WO2024032485A1

WO2024032485A1 - Non-phosphorylated and non-ubiquitinated crept protein and use thereof

Info

Publication number: WO2024032485A1
Application number: PCT/CN2023/111172
Authority: WO
Inventors: 常智杰; 任芳丽; 王银银; 宋云皓
Original assignee: 清华大学
Priority date: 2022-08-12
Filing date: 2023-08-04
Publication date: 2024-02-15
Also published as: CN116143907A

Abstract

The present invention relates to a non-phosphorylated and non-ubiquitinated CREPT protein and use thereof. Specifically, the present invention relates to a protein that is obtained by modifying CREPT or a homologous protein thereof. The modification enables the modified protein to maintain a non-phosphorylation and non-ubiquitination state so as not to be degraded when the protein is located in eukaryotic cells at the end of the G1 phase or during the G1/S transition phase, so that a double-MCM hexamer cannot be separated, thereby stopping the cell cycle, generating a genome stress response, and finally causing cell death. The present invention also relates to a method for screening a non-phosphorylated and non-ubiquitinated modifier of CREPT, a method for identifying whether a substance is a phosphorylation inhibitor of CREPT, a method for identifying eukaryotic cells at the end of the G1 phase or during the G1/S transition phase by using CREPT, and a method for inducing cancer cell apoptosis to treat cancers on the basis of non-phosphorylated and non-ubiquitinated CREPT.

Description

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

Technical field

The present invention relates to the field of molecular biology, and more specifically, to a CREPT protein that can maintain a non-phosphorylated and non-ubiquitinated state and its application.

Background technique

DNA is replicated in the S phase of the cell cycle. Errors in DNA replication will accumulate in stem cells in organisms and are closely related to cell death and individual aging. Therefore, DNA replication that determines cell fate needs to be tightly controlled. The DNA replication mechanism of eukaryotic cells is very complex. The starting point of DNA replication is located in the G1 phase of the cell cycle, and the pre-RC (pre-replication complex) protein is loaded on all potential starting points of the genome. First, the ORC complex (origin recognition complex, ORC1-6) with ATPase activity is recruited to the origin of replication. Further, the hexameric 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. The assembly of Pre-IC is triggered by DDK (DBF4-dependent kinase) and CDK (Cyclin-dependent kinase) during the G1/S transition period. DDK and CDK phosphorylate several proteins involved in DNA replication, such as MCM10, CDC45, RECQL4 (ATP-dependent DNA helicase Q4), treslin, GINS and TOPBP1 (DNAtopoisomerase 2-binding protein 1). In addition, DDK and CDK can also phosphorylate several bases in the MCM2-7 complex, causing helicase activation and unwinding of DNA. During helicase activation, the MCM2-7 double hexamer loaded on DNA separates into two separate hexamers that continue to unwind at both replication forks starting from the replication origin.

The cell cycle is precisely regulated by the degradation of Cyclin-related proteins and CDK inhibitors mediated by the ubiquitin-proteasome system (UPS). UPS achieves efficient protein degradation by adding ubiquitin to lysine (K) residues of substrate proteins for recognition by the proteasome. 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 that provides specificity for targets. In the G1/S phase, the RING-finger subfamily E3 composed of SKP1, CUL1 and F-box protein (FBP) is connected The SCF complex of the enzyme recognizes the substrate to be degraded. FBP primarily determines the target specificity of the SCF complex. The SCF complex prefers to bind to phosphodegrons, which are phosphorylated substrate motifs that create a surface for recognition by E3 ligases. FBP family member SKP2 (S-phase kinase-related protein 2) is one of the E3 ligases that regulates G1/S transition. The SKP2-CKS1-p27 complex mediates proteasomal degradation of the CDK inhibitor p27 and releases Cyclin E/CDK2 to promote G1/S transition.

The regulation of cell cycle is closely related to the occurrence and development of tumors. In previous studies, our laboratory discovered and cloned a gene related to cell cycle regulation and tumor formation, CREPT (Cell-cycle Related and Expression-elevated), in human cells. Protein in Tumor, Chinese patent number 200510135513.4, its corresponding protein sequence is shown in SEQ ID NO: 4). It was further found that this gene has very conserved homologous genes in a variety of eukaryotes, such as in cells of yeast, mouse, dog, cat, chicken, toad, zebrafish, Drosophila, nematode and Arabidopsis. CREPT protein is known to promote cell proliferation by regulating the expression of Cyclin D1 and B1.

However, the specific mechanism of how CREPT regulates the cell cycle is unclear.

Contents of the invention

The inventors of the present invention have discovered through extensive research that the CREPT protein in human cells promotes cells to enter the S1 phase through degradation during the G1/S transition phase, thus exerting a cell cycle regulatory effect.

Specifically, the inventors found that among multiple cells in the same culture, CREPT was expressed at a very low level in some cells, while maintaining high expression in other cells, and further found that this low expression phenomenon was due to It is caused by the degradation of CREPT during the G1/S transition period, and the degradation of CREPT is crucial for cells to enter the S phase from the G1 phase. The degradation of CREPT is mediated by ubiquitination modification, which depends on the phosphorylation of two sites of CREPT, S134 and S166. The inventor further discovered that if the 134 and 166 positions of the CREPT protein are kept in a non-phosphorylated state during the G1/S transition period and the protein is kept in a non-ubiquitinated state, the CREPT protein cannot be degraded. This kind of protein cannot be degraded. The CREPT protein cannot dissociate from the MCM complex, resulting in cell death.

In addition, the inventor also found that the homologous protein in non-human eukaryotic cells has the same or similar cell cycle regulatory effect and regulatory mechanism as human CREPT, and when the human CREPT protein is expressed in non-human eukaryotic cells, this Cell cycle regulation remains.

Based on the above findings, the inventor completed the present invention.

In a first aspect, the present invention provides a method for phosphorylating SEQ ID No: 4 at positions 134 and 166. The protein obtained by the inactivation modification. The phosphorylation inactivation modification allows the 134 and 166 positions to remain unphosphorylated and remain unphosphorylated when the protein is located in eukaryotic cells at the end of G1 phase or the G1/S transition phase. The protein remains in a non-ubiquitinated state, thereby preventing the protein from being degraded in the eukaryotic cell.

In a first aspect, the eukaryotic cell may be a human, yeast, mouse, canine, cat, chicken, toad, zebrafish, fruit fly, nematode or Arabidopsis thaliana cell, preferably a human cell, more preferably a human cancer cell cell.

In the first aspect, the phosphorylation inactivating modifications on the 134 and 166 positions may be amino acid mutations and/or chemical modifications.

In a first aspect, the phosphorylation-inactivating modification at the 134 and 166 positions may be a mutation of serine (S) to alanine (A).

In a first aspect, the present invention also provides a protein that has (i) more than 90% sequence identity and (ii) the same phosphorylation inactivating modifications at positions 134 and 166 with each of the above-mentioned proteins. In a second aspect, the present invention provides a protein having a tag sequence or a guide sequence connected to the N-terminal and/or C-terminal of the protein of the first aspect.

In a third aspect, the invention provides nucleic acids encoding the proteins of the first and second aspects.

In a fourth aspect, the present invention provides a vector comprising the nucleic acid of the third aspect.

In a fifth aspect, the invention provides cells comprising the vector of the fourth aspect.

In the sixth aspect, the present invention provides the use of the proteins, nucleic acids or vectors described in the first to fifth aspects to inhibit eukaryotic cell proliferation, inhibit eukaryotic cell DNA replication, regulate the cell cycle of eukaryotic cells or kill eukaryotic cells. Cellular reagent applications.

In a seventh aspect, the present invention provides the use of the protein, nucleic acid or vector described in the first to fifth aspects in the preparation of anti-cancer drugs.

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 using a method based on The gene editing technology of CRISPR/Cas9 edits the CRPET gene in the genome of the subject's cancer cells so that the cancer cells express the protein of SEQ ID NO:2. Wherein, the subject can be a mammal, preferably a human. Wherein, 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 cancer cells of the subject can be reduced or eliminated.

In an eighth aspect, the present invention provides a method for screening non-phosphorylated non-ubiquitinated modifiers of CREPT protein, wherein the modifier maintains the S134 and S166 sites of CREPT protein in a continuous non-phosphorylated state, thereby allowing The CREPT protein remains in a non-ubiquitinated state in cells and will not be degraded; the CREPT protein The amino acid sequence is SEQ ID No: 4, and the method includes:

i) Add candidate modifiers that mimic the non-phosphorylated state to eukaryotic cells expressing CREPT protein that are synchronized to G1 phase, then release and culture the eukaryotic cells, and examine the eukaryotic cells while they are alive Phosphorylation levels of S134 and S166 of CREPT protein;

or

ii) incubate the candidate modifier that simulates the non-phosphorylated state with the CREPT protein in vitro, and check the phosphorylation levels of the S134 and S166 sites of the CREPT protein under the catalytic conditions of Cyclin E/CDK2 kinase,

If the phosphorylation levels of the S134 and S166 sites 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 modifiers are screened as non-phosphorylated, non-ubiquitinated modifiers of the CREPT protein.

The present invention also provides a method for identifying whether a substance is a phosphorylation inhibitor of CREPT protein, wherein the inhibitor keeps the S134 and S166 sites of the CREPT protein in a continuous non-phosphorylated state, thereby causing the CREPT protein to function in cells. It maintains a non-ubiquitinated state and will not be degraded; the amino acid sequence of the CREPT protein is SEQ ID No: 4, and the method includes:

i) Add the substance to be identified to eukaryotic cells expressing CREPT protein that are synchronized to the G1 phase, then release and culture the eukaryotic cells, and examine the S134 and S134 of the CREPT protein while the eukaryotic cells are alive. Phosphorylation level at S166; or

ii) Incubate the substance to be identified with the CREPT protein in vitro, and check the phosphorylation levels of the S134 and S166 sites of the CREPT protein under the catalytic conditions of Cyclin E/CDK2 kinase;

If the phosphorylation levels of the S134 and S166 sites 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 substance is identified as a phosphorylation inhibitor of CREPT protein, which is otherwise not a phosphorylation inhibitor of CREPT protein.

In the above method, mass spectrometry or immunoprecipitation can be used to check the phosphorylation levels of S134 and S166 of the CREPT protein; preferably, the immunoprecipitation method can include: using the method to recognize the S134 and S166 of the CREPT protein. Spot phosphorylated anti-phosphorylated antibodies for immunoprecipitation.

The present invention also provides a method for identifying whether a substance is a phosphorylation inhibitor of CREPT protein, wherein the inhibitor keeps the S134 and S166 sites of the CREPT protein in a continuous non-phosphorylated state, thereby causing the CREPT protein to function in cells. Maintains a non-ubiquitinated state and will not be degraded; 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) using an immunoprecipitation method to check the eukaryotic cells The ubiquitination level of CREPT protein; compared with the ubiquitination level of CREPT protein in control cells that have not been treated with the substance, if the ubiquitination level of CREPT protein in the treated cells decreases, for example, decreases by more than 10% , more than 20%, more than 30% or more than 40%, then the substance is identified as a phosphorylation inhibitor of CREPT protein, otherwise the substance is not a phosphorylation inhibitor of CREPT protein. Before step i), the method may further include: using prediction tools SwissTargetPrediction and SEA to design the substance to be identified for CREPT. Additionally, 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.

In a ninth aspect, the invention provides homologous proteins of human CREPT derived from non-human eukaryotes, which have phosphorylation inactivation at the homology sites corresponding to positions 134 and 166 of SEQ ID No: 4 Modification, the phosphorylation inactivation modification makes the 134 and 166 positions corresponding to SEQ ID No: 4 when the homologous protein is located in the G1 end phase or G1/S transition phase cells of the eukaryotic organism. The homology site remains in a non-phosphorylated state and the homologous protein remains in a non-ubiquitinated state, so that the homologous protein will not be degraded in the cell, leading to cell cycle arrest and apoptosis. Death.

In a ninth aspect, the eukaryotic organism may be yeast, mouse, canine, cat, chicken, toad, zebrafish, Drosophila, nematode or Arabidopsis thaliana.

In the ninth aspect, the phosphorylation inactivation modification on the homology site corresponding to positions 134 and 166 of SEQ ID No: 4 may be amino acid mutation and/or chemical modification.

In the ninth aspect, the phosphorylation inactivation modification on the homology site corresponding to positions 134 and 166 of SEQ ID No: 4 may be to mutate serine to alanine.

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

In a tenth aspect, the invention provides a protein selected from:

i) A protein with a tag sequence or guide sequence connected to the N-terminus and/or C-terminus of the protein described in the ninth aspect; or

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.

In an eleventh aspect, the invention provides nucleic acids encoding proteins of the ninth and tenth aspects.

In a twelfth aspect, the present invention provides a vector comprising the nucleic acid according to the eleventh aspect.

In a thirteenth aspect, the present invention provides cells comprising the vector of the twelfth aspect.

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:

1) Prepare eukaryotic cells capable of endogenously expressing a detectably labeled human CREPT protein or its homologous protein in non-human eukaryotes, and culture the eukaryotic cells under conditions that allow the cell cycle to proceed;

2) using the detectable marker to observe or measure the expression level of the human CREPT protein or its homologous protein in the eukaryotic cell;

3) Identify cells with the lowest expression level of the human CREPT protein or its homologous protein as cells in the late G1 phase or G1/S transition phase;

Wherein, the sequence of the human CREPT protein is SEQ ID No: 4.

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

In a fourteenth aspect, the eukaryotic cell may be a human, yeast, mouse, canine, feline, chicken, toad, zebrafish, Drosophila, nematode or Arabidopsis thaliana cell.

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

1) Allowing an inhibitor selected from the group consisting of SKP2 inhibitors, CUL1 inhibitors, neddylation inhibitors and CDK2 inhibitors to enter eukaryotic cells expressing CREPT; and/or

2) Phosphorylation inactivation modification is performed on the 134th and 166th positions of the CREPT protein, so that when the modified protein is located in eukaryotic cells at the end of G1 phase or the G1/S transition phase, the 134th and 166th positions remain inactive. phosphorylation state and the modified protein remains in a non-ubiquitinated state, thereby preventing the modified protein from being degraded.

In this method, the eukaryotic cell can be a human, yeast, mouse, canine, cat, chicken, toad, zebrafish, fruit fly, nematode or Arabidopsis thaliana cell, preferably a human cell, more preferably a human cancer cell .

In this method, the phosphorylation inactivating modifications at the 134 and 166 positions may be amino acid mutations and/or chemical modifications. Preferably, the phosphorylation inactivating modifications at the 134 and 166 positions are Serine (S) is mutated into alanine (A).

In this method, the SKP2 inhibitor can be a double-stranded siRNA against SKP2, the sequence of which is AAUCUAAGCCUGGAAGGCCUGdTdT; the CUL1 inhibitor can be a double-stranded siRNA against CUL1, and the sequence of the neddylation inhibitor can be UAGACAUUGGGUUCGCCGUdTdT; It's MLN4924.

In a sixteenth aspect, the present invention also provides a protein selected from: 1) a protein obtained by mutating serine 166 of SEQ ID No: 4 to alanine; 2) a protein obtained by mutating SEQ ID No: 8 A protein obtained by mutating serine 136 of SEQ ID No: 8 to alanine; 3) A protein obtained by mutating serine 174 of SEQ ID No: 8 to alanine; and 4) Any one of 1) to 3) A protein with more than 90% sequence identity and the same alanine mutation. The invention also provides nucleic acids encoding the proteins and vectors containing the nucleic acids.

Description of drawings

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

Figures 1A-1E show that the expression levels of CREPT oscillate during the cell cycle; (Figure 1A) Representative fluorescence images of CREPT in tumor cells. White circles indicate cells without CREPT expression. Scale bar, 10 μm. (Figure 1B) Time-lapse microscopy live cell image of cells with GFP-CREPT knock-in. Scale bar, 10 μm. (Figure 1C) CREPT expression during the cell cycle; DLD1 cells were synchronized to G1/S phase and released with 2mM double thymidine block (DTB). Cell lysates were collected at the indicated time points and analyzed by immunoblotting. Detect Cyclin A/B1/E, SKP2 to confirm cell cycle progression. CREPT protein levels reach their lowest levels during the G1/S transition period. (Figure 1D) Representative fluorescence image of CREPT in DTB-synchronized DLD1 Fucci cells. Scale bar, 10 μm. (Fig. 1E) Quantification of CREPT, EdU and CDT1 expression levels in fluorescence images (Fig. 1D). Statistical significance (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001); P>0.05, not significant [n.s.], generated by t test.

Figures 2A-2K show that the degradation of CREPT during the G1/S transition phase is dependent on ubiquitin modification; (Figure 2A) CREPT expression in cycloheximide (CHX)-treated cells. CREPT degradation begins 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 h before the second thymidine release. (Fig. 2C) Quantitative immunoblotting results of CREPT in Fig. 2B. (Figure 2D) CREPT can be ubiquitinated. In vivo ubiquitination assays were performed in 293T cells transfected with the indicated plasmids. (Figure 2E) K11-polyubiquitination-mediated degradation of CREPT. In vivo ubiquitination assays were performed in 293T cells transfected with the indicated constructs. (Figure 2F) K11-type ubiquitination accelerates CREPT degradation. HeLa cells were transfected with HA-Ub-K11 plasmid and treated with CHX. (Figure 2G) CREPT immunoblot quantification results in Figure 2F. (Figure 2H) K48-type ubiquitination has no effect on CREPT degradation. HeLa cells were treated with CHX and transfected with HA-Ub-K48 plasmid. (Figure 2I) CREPT immunoblot quantification results in Figure 2H. (Figure 2J) K63 type ubiquitin Chemicalization has no effect on CREPT degradation. HeLa cells were treated with CHX and transfected with HA-Ub-K63 plasmid. (Figure 2K) CREPT immunoblot quantification results in Figure 2J. Statistical significance was calculated by t test (*P<0.05;**P<0.01;****P<0.0001).

Figure 3A-Figure 3M show that CRL1 ^SKP2 directly ubiquitinates CREPT during the G1/S transition phase; (Figure 3A) SKP2 has the highest binding affinity to CREPT. CREPT pull-down proteins in synchronized cells were analyzed by mass spectrometry. The proteins are ranked based on unique peptide 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) Interaction between exogenous CREPT and exogenous SKP2. Cell extracts from HEK293T cells expressing HA-CREPT and Flag-SKP2 were immunoprecipitated with anti-HA beads and subjected to immunoblot analysis with the indicated antibodies. (Figure 3C) Interaction between endogenous CREPT and endogenous SKP2. Cell extracts from DLD1 cells were immunoprecipitated with anti-CREPT beads and subjected to immunoblot analysis with the indicated antibodies. (Figure 3D) Purified prokaryotes expressing SKP2 and CREPT were used in in vitro co-IP assays followed by immunoblot analysis. (Figure 3E) SKP2 mediates ubiquitination of CREPT. In vivo ubiquitination assays were performed in 293T cells transfected with the indicated plasmids, with or without treatment of MG132. (Fig. 3F) and (Fig. 3G) Effect of overexpression of SKP2 on CREPT degradation. (Figure 3H) and (Figure 3I) Effect of knockdown of SKP2 on CREPT degradation. (Fig. 3J) and (Fig. 3K) SKP2 and CREPT levels after synchronization and release of cells into G1/S phase. (Fig. 3L) and (Fig. 3M) SKP2 and CREPT levels after synchronizing cells to M phase and releasing them.

Figures 4A-4G show that the S134A and S166A mutations of CREPT are unable to interact with Ub; (Figure 4A) CREPT is ubiquitinated in the CID domain. In vivo ubiquitination assays were performed in 293T cells transfected with the indicated plasmids and treated with MG132. (Figure 4B) CREPT modification was identified by mass spectrometry analysis. Modified mass spectrometry analysis was performed in 293T cells synchronized to G1/S phase, and the phosphorylation level of the serine (S) site on the CREPT protein was ranked according to #PSMs. (Figure 4C) Amino acid schematic diagram of CREPT and RTT103 in the CREPT 135-170 region. (Fig. 4D) The CREPT S134A/S166A mutant 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 indicated plasmids and harvested for co-IP assays followed by immunoblot analysis. (Figure 4F) Interaction between endogenous CREPT and endogenous CDK2/Cyclin E. Cell extracts from DLD1 cells were immunoprecipitated with anti-CREPT beads and subjected to immunoblot analysis with 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 anti-Flag antibodies from HEK293T cells transfected with the expression vectors of Flag-CDK2 and Flag-Cyclin E1. GST, GST-CREPT and GST-CREPT(S134A/S166A) were purified from prokaryotic expression systems using GST beads. Phosphorylation of GST or GST-tagged CREPT and its mutants was detected with a universal anti-phosphorylation antibody (top). CREPT(SA): CREPT(S134A/S166A).

Figures 5A-5G show that CREPT (S134A/S166A) mutation causes cell apoptosis; (Figure 5A) CREPT (S134A/S166A) mutant causes cell death. HeLa wild-type (Mock) and CREPT knockout (KO) cells were transfected with the indicated plasmids for 48 h and then stained with Annexin V and PI for FACS analysis. (Figure 5B) Statistical analysis of the flow cytometry data in Figure 5A. (Figure 5C) Cell death is triggered by apoptosis. HeLa wild-type (Mock) and CREPT knockout (KO) cells were transfected with the indicated plasmids for 48 hours and then subjected to Western blotting. (Figure 5D) Cell 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 indicated plasmids. (Figure 5E) CREPT(S134A/S166A) inhibits tumor growth. 1×10 ⁶ B16 cells overexpressing the indicated plasmids were injected into C57BL/6 mice (n=3). Mice were sacrificed on day 10 and tumor size was measured. (Fig. 5F) CREPT(S134A/S166A) mutant is lethal to S. cerevisiae. The growth sensitivity of WT, RTT103KO (Rtt103Δ) and human CREPT WT or mutant plasmids to temperature was compared in By4741 RTT103-KO cells. (Fig. 5G) Rtt103(S136A/S174A) mutant is lethal to S. cerevisiae. The growth sensitivity of WT, Rtt103Δ and yeast RTT103WT or mutant plasmids to temperature was compared in By4741RTT103-KO cells. CREPT(SA) represents CREPT(S134A/S166A) mutant, and CREPT(SE) represents CREPT(S134E/S166E) mutant.

Figures 6A-6G show that non-degraded mutant CREPT prevents cells from entering S phase; (Figure 6A) CREPT does not bind to the MCM complex during the G1/S transition phase. ChIP-MS analysis was performed in DLD1 cells to rank proteins according to #PSMs. Cell cycle synchronization with DTB. (Figure 6B) Interaction between exogenous CREPT and exogenous MCM5. Cell extracts from HEK293T cells expressing Myc-CREPT and Flag-MCM5 were immunoprecipitated with anti-Myc beads and subjected to immunoblot analysis with the indicated antibodies. (Figure 6C) Interaction between endogenous CREPT and endogenous MCM5. Cell extracts of DLD1 cells were cross-linked with 1% paraformaldehyde for 10 min. Then, immunoprecipitation was performed with anti-CREPT beads and immunoblot analysis was performed with the indicated antibodies. (Figure 6D) Interaction between CREPT and MCM5 during the cell cycle. DLD1 cells are synchronized to G2/M phase. Cell lysates were collected at the indicated times for endogenous co-IP assay. (Figure 6E) The CREPT(S134A/S166A) mutant pulled down more MCM protein. 293T cells were transfected with the indicated plasmids 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 overexpressing CREPT(S134A/S166A) mutants for 16 hours and then treated with pre- Use method to dye. Scale bar, 10 μm. CREPT(WT) represents CREPT wild-type plasmid, CREPT(SA) represents CREPT(S134A/S166A) mutant, and CREPT(SE) represents CREPT(S134E/S166E) mutant.

Figures 7A-7D show that non-degraded CREPT halts replication forks; (Figure 7A) Representative fluorescence images of CREPT in HeLa cells. Cells were transfected with indicated plasmids for 12 h. Immunofluorescence assay RPA2 quantitative results (right picture). 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. (Fig. 7C) DNA replication rates in WT and S134A/S166A mutants treated with HU. Asynchronous HeLa CREPT KO cells were first treated with 4mM HU for 4 hours. CIdU was then 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. (Fig. 7D) Percentage of aborted replication forks in WT and S134A/S166A mutants during HU block and release. DNA fiber assay was performed in HeLa CREPT KO cells to measure aborted replication forks. Statistical significance (*P<0.05; **P<0.01; ****P<0.0001) was calculated by t-test. CREPT(WT) represents CREPT wild-type plasmid, and CREPT(SA) represents CREPT(S134A/S166A) mutant.

Figure 8 shows a schematic diagram of the mechanism of action of CREPT in regulating the cell cycle.

Figures 9A-9G show that the expression level of CREPT oscillates in the cell cycle, wherein (Figure 9A) Schematic diagram of GFP-CREPT fusion protein knock-in into Hela cell line. GFP is fused after the ATG sequence in EXON1 of CREPT. (Fig. 9B) Western blotting confirmed that GFP-CREPT expression was knocked into cells. (Figure 9C) FACS analysis of synchronized DLD1 cells by PI staining at the indicated release time points. (Fig. 9D) CREPT expression during the cell cycle. HeLa cells are synchronized to G1/S phase and released via DTB. (Figure 9E) Real-time quantitative PCR analysis of CREPT mRNA expression at the indicated release time points after DTB treatment. (Figure 9F) Representative fluorescence image of CREPT in DTB-synchronized DLD1 Fucci cells. Scale bar 10 μm. (Figure 9G) Quantification results of CREPT and CDT1 expression levels in fluorescence images (Figure 9F). Statistically significant (****P<0.0001); P>0.05, not significant [n.s.]), calculated by t test.

Figures 10A-10D show that CREPT is degraded through the ubiquitin pathway during the G1/S transition phase; (Figure 10A) Quantitative immunoblotting results of CREPT in Figure 2A. (Figure 10B) FACS analysis of synchronized DLD1 cells with or without MG132 treatment. (Figure 10C) CREPT degradation is not mediated by autophagy. DLD1 cells were treated with the lysosomal inhibitors chloroquine or leupeptin 6 hours before the second thymidine release. (Figure 10D) HeLa cells were treated with the lysosomal inhibitors chloroquine or leupeptin 6 hours before the second thymidine release.

Figures 11A-11I show that CRL1 ^SKP2 directly ubiquitinates CREPT during the G1/S transition. (Figure 11A) GST-CREPT expression was verified. Gels with purified GST-CERPT protein were stained with Coomassie blue. (Fig. 11B) The expression of His-SKP1 and His-SKP2 was verified. Gels with purified His-SKP1 and His-SKP2 proteins were stained with Coomassie blue. (Fig. 11C) The expression of His-SKP2 was verified. Immunoassays were performed on purified SKP2 protein at different purification steps. (Figure 1 ID) FACS analysis of synchronized DLD1 cells by PI staining at the indicated release time points. (Figure 11E) Activation of cullin ligase is critical for CREPT accumulation. Immunoassay of CREPT in DLD1 cells treated with 1 μM neddylation inhibitor MLN4924 for 0-8 h. Among them, si Ctrl is the control cell, and si SKP2 is the cell in which SKP2 was downregulated with siRNA against SKP2 (Figure 11F). CUL1 interacts with CREPT. 293T cells were transfected with the indicated plasmids and harvested for co-immunoprecipitation (co-IP) assays followed by immunoblot analysis. (Figure 11G) CUL1 mediates ubiquitination of CREPT. In vivo ubiquitination assays were performed in 293T cells transfected with the indicated plasmids, with or without MG132 treatment. (Figure 11H) Overexpressed CUL1 accelerates CREPT degradation. CHX was added to HeLa cells with or without MG132 treatment at the times indicated. (Figure 11I) Knockdown of CUL1 prolonged CREPT half-life. HeLa cells were treated with CHX to knock down CUL1 or N/C via siRNA.

Figures 12A-12E show that the S134A and S166A mutations of CREPT are unable to interact with Ub; (Figure 12A) CREPT ubiquitination is independent of single lysine (K) mutations in the CID domain. In vivo ubiquitination assays were performed in 293T cells transfected with the indicated plasmids. (Figure 12B) CREPT ubiquitination does not depend on a single K mutation in the linker region. In vivo ubiquitination assays were performed in 293T cells transfected with the indicated plasmids. (Figure 12C) CREPT ubiquitination is independent of K. In vivo ubiquitination assays were performed in 293T cells transfected with the indicated plasmids 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 indicated plasmids. (Figure 12E) CREPT is phosphorylated in eukaryotic cells. Purified GST-CREPT in prokaryotic cells (E. coli) and eukaryotic cells (mammalian) was examined by Western blotting using a universal anti-phospho-antibody. p-CREPT(S/T/Y) stands for pan-phosphorylated antibody.

Figures 13A-13F show that CREPT (S134A/S166A) mutation leads to apoptosis; (Figure 13A) Cell growth inhibition and cell death caused by undegraded CREPT mutant protein. HeLa wild-type (Mock) and CREPT knockout (KO) cells were transfected with the indicated plasmids for 48 hours. (Figure 13B) Overexpression of CREPT (S134A/S166A) resulted in cell growth inhibition. Cell viability was determined by CCK-8 assay 48 hours after transfection of 293T and NCM460 cells with the indicated plasmids. (Fig. 13C) and (Fig. 13D) CREPT(S134A/S166A) mutations result in reduced clonogenicity. HeLa wild-type and CREPT knockout cells were transfected with the indicated plasmids for 6 h, and then 1000 cells were counted for clonogenic assay. (Figure 13E) CREPT(S134A/S166A) inhibits tumor growth. Pass 1×10 ⁶ pieces across the table B16 cells harboring the indicated plasmids were injected into C57BL/6 mice (n=3). Mice were sacrificed on day 10 and tumor size was measured. (Figure 13F) Comparison of the growth sensitivity of WT, RTT103KO (Rtt103Δ) and yeast RTT103WT or mutant plasmids to temperature in By4741RTT103WT cells. CREPT(SA): CREPT(S134A/S166A) mutation; CREPT(SE): CREPT(S134E/S166E) mutation.

Figures 14A-14G show that undegraded CREPT prevents cells from entering S phase; (Figure 14A) CREPT interacts with MCM2. 293T cells were transfected with the indicated plasmids and harvested for co-IP. (Figure 14B) CREPT interacts with MCM7. 293T cells were transfected with the indicated plasmids. (Figure 14C) In the presence of cross-linking, CREPT binds to endogenous MCM5. Endogenous co-IP assay in DLD1 cells. Cells were fixed with 1% paraformaldehyde. (Figure 14D) CREPT does not bind directly to chromatin. Representative fluorescence images of MCM5 and EdU in HeLa cells stained with direct fixation and pre-extraction methods. Scale bar, 10 μm. (Figure 14E) FACS analysis of CREPT KO HeLa cells transfected with the indicated plasmids for 12 hours. (Fig. 14F) MCM5 quantification results of immunofluorescence assay in Fig. 6G. (Figure 14G) Nucleus diameter in Figure 6G. WT: CREPT(WT); SA: CREPT(S134A/S166A).

Figures 15A-15D show that non-degraded CREPT halts replication forks; (Figure 15A) Representative fluorescent images of TUNEL signal in HeLa cells. Tunel staining of HeLa wild-type (Mock) and CREPT knockout (KO) cells when overexpressing the indicated plasmids, CREPT (SA): CREPT (S134A/S166A) mutation, CREPT (SE): CREPT (S134E/S166E) mutation . Scale bar, 10 μm. (Figure 15B) Representative fluorescence image of γH2AX in HeLa cells. HeLa CREPT knockout cells at 12 hours overexpression of the indicated plasmids, CREPT(SA): CREPT(S134A/S166A) mutation, CREPT(SE): CREPT(S134E/S166E) mutation. Scale bar, 10 μm. (Figure 15C) Quantitative values of γH2AX in cells stained by immunofluorescence. (Fig. 15D) Number of γH2AX foci of immunofluorescence stained cells. Statistical significance (****P<0.0001) was calculated by t-test.

Figure 16 is the screening results of small molecule inhibitors of CREPT phosphorylation and ubiquitination. #1 to #5 above indicate cells treated with candidate compounds #1 to #5, and the numbers in the middle (0.1 to 1.1) are the relative ubiquitination levels of each sample. SA: CREPT(S134A/S166A).

Figure 17 shows the effect of candidate compounds #1 to #5 on the proliferation of DLD1 cells (A) and MGC803 cells (B).

Detailed ways

definition

The term "human CREPT protein" or "CREPT protein" used herein refers to human wild-type CREPT protein unless otherwise specified.

The terms "CREPT protein variant" and "modified CREPT protein" used herein refer to protein variants obtained by subjecting the wild-type CREPT protein to amino acid mutations and/or chemical modifications.

The term "homologous protein" used herein refers to proteins with homologous amino acid sequences that perform the same or similar functions in different organisms.

The term "phosphorylation-inactivating modification" used in this article means that the modified amino acid residues in the protein can continue to maintain or simulate the non-phosphorylated state in eukaryotic cells. The residues modified by phosphorylation-inactivating cannot be truly modified. Kinase phosphorylation in nuclear cells.

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

After extensive research work, the inventor discovered the mechanism of action of CREPT protein in regulating cell cycle, and thus completed the present invention.

First, the inventors found that the expression level of CREPT protein was in an oscillating state during the cell cycle (Figure 1A). This was due to the degradation of CREPT protein during the G1/S transition phase, which caused its separation from the MCM complex. This drives the cell into S phase and begins DNA replication. Therefore, CREPT protein is at its lowest level during the G1/S transition phase of cells, and as cells enter S phase, the expression level of CREPT protein gradually recovers (Figure 1B and Figure 1D).

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

Wherein, the sequence of the human CREPT protein is SEQ ID No: 4.

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

In one embodiment, the eukaryotic cell is human, yeast, mouse, canine, feline, chicken, toad, zebra Fish, Drosophila, nematode or Arabidopsis thaliana cells, preferably human cells or yeast cells.

The inventor further discovered that this degradation of CREPT protein during the G1/S transition phase of cells relies on ubiquitination-mediated proteasomal degradation. The ubiquitination is catalyzed by the E3 ligase CRL1 ^SKP2 , and the recognition and catalysis of SKP2 Depends on the phosphorylation of CREPT protein at S134 and S166. The inventor simulated the phosphorylation and non-phosphorylation states of CREPT at sites 134 and 166 by mutating these two sites, and found that the CREPT protein that remained unphosphorylated at these two sites remained in a non-ubiquitinated state. , the non-phosphorylated and non-ubiquitinated S134A/S166A double mutant protein will not be degraded at the end of G1 or the G1/S transition phase. This non-degraded CREPT protein variant will not be separated from the MCM complex, resulting in cell Blocking in the late G1 phase or the G1/S transition phase with high expression of the MCM complex results in the cell cycle being unable to enter the S phase and stalling the progression of DNA replication forks, ultimately leading to cell death (Figure 5A-Figure 5G). The S134A mutation alone or the S166A mutant protein can affect ubiquitination to a certain extent (Fig. 4D). On the other hand, the S134E/S166E double mutant protein that simulates the phosphorylation state has no effect on cell ubiquitination and survival (Figure 4D, Figure 5A-Figure 5B), indicating that it is the same as the wild-type CREPT protein at the G1/S transition. degrade normally. The above proves that CREPT protein variants that maintain non-phosphorylation at positions 134 and 166 and maintain non-ubiquitination of the protein cannot be degraded at the end of G1 phase or the G1/S transition phase, leading to cell death.

It is known in the art that the recognition of substrates by ligases responsible for protein ubiquitination relies on the phosphorylation of specific sites on the substrate (usually serine, threonine, tyrosine), and the essence of phosphorylation lies in the protein's The charge changes. Therefore, by simulating the phosphorylation state of the corresponding site (ie, negatively charged), it is often possible to achieve a simulated phosphorylation state of the substrate protein. In this case, the phosphorylated protein can be recognized by ubiquitin ligase, thereby promoting protein degradation. Simulation of phosphorylation/non-phosphorylation is usually achieved using mutations or chemical modifications. For example, persistent activating mutations (i.e., mutations that mimic the phosphorylation state) include mutating residues to aspartic acid (D) or glutamic acid (E), since these two amino acids are the only negatively charged amino acids; The most common persistent inhibitory mutation (that is, a mutation that simulates non-phosphorylation) is to mutate serine to alanine (A), because alanine is positively charged and can continuously inhibit the activity of this residue site. In one aspect, activating chemical modifiers (i.e., chemical modifiers that mimic the phosphorylation state) may include phosphate donors such as acetyl phosphate, phosphoramide salts, carbamoyl phosphates, 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 the protein non-phosphorylated (Maiani et al., 2021; Simoneschi et al., 2021).

Based on the above findings of the inventor, those skilled in the art can reasonably infer that as long as CREPT's 134 If the residue at position 166 can remain non-phosphorylated at the end of G1 or the G1/S transition, the protein will not be ubiquitinated and thus cannot be degraded, leading to cell death.

Therefore, the present invention provides a non-phosphorylated and non-ubiquitinated CREPT protein variant. When the CREPT protein is located in a eukaryotic cell at the end of G1 or G1/S transition phase, 134 and 166 of the CREPT protein The site is able to remain unphosphorylated and the protein remains unubiquitinated, thereby preventing the protein variant from being degraded at this stage.

It is also known that deletion 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). This shows that CREPT plays an important core role in regulating the cell cycle. S residues 134 and 166 of CREPT are located in the connection region of CREPT, constitute a phosphorylated degron recognized by SKP2 ligase, and are highly conserved among homologous proteins (Figure 4C). The above shows that the phosphorylation status of residues 134 and 166 plays a crucial role in regulating the cell cycle.

The inventors also found that the effect of the S134A/S166A mutant form of human CREPT protein on inducing cell apoptosis also exists in other eukaryotes. The inventors exogenously expressed human CREPT S134A/S166A in Saccharomyces cerevisiae. As a result, the protein variant impaired the survival of yeast at different temperatures (Figure 13F). In order to exclude the influence of endogenous Rtt103 (homologous protein of CREPT) in yeast, human CREPT S134A/S166A was also exogenously expressed in the Rtt103-deficient yeast strain, and the results also significantly blocked the growth of yeast (Figure 5F). This shows that the cell cycle regulatory role and regulatory mechanism of CREPT protein and its homologous proteins are universally applicable in eukaryotes. It also shows that exogenous introduction of CREPT mutants can control the cell cycle of eukaryotic cells, preferably tumor cells, and Induces apoptosis.

Therefore, another aspect of the invention provides a protein obtained by phosphorylation inactivation modification of residues 134 and 166 in the CREPT protein sequence (SEQ ID NO: 4), the phosphorylation inactivation modification So that when the protein is located in eukaryotic cells in the late G1 phase or the G1/S transition phase, the 134 and 166 positions remain in a non-phosphorylated state and the protein remains in a non-ubiquitinated state, so that the protein in It is not degraded in eukaryotic cells.

In one embodiment, the eukaryotic cell is a human, yeast, mouse, canine, cat, chicken, toad, zebrafish, Drosophila, nematode or Arabidopsis thaliana cell, preferably a human cell, more preferably a human cancer cell .

In one embodiment, the phosphorylation-inactivating modifications at positions 134 and 166 are amino acid mutations and/or chemical modifications.

In one embodiment, the phosphorylation inactivation modification at positions 134 and 166 is to mutate serine (S) to alanine (A). At this time, the amino acid sequence of the protein is SEQ ID No: 2 , the S134A/S166A double mutant form of CREPT.

In one embodiment, a tag sequence or a guide sequence can be connected to the N-terminus and/or C-terminus of the protein. In one embodiment, the linkage is a covalent linkage. In one embodiment, the protein with a tag sequence or leader sequence is a fusion protein. In one embodiment, the protein with a tag sequence or leader 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, an epitope tag, or the like. 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 that are not based on endocytosis, and peptide sequences that are themselves prone to enter cells via endocytosis. or protein sequence.

The present invention also provides proteins that have the same phosphorylation inactivation modifications at positions 134 and 166 as the above-mentioned proteins and have a sequence identity of more than 90%, more than 95%, preferably more than 98% or more than 99%.

In another aspect, the invention provides nucleic acids encoding the above-mentioned proteins, vectors comprising the nucleic acids, and cells comprising the vectors.

The method of introducing the target protein (such as CREPT S134A/S166A of the present invention) into target cells (such as cancer cells) may include introducing a vector expressing the target protein into the target cells through transfection, infection or other means, or chemical modification may also be used mRNA (modRNA) to achieve the expression of target proteins in target cells. In addition, the target protein can be directly introduced into cells using, for example, the guide sequence described above. However, the present invention is not limited to this. For example, precision gene editing technology (such as prime editors) can be used to directly mutate target sites in the tumor genome (Anzalone, et al., 2019), such as mutating the corresponding bases of CREPT in the genome to make cells express CREPT S134A/ S166A.

In another aspect, the present invention provides the use of the above-mentioned protein, nucleic acid or vector in preparing reagents for inhibiting eukaryotic cell proliferation, inhibiting DNA replication of eukaryotic cells, regulating eukaryotic cell cycle or killing eukaryotic cells.

In one embodiment, the eukaryotic cell is a human, mouse, canine, feline, chicken, toad, zebrafish, Drosophila, nematode, yeast or Arabidopsis thaliana cell.

Since CREPT is highly expressed in most cancers (Li et al., 2021; Lu et al., 2012), on the other hand, the present invention provides the use of the above-mentioned proteins, nucleic acids or vectors in the preparation of anti-cancer drugs.

In yet another aspect, the present invention provides a method of treating cancer, the method comprising administering an effective amount of the above-mentioned protein, nucleic acid or vector to a human subject; alternatively, the method comprising utilizing a CRISPR/Cas9-based gene The editing technology edits the CRPET gene in the genome of the subject's cancer cells so that the cancer cells 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 includes introducing the above-described nucleic acid into a tumor cell. In one embodiment, expression of wild-type CREPT is reduced or eliminated in cancer cells of the subject before, during, or after administration of an effective amount of the protein, nucleic acid, or vector to the subject. In another aspect, the present invention provides a pharmaceutical composition, which includes: the above-mentioned protein, nucleic acid or carrier, and a pharmaceutically acceptable carrier, excipient or medium. In one embodiment, the pharmaceutical composition is used to treat cancer, such as liver, kidney, stomach or colorectal cancer.

Since the phosphorylation status 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-phosphorylation and non-ubiquitination of the CREPT protein. A method of modifying the agent or a method of identifying whether the substance is a phosphorylation inhibitor of the CREPT protein, wherein the modifying agent or inhibitor maintains the S134 and S166 sites of the CREPT protein in a continuous non-phosphorylated state, thereby making the CREPT protein Maintains a non-ubiquitinated state in cells and will not be degraded; especially when the CREPT protein is located in eukaryotic cells at the end of G1 phase or G1/S transition phase, the S134 and S166 sites can remain continuously non-phosphorylated state and the protein can maintain a non-ubiquitinated state, so that the protein will not be degraded; wherein, the amino acid sequence of the CREPT protein is SEQ ID No: 4.

In one embodiment, the method can be performed as follows: adding a candidate modifier that simulates a non-phosphorylated state or a substance to be identified into eukaryotic cells expressing CREPT protein that are synchronized to the G1 phase, and then releasing and culturing the eukaryotic cells, and the phosphorylation levels of S134 and S166 of the CREPT protein were examined when the eukaryotic cells were alive. In another embodiment, the method can be performed as follows: in vitro, a candidate modifier that simulates a non-phosphorylated state or a substance to be identified is incubated with the CREPT protein, and the results are examined under catalytic conditions of Cyclin E/CDK2 kinase. The phosphorylation levels of S134 and S166 of CREPT protein were described. In the above method, if the phosphorylation levels of S134 and S166 of the CREPT protein treated in step i) or ii) decrease relative to the respective untreated control, for example, by more than 10%, more than 20%, or 30% or more than 40%, preferably more than 50%, more than 60%, more than 70%, more than 80% or more than 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 check the phosphorylation status of the S134 and S166 sites of the CREPT protein. The immunoprecipitation method may include: using anti-phosphorylation antibodies that recognize the phosphorylation of S134 and S166 of the CREPT protein. Immunoprecipitation was performed with the body to quantify the phosphorylation level of the S134 site of CREPT protein. Specifically, anti-CREPT antibodies can be used to precipitate CREPT proteins, and the total amount of CREPT proteins can be measured as the background amount. For proteins immunoprecipitated with anti-CREPT antibodies, anti-phosphorylated antibodies can be used to detect phosphorylated proteins and detect them. For quantification, the phosphorylation level can be expressed as the amount of phosphorylated protein relative to the background amount of CREPT protein, and can be normalized to a control.

The invention also relates to the following compounds:

As well as the use of the compound in preparing phosphorylation inhibitors of S134 and S166 sites of CREPT protein or ubiquitination inhibitors of CREPT protein, and its use in preparing drugs for treating cancer. In one embodiment, the cancer is melanoma, liver cancer, kidney cancer, gastric cancer, or colorectal cancer.

Since the phosphorylation of S134 and S166 of CREPT protein is a necessary condition for ubiquitination of the protein, the ubiquitination level can be used to identify phosphorylation ubiquitination inhibitors of 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 keeps the S134 and S166 sites of the CREPT protein in a continuous non-phosphorylated state, thereby making the CREPT protein Maintains a non-ubiquitinated state in cells and will not be degraded; the amino acid sequence of the CREPT protein is SEQ ID No: 4, and the method includes: i) adding the substance to be identified into eukaryotic cells expressing the CREPT protein and Culturing the eukaryotic cells, and ii) using an immunoprecipitation method to examine the ubiquitination level of the CREPT protein in the eukaryotic cells; compared with the ubiquitination level of the CREPT protein in control cells not treated with the substance, 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%, the substance is identified as a phosphorylation inhibitor of CREPT protein, otherwise Said substance is not a phosphorylation inhibitor of CREPT protein. Before step i), the method may further include: using prediction tools SwissTargetPrediction and SEA to design the substance to be identified for CREPT. Additionally, 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. The ubiquitination level can be a relative value of the amount of ubiquitinated protein relative to the total amount of CREPT protein and can be normalized to a control.

In addition, S134 and S166 in CREPT correspond to S136 and S174 in yeast Rtt103 (see Figure 4C), and the inventors found that overexpression of the Rtt103S136A/S174A double mutant protein resulted in a lethal phenotype of Rtt103-deficient yeast (Figure 5G). These results indicate that homologous proteins of human CREPT protein in other eukaryotic cell organisms have the same or similar mechanism of action in regulating the cell cycle.

Therefore, in yet another aspect, the invention provides homologous proteins of human CREPT derived from non-human eukaryotes, which have a loss of phosphorylation at homology sites corresponding to positions 134 and 166 of the human CREPT protein. Inactive modification, the phosphorylation inactivating modification enables the homologous site to remain in an unphosphorylated state and the homologous site remains unphosphorylated when the homologous protein is located in cells at the end of G1 phase or G1/S transition phase of the eukaryotic organism. The homologous protein remains in a non-ubiquitinated state, so that the homologous protein will not be degraded in the cell, leading to cell cycle arrest and apoptosis.

In one embodiment, the eukaryotic organism is yeast, mouse, canine, feline, chicken, toad, zebrafish, Drosophila, nematode, or Arabidopsis thaliana.

In one embodiment, the phosphorylation-inactivating modifications on the homology sites corresponding to positions 134 and 166 of the human CREPT protein are amino acid mutations and/or chemical modifications. In one embodiment, the phosphorylation-inactivating modification at the homology site corresponding to positions 134 and 166 of the human CREPT protein is to mutate serine to alanine. In one embodiment, the homologous protein is Saccharomyces cerevisiae Rtt103 S136A/S174A double mutant protein, whose amino acid sequence is SEQ ID No: 6.

In one embodiment, the N-terminus and/or C-terminus of the homologous protein can be connected with a tag sequence or a leader sequence to form, for example, a fusion protein or a conjugated protein.

In one embodiment, it is also provided that the protein has the same phosphorylation inactivation modification at the homology site and has a sequence identity of more than 90%, more than 95%, preferably more than 98% or more than 99%. of protein.

In one embodiment, the present invention provides nucleic acids encoding the above-mentioned homologous proteins, vectors containing the nucleic acids, and cells containing the vectors.

In one embodiment, the present invention provides the use of the above homologous proteins, nucleic acids or vectors in preparing reagents for inhibiting eukaryotic cell proliferation, inhibiting DNA replication of eukaryotic cells, regulating eukaryotic cell cycle or killing eukaryotic cells.

In one embodiment, the eukaryotic cell is a human, yeast, mouse, canine, feline, chicken, toad, zebrafish, Drosophila, nematode, or Arabidopsis thaliana cell.

In addition to phosphorylation inactivation modifications at positions 134 and 166 of the CREPT protein, the inventors also found that even without modifying CREPT, knocking down or depleting them using siRNA targeting SKP2 or CUL1 can also Significantly inhibited the degradation of wild-type CREPT protein in cells (see Figure 3H and Figure 3I, Figure 11E and Figure 11I).

Therefore, on the other hand, the present invention provides a method for inhibiting the degradation of CREPT protein in eukaryotic cells, the method comprising: 1) using an inhibitor selected from the group consisting of SKP2 inhibitors, CUL1 inhibitors, neddylation inhibitors and CDK2 inhibitors The inhibitor enters the eukaryotic cells expressing CREPT; and/or 2) phosphorylation and inactivation modification of the 134th and 166th positions of the CREPT protein, so that when the modified protein is located at the end of G1 phase or the G1/S transition phase, In eukaryotic cells, the 134 and 166 sites 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 cell may be a human, yeast, mouse, canine, cat, chicken, toad, zebrafish, Drosophila, nematode or Arabidopsis thaliana cell, preferably a human cell, more preferably a human cancer cell cell. In one embodiment, the phosphorylation inactivating modification at the 134 and 166 positions may be an amino acid mutation and/or a chemical modification, preferably, the mutation of serine (S) to alanine (A). In one embodiment, the SKP2 inhibitor can be a double-stranded siRNA directed against SKP2, and its sequence can be AAUCUAAGCCUGGAAGGCCUGdTdT; the CUL1 inhibitor can be a double-stranded siRNA directed against CUL1, and its sequence can be UAGACAUUGGGUUCGCCGUdTdT; the neddylation inhibitor can be MLN4924.

In addition, as shown in Figure 4D, the single mutant proteins S134A or S166A of CREPT can inhibit ubiquitination to a certain extent. Therefore, it can be understood that similar single mutations of these single mutant proteins and homologous proteins in other eukaryotic cells All have the function of inhibiting CREPT degradation.

Therefore, in yet another aspect, the present invention also provides the following proteins: 1) a protein obtained by mutating serine 166 of SEQ ID No: 4 to alanine; 2) a protein obtained by mutating serine 136 of SEQ ID No: 8 A protein obtained by mutating serine 174 of SEQ ID No: 8 to alanine; 3) A protein obtained by mutating serine 174 of SEQ ID No: 8 to alanine; and 4) A protein having 90% affinity with any one of 1) to 3) Proteins with more than % sequence identity and the same alanine mutation. In addition, the present invention also provides nucleic acids encoding the proteins and vectors containing the nucleic acids.

Sequence description:

SEQ ID NO:1 Nucleic acid sequence encoding CREPT S134A/S166A double mutant protein;

SEQ ID NO:2CREPT Amino acid sequence of S134A/S166A double mutant protein;

SEQ ID NO:3 Coding nucleic acid sequence of wild-type CREPT;

SEQ ID NO:4 Amino acid sequence of wild-type CREPT;

SEQ ID NO:5 Nucleic acid sequence encoding Rtt103 S136A/S174A double mutant protein;

SEQ ID NO: 6Rtt103 Amino acid sequence of S136A/S174A double mutant protein;

SEQ ID NO:7 Coding nucleic acid sequence of wild-type Rtt103;

SEQ ID NO:8 Amino acid sequence of wild-type Rtt103.

Example

Example 1. CREPT degrades in the G1/S transition phase and recovers in the S phase

Immunofluorescence (IF) staining experiments were performed on DLD1 and HeLa cells to determine the expression pattern of CREPT in tumor cells. It was found that most tumor cells expressed abundant CREPT, but a few tumor cells were CREPT negative (Figure 1A, see dotted circle). Moreover, the nuclei of CREPT-negative tumor cells were slightly larger and evenly stained with DAPI (Figure 1A, DAPI staining). Therefore, CREPT-negative tumor cells may be due to the loss of CREPT during specific cell cycle stages. To test 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 recovered (Figure 1B). This suggests that CREPT protein levels oscillate during the tumor cell cycle.

To further confirm the oscillation of CREPT protein in the cell cycle, double thymidine block (DTB) was used to synchronize DLD1 and HeLa cells to the G1/S transition phase and release them to different cell cycle time points. Fluorescence-activated cell sorting (FACS) analysis showed that more than 90% of cells were synchronized in G1 phase after DTB treatment (Fig. 9C). Western blotting showed that CREPT protein was almost undetectable during the G1/S transition, but it increased during S phase (Fig. 1C and Fig. 9D, lane 1, 0 and 1 h). This change in CREPT protein was accompanied by opposite trends for Cyclin E and SKP2 but similar to the expression patterns of Cyclin A and Cyclin B1 (Figure 1C and Figure 9D). At the same time, the mRNA level of CREPT remained unchanged from G1/S transition to S phase (Fig. 9E). These results indicate that CREPT protein is degraded during the G1/S transition phase of the tumor cell cycle.

To directly visualize specific time points of CREPT disappearance, a fluorescent 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 (markers of G1/S phase) (Figure 1D, 0 h). As the tumor cell cycle enters the S phase, CREPT protein gradually increases (Figure 1D, 1 hour later). 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 CREPT protein is at its lowest level during the G1/S transition phase of tumor cells.

Example 2. GREPT is degraded by ubiquitination of SKP2 during the G1/S transition phase

In order to elucidate the degradation mechanism of CREPT during the G1/S transition period, the inventors first used cycloheximide (CHX) to block protein synthesis to check the protein stability of CREPT. The results showed that CREPT protein decreased after CHX treatment (Figure 2A, Figure 10A, 10-12h), indicating that CREPT protein was degraded. Furthermore, when MG132, an inhibitor of proteasome-induced degradation, was added during cell cycle synchronization, CREPT protein remained at relatively high levels during the G1/S transition phase (Figure 2B, lanes 7 vs 1, Figure 2C, Figure 10B). These results indicate that the decrease in CREPT expression is due to the induction of its degradation by the proteasome.

In order to confirm whether the degradation of CREPT depends on ubiquitin, the inventors performed a co-immunoprecipitation (Co-IP) experiment to detect the interaction between CREPT and ubiquitin. The results showed that antibodies against Myc precipitated Myc-CREPT and HA-ubiquitin, and MG132 increased the level of precipitated HA-ubiquitin (Fig. 2D), indicating that CREPT can be modified by ubiquitin. Further experiments to characterize the type of ubiquitin linkage found that CREPT was mainly ubiquitinated via K11 linkage rather than K48 linkage (Figure 2E). Notably, K63 ubiquitin was also able to moderately induce CREPT ubiquitination (see Figure 2E, lanes 6 and 12). This result was unexpected because K48 ubiquitin has been widely reported to mediate protein degradation. The inventors further overexpressed different types of ubiquitin together with CREPT in the presence of CHX. The results showed that CREPT was reduced in the presence of K11 but not K48 or K63 ubiquitin (Figure 2F-Figure 2K). In addition, the inventors treated DLD1 and HeLa cells with autophagy inhibitors CQ and LEU to synchronize DLD1 and HeLa cells to the G1/S phase. The results showed that under CQ or LEU treatment, CREPT protein remained degraded 0 hours after DTR (Figure 10C-Figure 10D). These results indicate that the degradation of CREPT during the G1/S transition depends on K11-linked ubiquitination.

To identify E3 ligases targeting CREPT, the inventors synchronized cells to 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 that potentially interact with CREPT, SKP2 has the highest probability of binding to CREPT (Fig. 3A). To find out whether SKP2 is the E3 ligase used for CREPT degradation, the inventors verified their interaction under different conditions. IP experiments showed that antibodies against HA precipitated HA-CREPT and Flag-SKP2 (Figure 3B), indicating that HA-CREPT interacts with Flag-SKP2. Importantly, antibodies against CREPT were observed to precipitate endogenous SKP2 in DLD1 cells (Fig. 3C), indicating 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). The GST sedimentation experiment showed that antibodies against GST simultaneously sedimented GST-CREPT and His-SKP2, indicating that SKP2 directly binds to CREPT (Figure 3D). These results indicate that CREPT and SKP2 Direct interactions both in vivo and in vitro.

To demonstrate the role of SKP2 in CREPT ubiquitination, the inventors examined ubiquitinated CREPT levels. In vivo ubiquitination assay showed that overexpression of SKP2 enhanced polyubiquitination of Myc-CREPT (Fig. 3E). To find out 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 at 8 h after CHX treatment, compared with 10 h in control cells, indicating that overexpression of SKP2 resulted in accelerated degradation of CREPT by 2 h (Figure 3F—figure supplement 3G, 10-12 hours). Notably, addition of MG132 impaired CREPT degradation (Fig. 3F, 12 h of MG132), indicating that SKP2-induced CREPT degradation is proteasome dependent. To confirm the role of SKP2 in CREPT degradation in vivo, endogenous SKP2 was depleted using siRNA in HeLa cells. The results showed that in SKP2-depleted cells, CREPT protein levels remained stable at different time points after CHX treatment (Figure 3H-Figure 3I). These indicate that CREPT ubiquitination is mediated by the F-box family E3 ligase SKP2.

In order to find out the role of SKP2 in CREPT degradation during the tumor cell cycle, the inventors synchronized the cells to the G1/S phase and precipitated the CREPT complex at different release time points. The results showed that during the cell cycle progression, SKP2 was expressed from the G1/S transition phase to the S phase, while the expression of CREPT was lowest in the G1/S phase and then gradually recovered (Fig. 3J, lysate). Notably, antibodies against CREPT strongly precipitated SKP2 from 0 to 4 hours after release, but not at 10 hours, indicating that SKP2 interacts with CREPT specifically in the G1/S phase rather than the G2/M phase ( Figure 3J-Figure 3K, 0-8 hours).

To further determine the time point of CREPT/SKP2 interaction in the tumor cell cycle, cells were synchronized to G2/M phase. Co-IP experiments showed that SKP2 was present at 10 h after release, but CREPT expression was at its lowest level (Fig. 3J). It was also observed that the interaction of CREPT and SKP2 appeared at 10 hours after cell release and continued until 18 hours (Figure 3L-Figure 3M, 10-18h). Flow cytometry analysis showed that cells were still in the G1/S phase 10 hours after release (Fig. 11D). These results indicate that SKP2 interacts with CREPT during the G1/S transition phase and induces its degradation.

Since SKP2 belongs to the F-box family of SCF complex proteins, to find out whether the Cul1-RING E3 ubiquitin ligase (CRL1) complex is involved in CREPT degradation, the inventors used the neddylation inhibitor MLN4924, which inhibits all Cullin-RING ligases. Activation of the complex. Results CREPT protein levels increased in control cells (si Ctrl) but not in MLN4924-treated SKP2-depleted cells (si SKP2) (Fig. 11E). Figure 11E shows that Cullin-RING ligase was gradually inactivated by MLN4924 over time (0-6 hours) in control cells. Although SKP2 always maintains a significant expression level, the ubiquitination level of CREPT gradually decreases and its degradation is inhibited, and its level gradually increases over time; while in cells depleted of SKP2 with siRNA, the ubiquitination and degradation of CREPT are always inhibited. , so it always remains at a significant level. The above results indicate that the deletion of SKP2 can significantly inhibit the ubiquitination and degradation of CREPT.

Correspondingly, immunoprecipitation assay results found that Flag-Cul1 interacted with HA-CREPT (Fig. 11F), and overexpression of Cul1 increased the ubiquitination level of CREPT with or without MG132 treatment (Fig. 11G, see HA band). Consistently, overexpression of Cul1 accelerated CREPT degradation by 2 hours (Fig. 11H), but deletion of Cul1 with siRNA protected CREPT from degradation (Fig. 11I). These results indicate that CRL1 ^SKP2 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.

Example 3. Ubiquitination of CREPT depends on phosphorylation of S134 and S166

To identify the ubiquitin conjugation site on the CREPT protein, the inventors expressed Myc-tagged CID domain (Myc-CREPT-CID) and CCT domain (Myc) using HA-Ub in 293T cells in the presence of MG132. -CREPT-CCT). Western blot analysis showed that Myc-CREPT-CID was ubiquitinated, but Myc-CREPT-CCT was not (Fig. 4A), indicating that ubiquitination occurs within the CID domain. The inventors then mutated all single lysine (K) residues in the CID domain to arginine (R) to map ubiquitination sites, but Western blot analysis showed that all mutations failed to impair ubiquitination levels. (Figures 12A and 12B). The K56R mutation increased ubiquitination levels (Fig. 12A, lane 9), indicating that K56 is an inhibitory residue for CREPT ubiquitination. When all K residues were mutated, Myc-CREPT-CID remained at high ubiquitination levels (Fig. 12C, last lane), however, when all K residues but not K56 were mutated, ubiquitination was at baseline levels ( Figure 12C, lane 4). These results suggest that ubiquitination may occur on other residues, with K56 being the inhibitory residue.

To search for other possible ubiquitin-linking residues, the inventors also created mutations on threonine (T) and cysteine (C) residues in the CID domain, but CREPT ubiquitination levels were significantly lower with these mutations. remains unchanged (Figure 12D). These negative results prompted the inventors to verify whether CREPT is a substrate with phosphorylated degron, because the SCF complex prefers to bind to phosphorylated degron for ubiquitination. To this end, the inventors analyzed the phosphorylation modification of CREPT through mass spectrometry analysis and found that serine 134 (S134) and serine 166 (S166) sites are highly phosphorylated (Figure 4B). Notably, these two S residues are located in the linker region of CREPT and are highly conserved between CREPT and its ortholog Rtt103 in S. cerevisiae (Fig. 4C). To verify whether phosphorylation of S134 and S166 regulates CREPT ubiquitination, the inventors generated different mutants, including S134A, S166A and S134A/S166A double mutations to simulate the non-phosphorylated state of loss of function, and S134E, S166E and S134E/S166E double mutations to simulate continuous phosphorylation. Western blot analysis showed that mutations of S134A, S166A, and S134A/S166A impaired ubiquitination, but other mutations had no effect on ubiquitination (Fig. 4D). This result indicates that phosphorylation of S134 and S166 is essential for ubiquitination of CREPT. In order to confirm whether these two S residues are critical for SKP2 recognition, the inventors performed IP experiments. The results showed that the Myc antibody precipitated the Myc-CREPT(S134E/S166E)/Flag-SKP2 complex, but did not precipitate Myc- CREPT(S134A/S166A)/Flag-SKP2 complex (Fig. 4E), indicating that CREPT(S134A/S166A) failed to interact with SKP2. These results indicate that S134 and S166 are involved in forming the phosphorylated degron of CREPT.

In addition, the amino acid sequence analysis of CREPT showed that both the S134 and S166 sites may be the recognition sites of the Cyclin E-CDK2 complex with sp sequence. CREPT was also observed to interact with Cyclin E1 and CDK2 in IP experiments (Fig. 4F). In vitro kinase assay showed that GST-CREPT was phosphorylated by CDK2 and Cyclin E1, but GST-CREPT (S134A/S166A) was not phosphorylated (Fig. 4G). It was also observed that GST-CREPT was phosphorylated in mammalian cells but not in E. coli (Fig. 12E). These results indicate that the degradation of CREPT in G1/S phase depends on the phosphorylated degron recognized by CRL ^SKP2 at S134 and S166.

Example 4. Undegraded CREPT protein variants cause apoptosis

The inventors found that the mutant protein CREPT (S134A/S166A) always induced cell death (Figure 13A, top panel). 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, whereas overexpression of CREPT(S134A/S166A) resulted in a significant increase in cell death (Fig. 13A, lower panel). FACS analysis showed that expression of CREPT (S134A/S166A) resulted in a significant increase in the death of wild-type cells and CREPT-deficient cells, while wild-type CREPT and CREPT (S134E/S166E) had no effect (Figure 5A-Figure 5B). Moreover, the death rate of CREPT-deficient cells induced by CREPT (S134A/S166A) was much higher than that of wild-type cells induced by CREPT (Figure 5A-Figure 5B). Correspondingly, when CREPT (S134A/S166A) was expressed in wild-type cells or CREPT-deficient cells, cell growth rate (Fig. 5D) and colony formation ability (Fig. 5D and Fig. 13C) were significantly impaired. Western blot analysis showed that overexpression of CREPT (S134A/S166A) induced the expression of the cleaved form of caspase 7, a typical apoptotic executor (Fig. 5C). The above results indicate that undegraded CREPT variants induce apoptosis.

In order to confirm whether the apoptosis-inducing effect of S134A/S166A mutation also exists in other species, we found Akito exogenously expressed CREPT mutant protein in Saccharomyces cerevisiae. As a result, it was observed that CREPT(S134A/S166A) significantly blocked the growth of yeast at different temperatures (Figure 13F). Exogenous expression of human p15RS had no effect on yeast survival (Fig. 13F, bottom). In order to eliminate the influence of endogenous Rtt103 (homologous protein 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), indicating that CREPT(S134A/S166A) is also lethal in yeast. In addition, p15RS also appears to inhibit the growth of Rtt103-deficient yeast, which echoes the inhibitory effect of p15RS in mammalian cells. The above results indicate that double mutations of S134A and S166A in CREPT can induce cell death in mammalian and yeast cells.

Since S134 and S166 in CREPT correspond to S136 and S174 in yeast Rtt103 (see Figure 4C), the inventors generated the corresponding yeast mutant proteins and observed that overexpression of Rtt103 (S136A/S174A) resulted in Rtt103-deficient yeast. lethal phenotype (Figure 5G). These results indicate that disruption of the phosphorylated degron in CREPT and RTT103 leads to cell death.

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

Previous results indicate that the phosphorylated degron of CREPT is critical for the G1/S transition. In order to investigate the mechanism of CREPT phosphorylation on the G1/S transition, the inventors identified proteins that cannot interact with CREPT in the G1/S phase. Specifically, double thymidine blockade was used to synchronize the DLD1 cell line to G1/S. phase, and analyzed the proteins that interact with CREPT through chromatin immunoprecipitation mass spectrometry (ChIP-MS) experiments. Comparing the differences in CREPT-precipitated proteins between asynchronous and synchronous DLD1 cells, it was found that MCM hexameric proteins MCM5 and MCM7 It does not appear to interact with CREPT in the G1/S phase, but it still maintains interaction with CREPT in other phases (Fig. 6A). This result indicates that CREPT may dissociate from the MCM hexamer during the G1/S transition period, which may be due to the degradation of CREPT.

The MCM hexamer contains 6 MCMs, including MCM2 to MCM7. To verify the interaction between CREPT and MCM hexamer, Myc-CREPT and Flag-MCM5 were overexpressed in 293T cells. IP experiments showed that Myc-CREPT and Flag-MCM5 interacted strongly in unsynchronized 293T cells (Figure 6B). Myc-CREPT was also observed to interact with Flag-MCM7 (Fig. 14A) and Myc-CREPT with Flag-MCM2 (Fig. 14B), indicating that CREPT binds to the MCM hexamer. To confirm the endogenous interaction, the inventors performed co-IP experiments using antibodies against CREPT under different cross-linking conditions (Figure 14C). The results showed that CREPT and MCM5 interacted under 10 min of cross-linking (Figure 6C). It was also observed that CREPT and MCM5 interact strongly when synchronized cells were released for 0 to 8 h, but their interaction appeared Attenuated 10 hours after release (Fig. 6D). Notably, CREPT was maintained at minimal levels at 10 hours after releasing cells, corresponding to the emerging SKP2 and Cyclin E1 (Fig. 6D, lysates). These observations suggest that the CREPT/MCM interaction is impaired during the G1/S phase due to CREPT degradation. The inventor further used undegraded mutant CREPT to conduct IP experiments, and the results showed that the interaction between Myc-CREPT (S134A/S166A) and Flag-MCM5 was greater than the interaction between WT protein and Myc-CREPT (S133E/S166E) and Flag-MCM5. stronger (Fig. 6E). This result indicates that the degradation of CREPT leads to its dissociation from the MCM hexamer.

To decipher CREPT/MCM interactions and dissociation, the inventors examined the occupancy of MCM hexamers on chromatin DNA. To this end, different fixation strategies were used to stain MCM5 and CREPT proteins. The results showed that MCM5 stained uniformly in cells when directly fixed with 1% paraformaldehyde (Figure 14D, top panel), but became negative in some cells when pre-extracted with TritonX100 before fixation (Figure 14D, bottom panel). picture). CREPT was not stained in pre-extracted cells, which means that CREPT does not bind directly to DNA. To find out whether the degradation of CREPT regulates the occupancy of MCM hexamers on chromatin DNA, the non-degraded CREPT mutant protein Myc-CREPT (S134A/S166A) was overexpressed and MCM5 was stained under pre-extraction conditions. The results show that when Myc-CREPT (S134A/S166A) is overexpressed, MCM5 is strongly stained (Figure 6F and Figure 6G), which means that double MCM hexamers are loaded onto chromatin DNA and the cell cycle is arrested at the end of G1 phase or G1/S phase. FACS analysis confirmed that expression of CREPT (S134A/S166A) resulted in a significant reduction of cells in S phase (Fig. 14E). Notably, the nuclei of all MCM5-positive cells were enlarged and showed EdU-negative staining in cells overexpressing Myc-CREPT (S134A/S166A) (Fig. 6G, see EdU staining). These results indicate that all MCM5 strongly positive cells with Myc-CREPT (S134A/S166A) overexpression were cell cycle arrested before entering the S phase. Taken together, all results indicate that degradation of CREPT dissociates MCM hexamers and that failure of this dissociation arrests the cell cycle before entering S phase.

Example 6. Undegraded CREPT variants abort DNA replication forks

Undegraded CREPT mutants cause cell death and cell cycle arrest by remaining bound to MCM hexamers, so the inventors verified whether the CREPT-MCM complex induces genomic stress during the G1/S transition phase. Since genomic stress causes DNA damage, the inventors performed TUNEL staining experiments to examine DNA strand breaks. The results showed that overexpression of CREPT (S134A/S166A) resulted in significant TUNEL signals in WT cells and CREPT-deficient cells, while overexpression of WT protein and CREPT (S134E/S166E) showed negative signals (Fig. 15A). Notably, overexpression of CREPT (S134A/S166A) produced a much stronger TUNEL signal in CREPT-deficient cells than in WT cells (Fig. 15A, compare KO with Mock). Mutually Correspondingly, γH2AX foci were also observed to appear in CREPT (S134A/S166A) overexpressing cells (Fig. 15B). These results indicate that undegraded CREPT variants cause DNA damage.

To confirm whether this 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. change. IF staining with an antibody against total RPA2 showed that overexpression of CREPT (S134A/S166A) in HeLa cells resulted in increased RPA2 foci and increased protein levels (Fig. 7A, see ctrl). When cells were treated with HU, an inhibitor of dNTP synthesis that blocks DNA replication, RPR2 foci were greatly increased (Fig. 7A, compare HU and ctrl). Upon further examination of phosphorylated RPA2 (p-RPA2), it was observed that positive foci appeared in cells overexpressing CREPT (S134A/S166A) and were greatly increased under HU treatment (Fig. 7B). These results indicate that undegraded CREPT protein variants contribute to genomic stress.

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 with disruption of DNA replication. Newly synthesized DNA fibers were examined for this purpose. Cells were released after HU blockade and CIdU incorporation was allowed. IF staining results showed that when CREPT (S134A/S166A) was expressed for 12 hours, the length of CIdU-labeled DNA fiber tracks was shortened (Figure 7C). To further confirm this result, double IdU and CIdU labeling experiments were performed. The results showed that the length of IdU-labeled fibers was reduced in CREPT (S134A/S166A) cells compared with WT cells (Fig. 7D). Consistently, CIdU-labeled fibers, which represent replication rates under normal conditions, were shorter in CREPT (S134A/S166A) cells (Fig. 7D). Quantitative analysis showed that more replication-arrested fibers (CIdU ⁺ IdU ^- trajectories) were induced in CREPT(S134A/S166A)-overexpressing cells compared with WT cells (Fig. 7D). These results suggest that non-degraded CREPT variants may cause DNA synthesis defects by affecting stalled replication forks.

Materials and methods

The materials and methods used in the above examples are described below.

Plasmids and siRNA

HA-CREPT, Flag-CREPT, Myc-CREPT, Myc-CREPT-CID, Myc-CREPT-CCT, GSTCREPT, GFP-P15RS and Myc-P15RS plasmids were constructed by the inventor's laboratory. Flag-MCM2 plasmid was a gift from Dr. Kong Daochun (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) was purchased from Addgene. Flag-SKP1、 Flag-SKP2, Flag-CUL1, Flag-MCM5 and Flag-MCM7 were generated from cDNA. CREPT mutated plasmids were generated by site-directed mutagenesis (Muta-direct ^™ , SBS Genetech). SKP2 and CUL1 siRNA duplexes were transfected by Lipofectamine RNAi MAX (Invitrogen), and the oligonucleotide sequences were AAUCUAAGCCUGGAAGGCCUGdTdT and UAGACAUUGGGUUCGCCGUdTdT, respectively.

CRISPR-Cas9 knock-in cell lines

HeLa cells were used to construct CRISPR-Cas9 knock-in cell lines. Short guide RNA (sgRNA) oligonucleotides were designed and optimized according to Zhang Feng's laboratory guidance (http://crispr.mit.edu/). The sgRNA sequence is CTCCTTCTCTGAGTCGGCGC. Annealed sgRNA and BbsI-digested Px458 vector were ligated by solution I (Takara) to construct Cas9 DNA shearing plasmid. On the other hand, the coding sequence of GFP was cloned and ligated into the PCDNA 3.1-HA vector to construct a GFP transcription plasmid. HeLa cells were co-transfected with Cas9 DNA cleavage and GFP transcription plasmids. GFP-positive HeLa cells were sorted by flow cytometry, and individual cells were seeded in 96-well plates to select DNA recombinant clones. One week later, cells were selected for expression of the genomically inserted GFP.

cell cycle synchronization

Cells were synchronized in G1/S phase by double thymidine block (DTB). Cells were treated with 2mM thymidine for at least 18 hours, released in fresh medium for 8 hours, and then treated with 2mM thymidine for at least another 16 hours. Cells were synchronized in G2/M phase by thymidine-nocodazole block. Cells were treated with 2mM 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 times indicated. Verify the cell cycle stage of harvested cells by flow cytometric analysis. For MG132 treatment, MG132 was added to DLD1 cells 4 hours before harvest.

In vitro protein assay

Because SKP2 protein is unstable in prokaryotic expression system. In vitro protein interaction assay was performed using pET22b-SKP1, pET30A-SKP2 and GST-CREPT proteins. Immunoprecipitation assay verified the interaction between GST-CREPT and pET30A-SKP2.

Purified GST, GST-CREPT (wild type) or GST-CREPT (S134A/S166A) were used for in vitro kinase assay. The protein was dissolved in kinase buffer (10mM HEPES (pH7.5), 50mM NaCl, 2mM MgCl) at 30°C. ₂ , 1mM dithiothreitol, 1mM EGTA and 0.1mM ATP) and incubated with Myc-Cyclin E/Myc-CDK2 protein for 30 minutes. Stop the reaction using SDS loading buffer. Phosphorylation of CREPT was detected by Western blotting.

DNA fiber analysis

DNA fiber experiments were performed as previously described (Genois et al., 2021). Briefly, DLD1 cells were first labeled with 50mM CldU, washed twice with PBS and labeled with 250mM IdU. Harvest the cells and suspend them in cold PBS to a concentration of 1 to 1.5 × ¹⁰ cells/ml, then mix 3ul of cell solution with 7ul of spreading buffer (0.5% SDS, 200mM Tris-HCl pH7.4, 50mM EDTA) , and spread on silanized glass slides. Tilt the slide at a 30-60° angle to spread the fibers and leave 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 min and blocked with 3% BSA for 60 min. CldU and IdU were detected with rat anti-BrdU and mouse anti-BrdU for 2 hours at room temperature, followed by conjugation with Alexa488 anti-mouse and Cy3 anti-rat secondary antibodies for 1 hour at room temperature. Slides were fixed with Prolong Gold Antifade Reagent. Fibers were imaged on an Olympus FV3000 confocal microscope using a 60X objective.

Immunofluorescence assay

In the pre-extraction method, to extract soluble proteins, live cells were first treated with 1 mL of permeabilization buffer (containing 0.2% TritonX-100, 20mM HEPES pH=7.4, 100mM NaCl, and 300mM sucrose) for 5 minutes on ice. After removing the permeabilization buffer, cells were fixed with 2% paraformaldehyde for 10 min at room temperature. In the direct fixation method, cells are first fixed with 2% paraformaldehyde for 10 min at room temperature. Then, cells were permeabilized with 0.3% Triton X100 in PBS for 15 min and blocked with 10% BSA in PBST. Fixed cells were incubated with the indicated primary antibodies for 2 h at room temperature or overnight at 4°C. After incubation with fluorescent secondary antibodies and mounting in Prolong Gold, cells were imaged on an Olympus FV3000 confocal microscope using a 60X objective.

Example 7. Screening of CREPT phosphorylation ubiquitination inhibitors

7.1 Prediction of small molecule compounds as potential CREPT phosphorylation ubiquitination inhibitors

The prediction tool SwissTargetPrediction (http://www.swisstargetprediction.ch/) and SEA (Similarity ensemble approach; https://sea.bkslab.org/) were used to jointly predict the small molecule phosphorylation inhibitor of CREPT, and 5 was obtained and synthesized. candidate small molecule compounds #1 to #5. Since the 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.

7.2 Effect of candidate small molecule compounds on CREPT ubiquitination

1) Divide 293T cells into 6cm culture dishes and culture them at 37°C for 24 hours. The plasmids specified in Figure 16 (HA-Ub and/or Myc-CREPT) were transfected into the cells and replaced with fresh medium after 5 hours.

2) 24 hours after transfection, use 1ml RIPA lysis buffer to collect cells and lyse them at 4°C for 1 hour. Centrifuge at 13000rpm and 4℃ for 10 minutes. Take 800μl supernatant, add 50μl protein plus beads and 5μl anti-myc antibody, label it as IP sample, and incubate overnight at 4°C with rotation. Add 50 μl of the supernatant to an equal volume of 2x loading buffer and label it as a lysate sample.

3) IP samples were centrifuged and eluted using cell lysis buffer 4 times, 10 minutes each time. Add 50 μl of 2x loading buffer to the eluted IP sample. Boil IP and lysate samples at 100°C for 10 minutes. SDS PAGE gel running test.

The results are shown in Figure 16. The CREPT wild-type protein without candidate inhibitor treatment can be ubiquitinated by ubiquitin (Ub) (the third lane of Figure 16). The negative control CREPT mutant SA control group cannot detect ubiquitin. Vegetarianization. In the bands to which candidate compounds #1 to #5 were added, ubiquitination of #4 was significantly attenuated (approximately 20% decrease in ubiquitination level relative to the untreated wild-type protein).

The above results demonstrate that compound #4 is a potent inhibitor of CREPT phosphorylation and ubiquitination. The structural formula of compound #4 is as follows:

7.3 Effect of candidate small molecule compounds on cell proliferation

1) Take DLD1 (human colorectal adenocarcinoma epithelial cells) or MGC803 (human gastric cancer cells) in the logarithmic growth phase, digest it with 0.25% trypsin and pipet gently to turn it into single cells, count the viable cells, and use The cell density was adjusted to 1×10 ⁴ cells/L in DMEM culture medium containing 10% fetal calf serum.

2) After adding 10 mL of culture medium and 10 mL of cell diluent in a 1:1 ratio, add 0.2 mL of the mixed solution to each well of the 96-well plate, with a total of 3 replicate wells. Place in a 37°C, 5% _CO2 incubator for 12 hours.

3) Take the above five candidate small molecule compounds #1 to #5 and dissolve them in DMSO. The initial screening concentration of each compound is 10 μM (DLD1 cells) or 5 μM (MGC803 cells). Each compound was run in triplicate; each compound was incubated for 3 days at a concentration of 10 μM (DLD1 cells) or 5 μM (MGC803 cells), and cell proliferation was measured using CCK. Before measurement, each well was replaced with 10 μl of mixed CCK-8 solution and 90 μl of complete culture medium (the wells with corresponding amounts of CCK-8 solution and cell culture medium were added as blank controls). Incubate at 37°C for 3 hours. Measurement Determine the absorbance at a wavelength of 450nm. The results were calculated and statistically drawn and graphed. The results are shown in Figure 17 A (DLD1 cells) and B (MGC803 cells).

It can be seen that compound #4, which is an inhibitor of CREPT phosphorylation and ubiquitination, significantly inhibited cell proliferation, which shows that the test concentration of compound #4 inhibited the degradation of CREPT to a certain extent, leading to apoptosis in some cells. This result is consistent with The results of Examples 2-4 are consistent.

The technical ideas and specific implementations of the present invention have been described above, but it should be understood that the above specific implementations do not limit the scope of the present invention in any way. Those skilled in the art can understand that various modifications and/or changes can be made to the invention shown in the specific embodiments without departing from the essence of the invention, and the modified and/or changed embodiments are also covered by this invention. within the scope of the invention. Accordingly, the embodiments of the present invention are illustrative only and not restrictive.

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Claims

A protein obtained by phosphorylation inactivation modification at positions 134 and 166 of SEQ ID No: 4. The phosphorylation inactivation modification enables the true expression of the protein when it is at the end of G1 phase or the G1/S transition phase. In nuclear cells, the 134 and 166 positions remain in a non-phosphorylated state and the protein remains in a non-ubiquitinated state, thereby preventing the protein from being degraded in the eukaryotic cells, thereby causing cell cycle arrest. and apoptosis.
The protein of claim 1, wherein the eukaryotic cell is a human, yeast, mouse, dog, cat, chicken, toad, zebrafish, Drosophila, nematode or Arabidopsis thaliana cell, preferably a human cell, Human cancer cells are more preferred.
The protein of claim 1, wherein the phosphorylation inactivating modifications at positions 134 and 166 are amino acid mutations and/or chemical modifications.
The protein of claim 1, wherein the phosphorylation-inactivating modification at the 134 and 166 positions is to mutate serine (S) to alanine (A).
A protein having (i) more than 90% sequence identity and (ii) the same phosphorylation inactivation modifications at positions 134 and 166 as the protein according to any one of claims 1 to 4.
A protein with a tag sequence or a guide sequence connected to the N-terminus and/or C-terminus of the protein according to any one of claims 1 to 5.
Nucleic acid encoding the protein of claim 4.
A vector comprising the nucleic acid of claim 7.
A cell comprising the vector of claim 8.
The protein according to any one of claims 1 to 6, the nucleic acid according to claim 7 or the vector according to claim 8 can be used in the preparation of inhibiting eukaryotic cell proliferation, inhibiting DNA replication of eukaryotic cells and regulating eukaryotic cells. Applications in the cell cycle or as reagents to kill eukaryotic cells.
Use of the protein according to any one of claims 1 to 6, the nucleic acid according to claim 7 or the vector according to claim 8 in the preparation of anti-cancer drugs.
A method of treating cancer, the method comprising:

i) administering to a subject an effective amount of the protein of any one of claims 1-6, the nucleic acid of claim 7, or the vector of claim 8; or

ii) Use CRISPR/Cas9-based gene editing technology to edit CRPET in the genome of cancer cells of subjects Gene, so that the cancer cell expresses the protein of claim 4.
The method of claim 12, wherein the subject is a mammal, preferably a human.
The method of claim 12, wherein i) further comprises reducing or eliminating cancer cells in the subject before, during or after administering an effective amount of the protein, nucleic acid or vector to the subject Expression of wild-type CREPT.
Homologous proteins of human CREPT derived from non-human eukaryotes, which have phosphorylation-inactivating modifications at homology sites corresponding to positions 134 and 166 of SEQ ID No: 4, said phosphorylation-inactivating The modification is such that when the homologous protein is located in cells in the G1 end phase or G1/S transition phase of the eukaryotic organism, the homology sites corresponding to positions 134 and 166 of SEQ ID No: 4 are maintained. Non-phosphorylated state and the homologous protein remains in a non-ubiquitinated state, so that the homologous protein will not be degraded in the cell, thereby leading to cell cycle arrest and apoptosis.
The homologous protein of claim 15, wherein the eukaryotic organism is yeast, mouse, dog, cat, chicken, toad, zebrafish, fruit fly, nematode or Arabidopsis thaliana.
The homologous protein of claim 15, wherein the phosphorylation inactivation modification on the homology site corresponding to positions 134 and 166 of SEQ ID No: 4 is an amino acid mutation and/or chemical Grooming.
The homologous protein of claim 15, wherein the phosphorylation inactivation modification on the homology site corresponding to positions 134 and 166 of SEQ ID No: 4 is to mutate serine to alanine acid.
The homologous protein as claimed in claim 15, its amino acid sequence is SEQ ID No: 6.
A protein selected from:

i) A protein with a tag sequence or guide sequence connected to the N-terminus and/or C-terminus of the protein according to any one of claims 15 to 19; or

ii) A protein that has more than 90% sequence identity with the protein of any one of claims 15 to 19 and has the same phosphorylation inactivation modification at the homology site.
Nucleic acid encoding the homologous protein of claim 18 or 19.
A vector comprising the nucleic acid of claim 21.
A cell comprising the vector of claim 22.
A method for identifying eukaryotic cells in late G1 phase or G1/S transition phase, the method comprising:

1) Prepare eukaryotic cells capable of endogenously expressing a detectably labeled human CREPT protein or its homologous protein in non-human eukaryotes, and culture the eukaryotic cells under conditions that allow the cell cycle to proceed;

2) using the detectable marker to observe or measure the expression level of the human CREPT protein or the homologous protein in the eukaryotic cell;

3) Identify cells with the lowest expression level of the human CREPT protein or the homologous protein as cells in the late G1 phase or the G1/S transition phase;

Wherein, the sequence of the human CREPT protein is SEQ ID No: 4.
The method of claim 24, wherein the detectable label is an isotope label, a fluorescent label or a quantum dot label or a label that can further be combined with an isotope label, a fluorescent label or a quantum dot label, preferably GFP.
The method of claim 24, wherein the eukaryotic cell is a human, yeast, mouse, canine, cat, chicken, toad, zebrafish, Drosophila, nematode or Arabidopsis thaliana cell.
A method for screening non-phosphorylation and non-ubiquitination modifiers of CREPT protein, wherein the modifier keeps the S134 and S166 sites of the CREPT protein in a continuous non-phosphorylated state, thereby allowing the CREPT protein to function in eukaryotic cells It maintains a non-ubiquitinated state and will not be degraded; the amino acid sequence of the CREPT protein is SEQ ID No: 4, and the method includes:

i) Add candidate modifiers that mimic the non-phosphorylated state to eukaryotic cells expressing CREPT protein that are synchronized to G1 phase, then release and culture the eukaryotic cells, and examine the eukaryotic cells while they are alive Phosphorylation levels of S134 and S166 of CREPT protein;

or

ii) incubate the candidate modifier that simulates the non-phosphorylated state with the CREPT protein in vitro, and check the phosphorylation levels of the S134 and S166 sites of the CREPT protein under the catalytic conditions of Cyclin E/CDK2 kinase,

If the phosphorylation levels of the S134 and S166 sites 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 modifiers are screened as non-phosphorylated, non-ubiquitinated modifiers of the CREPT protein.
A method for identifying whether a substance is a phosphorylation inhibitor of CREPT protein, wherein the inhibitor keeps the S134 and S166 sites of the CREPT protein in a continuous non-phosphorylated state, thereby maintaining the non-ubiquitin of the CREPT protein in cells. The amino acid sequence of the CREPT protein is SEQ ID No: 4, and the method includes:

i) Add the substance to be identified to eukaryotic cells expressing CREPT protein that are synchronized to the G1 phase, then release and culture the eukaryotic cells, and examine the S134 of the CREPT protein while the eukaryotic cells are alive and the phosphorylation level of S166; or

ii) Incubate the substance to be identified with the CREPT protein in vitro, and check the phosphorylation levels of the S134 and S166 sites of the CREPT protein under the catalytic conditions of Cyclin E/CDK2 kinase;

If the phosphorylation levels of the S134 and S166 sites 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 substance is identified as a phosphorylation inhibitor of CREPT protein, which is otherwise not a phosphorylation inhibitor of CREPT protein.
The method of claim 27 or 28, wherein the step of checking the phosphorylation levels of S134 and S166 sites of the CREPT protein is performed using mass spectrometry or immunoprecipitation.
The method of claim 29, wherein the immunoprecipitation method includes performing immunoprecipitation using an anti-phosphorylated antibody that recognizes the phosphorylation of S134 and S166 sites of the CREPT protein.
A method for identifying whether a substance is a phosphorylation inhibitor of CREPT protein, wherein the inhibitor keeps the S134 and S166 sites of the CREPT protein in a continuous non-phosphorylated state, thereby maintaining the non-ubiquitin of the CREPT protein in cells. 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 the CREPT protein and culturing the eukaryotic cells, and

ii) using an immunoprecipitation method to examine the ubiquitination level of the CREPT protein of the eukaryotic cells;

Compared with the ubiquitination level of CREPT protein in control cells that have not been treated with the substance, if the ubiquitination level of CREPT protein in the treated cells decreases, for example, decreases by more than 10%, more than 20%, or more than 30% or more than 40%, then the substance is identified as a phosphorylation inhibitor of CREPT protein, otherwise the substance is not a phosphorylation inhibitor of CREPT protein.
The method of claim 30, wherein before step i), the method further comprises: using prediction tools SwissTargetPrediction and SEA to design the substance to be identified for CREPT.
The method of claim 30, wherein step ii) includes quantifying the ubiquitination level of the CREPT protein using an anti-CREPT antibody that recognizes the CREPT protein and an ubiquitin antibody that recognizes ubiquitin.
A method for inhibiting the degradation of CREPT protein in eukaryotic cells, the method comprising:

1) Allowing an inhibitor selected from the group consisting of SKP2 inhibitors, CUL1 inhibitors, neddylation inhibitors and CDK2 inhibitors to enter eukaryotic cells expressing CREPT; and/or

2) Phosphorylation and inactivation modification of positions 134 and 166 of the CREPT protein, so that when the modified protein When the protein is located in eukaryotic cells at the end of G1 phase or G1/S transition phase, the 134 and 166 sites remain in an unphosphorylated state and the modified protein remains in a non-ubiquitinated state, thereby making the modified protein The protein is not degraded.
The method of claim 34, wherein the eukaryotic cell is a human, yeast, mouse, dog, cat, chicken, toad, zebrafish, fruit fly, nematode or Arabidopsis thaliana cell, preferably a human cell, Human cancer cells are more preferred.
The method of claim 34, wherein the phosphorylation inactivating modifications at positions 134 and 166 are amino acid mutations and/or chemical modifications.
The method of claim 34, wherein the phosphorylation-inactivating modifications at positions 134 and 166 are mutations of serine (S) to alanine (A).
The method of claim 34, wherein the SKP2 inhibitor is a double-stranded siRNA directed against SKP2 and its sequence is AAUCUAAGCCUGGAAGGCCUGdTdT; the CUL1 inhibitor is a double-stranded siRNA directed against CUL1 and its sequence is UAGACAUUGGGUUCGCCGUdTdT; the neddylation The inhibitor is MLN4924.
A protein selected from:

1) Protein obtained by mutating serine 166 of SEQ ID No: 4 to alanine;

2) Protein obtained by mutating serine at position 136 of SEQ ID No:8 to alanine;

3) Protein obtained by mutating serine 174 of SEQ ID No: 8 to alanine;

4) Protein obtained by mutating serine 134 and serine 166 of SEQ ID No: 4 to cysteine;

5) Protein obtained by mutating serine 166 of SEQ ID No: 4 to cysteine;

6) Protein obtained by mutating serine at position 136 of SEQ ID No:8 to cysteine;

7) Protein obtained by mutating serine at position 174 of SEQ ID No:8 to cysteine;

8) A protein obtained by mutating both serine 136 and serine 174 of SEQ ID No: 8 to cysteine; and

9) A protein that has more than 90% sequence identity with any protein in 1) to 8) and has the same alanine or cysteine mutation.
Nucleic acid encoding the protein of claim 39.
A vector comprising the nucleic acid of claim 40.