and does not degrade in eukaryotic cells; the amino acid sequence of the CREPT protein is SEQ ID No: 4, wherein the eukaryotic cells are human cancer cells or yeast cells, and the method includes: i) A candidate modifier simulating a non-phosphorylated state was added to eukaryotic cells expressing CREPT protein that were synchronized to the G1 phase, and then the eukaryotic cells were released and cultured. The phosphorylation levels of the S134 and S166 sites of the CREPT protein were examined while the eukaryotic cells were alive. or ii) In vitro, candidate modifiers simulating the non-phosphorylated state were incubated with the CREPT protein, and the phosphorylation levels at S134 and S166 sites of the CREPT protein were examined under Cyclin E / CDK2 kinase catalysis. If the phosphorylation levels at S134 and S166 sites of the CREPT protein treated in step i) or ii) decrease relative to the untreated control, the candidate modifier is screened as a non-phosphorylated, non-ubiquitinated modifier for the CREPT protein.

16. A method for identifying whether a substance is a phosphorylation inhibitor of CREPT protein, wherein, The inhibitor maintains the S134 and S166 sites of the CREPT protein in a persistently non-phosphorylated state, thereby keeping the CREPT protein in a non-ubiquitinated state and preventing its degradation in eukaryotic cells; the amino acid sequence of the CREPT protein is SEQ ID No: 4, wherein the eukaryotic cells are human cancer cells or yeast cells, and the method includes: i) The substance to be identified is added to eukaryotic cells expressing CREPT protein that have been synchronized to the G1 phase, then the eukaryotic cells are released and cultured, and the phosphorylation levels of the CREPT protein at sites S134 and S166 are examined while the eukaryotic cells are still viable; or ii) The substance to be identified was incubated with CREPT protein in vitro, and the phosphorylation levels of the S134 and S166 sites of the CREPT protein were examined under the catalytic conditions of Cyclin E / CDK2 kinase. If the phosphorylation levels at sites S134 and S166 of the CREPT protein treated in step i) or ii) decrease relative to the untreated control, the substance is identified as a phosphorylation inhibitor of the CREPT protein; otherwise, the substance is not a phosphorylation inhibitor of the CREPT protein.

17. The method of claim 15 or 16, wherein, The phosphorylation levels at S134 and S166 sites of the CREPT protein were examined using mass spectrometry or immunoprecipitation.

18. The method of claim 15 or 16, wherein, The decrease in phosphorylation levels at S134 and S166 sites of the CREPT protein treated in step i) or ii) relative to the untreated control is defined as a decrease of more than 10%.

19. The method of claim 15 or 16, wherein, The decrease in phosphorylation levels at S134 and S166 sites of the CREPT protein treated in step i) or ii) relative to the untreated control is defined as a decrease of more than 20%.

20. The method of claim 15 or 16, wherein, The decrease in phosphorylation levels at S134 and S166 sites of the CREPT protein treated in step i) or ii) relative to the untreated control is defined as a decrease of more than 30%.

21. The method of claim 15 or 16, wherein, The decrease in phosphorylation levels at S134 and S166 sites of the CREPT protein treated in step i) or ii) relative to the untreated control is defined as a decrease of more than 40%.

22. The method of claim 17, wherein, The immunoprecipitation method includes performing immunoprecipitation using antiphosphorylated antibodies that recognize phosphorylated sites S134 and S166 of the CREPT protein.

23. A method for identifying whether a substance is a phosphorylation-ubiquitination inhibitor of CREPT protein, wherein, The inhibitor maintains the S134 and S166 sites of the CREPT protein in a persistently non-phosphorylated state, thereby keeping the CREPT protein in a non-ubiquitinated state and preventing its degradation in eukaryotic cells; the amino acid sequence of the CREPT protein is SEQ ID No: 4, wherein the eukaryotic cells are human cancer cells or yeast cells, and the method includes: i) The substance to be identified was added to eukaryotic cells expressing the CREPT protein and the eukaryotic cells were cultured. ii) The ubiquitination level of the CREPT protein in the eukaryotic cells was examined using immunoprecipitation. If the ubiquitination level of CREPT protein in the treated eukaryotic cells is decreased compared to the ubiquitination level of CREPT protein in the control eukaryotic cells that have not been treated with the substance, then the substance is identified as an inhibitor of phosphorylation-ubiquitination of CREPT protein; otherwise, the substance is not an inhibitor of phosphorylation-ubiquitination of CREPT protein.

24. The method of claim 23, wherein, The decrease in ubiquitination level of CREPT protein in the treated cells is defined as a decrease of more than 10%.

25. The method of claim 23, wherein, The decrease in ubiquitination level of CREPT protein in the treated cells is defined as a decrease of more than 20%.

26. The method of claim 23, wherein, The decrease in ubiquitination level of CREPT protein in the treated cells is defined as a decrease of more than 30%.

27. The method of claim 23, wherein, The decrease in ubiquitination level of CREPT protein in the treated cells is defined as a decrease of more than 40%.

28. The method of claim 23, wherein, Prior to step i), the method further includes: using the prediction tools SwissTargetPrediction and SEA to design the substance to be identified for CREPT.

29. The method of claim 23, wherein, Step ii) includes quantifying the ubiquitination level of the CREPT protein using an anti-CREPT antibody that recognizes the CREPT protein and a ubiquitin antibody that recognizes ubiquitin.

30. A non-therapeutic method for inhibiting the degradation of CREPT protein in eukaryotic cells, said method comprising: Amino acid mutations were made at positions 134 and 166 of the CREPT protein of SEQ ID NO: 4 to obtain SEQ ID NO: 2, such that when the mutated protein is located in a eukaryotic cell in the G1 terminal phase or the G1 / S transition phase, positions 134 and 166 remain in a non-phosphorylated state and the mutated protein remains in a non-ubiquitinated state, thereby preventing the mutated protein from being degraded. The eukaryotic cells mentioned are human cancer cells or yeast cells.

31. The non-treatment method of claim 30, further comprising introducing an inhibitor selected from SKP2 inhibitors, CUL1 inhibitors, and neddylation inhibitors into the eukaryotic cells expressing CREPT.

32. The non-treatment method as described in claim 31, wherein, The SKP2 inhibitor is a double-stranded siRNA targeting SKP2, with the sequence AAUCUAAGCCUGGAAGGCCUGdTdT; the CUL1 inhibitor is a double-stranded siRNA targeting CUL1, with the sequence UAGACAUUGGGUUCGCCGUdTdT; and the neddylation inhibitor is MLN4924.

33. A protein selected from: 1) A protein obtained by mutating serine at position 166 of SEQ ID No: 4 to alanine; 2) A protein obtained by mutating serine at position 136 of SEQ ID No: 8 to alanine; and 3) The protein obtained by mutating serine at position 174 of SEQ ID No: 8 to alanine.

34. A nucleic acid encoding the protein of claim 33.

35. A vector comprising the nucleic acid of claim 34.

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