WO2024032485A1 - Protéine crept non phosphorylée et non ubiquitinée et son utilisation - Google Patents

Protéine crept non phosphorylée et non ubiquitinée et son utilisation Download PDF

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WO2024032485A1
WO2024032485A1 PCT/CN2023/111172 CN2023111172W WO2024032485A1 WO 2024032485 A1 WO2024032485 A1 WO 2024032485A1 CN 2023111172 W CN2023111172 W CN 2023111172W WO 2024032485 A1 WO2024032485 A1 WO 2024032485A1
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protein
crept
cells
phosphorylation
cell
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常智杰
任芳丽
王银银
宋云皓
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清华大学
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Definitions

  • 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.
  • 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.
  • ORC complex oil recognition complex
  • 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).
  • MCM10, CDC45, RECQL4 ATP-dependent DNA helicase Q4
  • treslin GINS
  • TOPBP1 DNAtopoisomerase 2-binding protein 1
  • DDK and CDK can also phosphorylate several bases in the MCM2-7 complex, causing helicase activation and unwinding of DNA.
  • 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.
  • UPS ubiquitin-proteasome system
  • E1 ubiquitin-activating enzymes
  • E2 ubiquitin-conjugating enzymes
  • E3 ubiquitin ligases
  • 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.
  • CREPT Cell-cycle Related and Expression-elevated
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the phosphorylation inactivating modifications on the 134 and 166 positions may be amino acid mutations and/or chemical modifications.
  • the phosphorylation-inactivating modification at the 134 and 166 positions may be a mutation of serine (S) to alanine (A).
  • 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.
  • 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.
  • the invention provides nucleic acids encoding the proteins of the first and second aspects.
  • the present invention provides a vector comprising the nucleic acid of the third aspect.
  • the invention provides cells comprising the vector of the fourth 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 are described in the first to fifth aspects.
  • 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.
  • 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.
  • the subject can be a mammal, preferably a human.
  • the expression of wild-type CREPT in the cancer cells of the subject can be reduced or eliminated.
  • 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:
  • 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:
  • 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.
  • 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.
  • 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.
  • step i) 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 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.
  • the eukaryotic organism may be yeast, mouse, canine, cat, chicken, toad, zebrafish, Drosophila, nematode or Arabidopsis thaliana.
  • 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.
  • 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.
  • amino acid sequence of the homologous protein may be SEQ ID No: 6.
  • the invention provides a protein selected from:
  • the invention provides nucleic acids encoding proteins of the ninth and tenth aspects.
  • the present invention provides a vector comprising the nucleic acid according to the eleventh aspect.
  • the present invention provides cells comprising the vector of the twelfth 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:
  • sequence of the human CREPT protein is SEQ ID No: 4.
  • 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.
  • the eukaryotic cell may be a human, yeast, mouse, canine, feline, chicken, toad, zebrafish, Drosophila, nematode or Arabidopsis thaliana cell.
  • the present invention provides a method for inhibiting the degradation of CREPT protein in eukaryotic cells, the method comprising:
  • 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.
  • 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 .
  • the phosphorylation inactivating modifications at the 134 and 166 positions may be amino acid mutations and/or chemical modifications.
  • the phosphorylation inactivating modifications at the 134 and 166 positions are Serine (S) is mutated into alanine (A).
  • 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.
  • 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.
  • 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 ⁇ m.
  • Figure 1B Time-lapse microscopy live cell image of cells with GFP-CREPT knock-in. Scale bar, 10 ⁇ m.
  • 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.
  • FIG. 1D Representative fluorescence image of CREPT in DTB-synchronized DLD1 Fucci cells. Scale bar, 10 ⁇ m.
  • 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 ⁇ g/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.
  • FIG. 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 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.
  • FIG. 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.
  • FIG. 3H 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;
  • 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 5F CREPT(S134A/S166A) mutant is lethal to S. cerevisiae.
  • 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.
  • CREPT(WT) represents CREPT wild-type plasmid
  • CREPT(SA) represents CREPT(S134A/S166A) mutant
  • 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 ⁇ m.
  • Figure 7B Representative fluorescence image of CREPT in HeLa cells. Quantitative results of p-RPA2 by immunofluorescence assay (right panel). Scale bar, 10 ⁇ m.
  • 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 ⁇ m.
  • 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
  • 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. (Fig. 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.
  • FIG. 9F Representative fluorescence image of CREPT in DTB-synchronized DLD1 Fucci cells. Scale bar 10 ⁇ m.
  • 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.
  • 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.
  • CREPT 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.
  • Figures 14A-14G show that undegraded CREPT prevents cells from entering S phase;
  • 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.
  • CREPT does not bind directly to chromatin.
  • FIG. 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 ⁇ m.
  • Figure 15B Representative fluorescence image of ⁇ H2AX in HeLa cells.
  • 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.
  • Figure 17 shows the effect of candidate compounds #1 to #5 on the proliferation of DLD1 cells (A) and MGC803 cells (B).
  • human CREPT protein or “CREPT protein” used herein refers to human wild-type CREPT protein unless otherwise specified.
  • 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.
  • homologous protein refers to proteins with homologous amino acid sequences that perform the same or similar functions in different organisms.
  • 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.
  • a method for identifying eukaryotic cells in the late G1 phase or the G1/S transition phase comprising:
  • sequence of the human CREPT protein is SEQ ID No: 4.
  • 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.
  • 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).
  • 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.
  • Figure 4D Figure 5A- Figure 5B
  • 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.
  • persistent activating mutations 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.
  • activating chemical modifiers may include phosphate donors such as acetyl phosphate, phosphoramide salts, carbamoyl phosphates, and sodium pyrophosphate, as well as beryllium trifluoride.
  • phosphate donors such as acetyl phosphate, phosphoramide salts, carbamoyl phosphates, and sodium pyrophosphate, as well as beryllium trifluoride.
  • 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).
  • the present invention provides a non-phosphorylated and non-ubiquitinated CREPT protein variant.
  • 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.
  • 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.
  • the protein variant impaired the survival of yeast at different temperatures ( Figure 13F).
  • 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).
  • 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.
  • 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.
  • 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 .
  • the phosphorylation-inactivating modifications at positions 134 and 166 are amino acid mutations and/or chemical modifications.
  • the phosphorylation inactivation modification at positions 134 and 166 is to mutate serine (S) to alanine (A).
  • the amino acid sequence of the protein is SEQ ID No: 2 , the S134A/S166A double mutant form of CREPT.
  • a tag sequence or a guide sequence can be connected to the N-terminus and/or C-terminus of the protein.
  • the linkage is a covalent linkage.
  • the protein with a tag sequence or leader sequence is a fusion protein.
  • the protein with a tag sequence or leader sequence is a conjugated protein.
  • 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.
  • 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%.
  • 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 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.
  • the target protein can be directly introduced into cells using, for example, the guide sequence described above.
  • the present invention is not limited to this.
  • precision gene editing technology such as prime editors
  • 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.
  • the eukaryotic cell is a human, mouse, canine, feline, chicken, toad, zebrafish, Drosophila, nematode, yeast or Arabidopsis thaliana cell.
  • the present invention provides the use of the above-mentioned proteins, nucleic acids or vectors in the preparation of anti-cancer drugs.
  • 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.
  • the cancer is liver cancer, kidney cancer, gastric cancer, or colorectal cancer.
  • the method includes introducing the above-described nucleic acid into a tumor cell.
  • 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.
  • 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.
  • the pharmaceutical composition is used to treat cancer, such as liver, kidney, stomach or colorectal cancer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • the cancer is melanoma, liver cancer, kidney cancer, gastric cancer, or colorectal cancer.
  • 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
  • step ii) 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.
  • 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).
  • 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.
  • the eukaryotic organism is yeast, mouse, canine, feline, chicken, toad, zebrafish, Drosophila, nematode, or Arabidopsis thaliana.
  • 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.
  • 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.
  • 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.
  • the present invention provides nucleic acids encoding the above-mentioned homologous proteins, vectors containing the nucleic acids, and cells containing the vectors.
  • 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.
  • the eukaryotic cell is a human, yeast, mouse, canine, feline, chicken, toad, zebrafish, Drosophila, nematode, or Arabidopsis thaliana cell.
  • 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,
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the present invention also provides nucleic acids encoding the proteins and vectors containing the nucleic acids.
  • 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: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:8 Amino acid sequence of wild-type Rtt103.
  • Example 1 CREPT degrades in the G1/S transition phase and recovers in the S phase
  • 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.
  • double thymidine block 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).
  • the mRNA level of CREPT remained unchanged from G1/S transition to S phase (Fig. 9E).
  • 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).
  • the inventors treated DLD1 and HeLa cells with autophagy inhibitors CQ and LEU to synchronize DLD1 and HeLa cells to the G1/S phase.
  • E3 ligases targeting CREPT 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.
  • SKP2 belongs to the F-box family of SCF complex proteins
  • CTL1-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.
  • SKP2 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.
  • Myc-CREPT-CID Myc-tagged CID domain
  • Myc CCT domain
  • 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.
  • S134 and S166 regulates CREPT ubiquitination are located in the linker region of CREPT and are highly conserved between CREPT and its ortholog Rtt103 in S. cerevisiae (Fig. 4C).
  • 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.
  • CREPT 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).
  • the MCM hexamer contains 6 MCMs, including MCM2 to MCM7.
  • 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.
  • the inventors performed co-IP experiments using antibodies against CREPT under different cross-linking conditions (Figure 14C).
  • 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.
  • RPA2 replication protein A2
  • ssDNA single-stranded DNA
  • Fig. 7A see ctrl
  • Fig. 7A see ctrl
  • 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 TM , SBS Genetech).
  • SKP2 and CUL1 siRNA duplexes were transfected by Lipofectamine RNAi MAX (Invitrogen), and the oligonucleotide sequences were AAUCUAAGCCUGGAAGGCCUGdTdT and UAGACAUUGGGUUCGCCGUdTdT, respectively.
  • sgRNA Short guide RNA
  • sgRNA sequence is CTCCTTCTCTGAGTCGGCGC.
  • Annealed sgRNA and BbsI-digested Px458 vector were ligated by solution I (Takara) to construct Cas9 DNA shearing plasmid.
  • 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.
  • 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.
  • DTB double thymidine block
  • 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.
  • 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 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.
  • IP samples were centrifuged and eluted using cell lysis buffer 4 times, 10 minutes each time. Add 50 ⁇ l 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).
  • compound #4 is a potent inhibitor of CREPT phosphorylation and ubiquitination.
  • the structural formula of compound #4 is as follows:
  • DLD1 human colorectal adenocarcinoma epithelial cells
  • MGC803 human gastric cancer cells
  • 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.

Abstract

La présente invention concerne une protéine CREPT non phosphorylée et non ubiquitinée et son utilisation. Plus particulièrement, la présente invention concerne une protéine qui est obtenue par modification d'une CREPT ou d'une protéine homologue de celle-ci. La modification permet à la protéine modifiée de maintenir un état de non-phosphorylation et de non-ubiquitination de façon à ne pas être dégradée lorsque la protéine est située dans des cellules eucaryotes à la fin de la phase G1 ou pendant la phase de transition G1/S, de telle sorte qu'un hexamère double-MCM ne peut pas être séparé, ce qui permet d'arrêter le cycle cellulaire, de générer une réponse au stress du génome, et enfin de provoquer la mort cellulaire. La présente invention concerne également un procédé de criblage d'un modificateur non phosphorylé et non ubiquitiné de CREPT, un procédé d'identification pour déterminer si une substance est un inhibiteur de phosphorylation de CREPT, un procédé d'identification de cellules eucaryotes à la fin de la phase G1 ou pendant la phase de transition G1/S à l'aide de CREPT, et un procédé d'induction de l'apoptose de cellules cancéreuses pour traiter des cancers sur la base d'une CREPT non phosphorylée et non ubiquitinée.
PCT/CN2023/111172 2022-08-12 2023-08-04 Protéine crept non phosphorylée et non ubiquitinée et son utilisation WO2024032485A1 (fr)

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