Biomarker, detection tool and treatment target for microtubule defects and related diseases thereof

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a biomarker, a detection tool and a treatment target for microtubule defects and related diseases thereof. The invention provides a biomarker for indicating microtubule defects and related diseases and a detection tool, and provides application of a substance for detecting phosphorylation of gamma-microtubule protein in preparation of products for indicating microtubule nucleation defects, indicating mitosis extension and indicating spindle body defects. A gamma-microtubulin antibody with a phosphorylated Ser364 site is used as a detection tool for microtubule defects and related diseases (such as cancers such as oral cancer and neurodegenerative diseases). The invention also provides that the gamma-microtubulin Ser364 site can be used as a treatment target of microtubule dysfunction related diseases. The biomarker, the detection tool and the treatment target spot are novel in conception and high in practicability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a biomarker, a detection tool and a treatment target for microtubule defects and related diseases. Background Art

[0002] Microtubules are important components of the cytoskeleton and play a key role in cell physiological activities such as mitosis and cell movement. They are also the main structure of the mitotic spindle. Microtubule nucleation is the initial stage of microtubule formation. At this stage, tubulin molecules interact with each other to form the "core" of microtubule organization. In human cells, the γ-tubulin ring complex (γ-TuRC) is the main microtubule nucleator, and γ-tubulin is one of the core components of the γ-tubulin ring complex. Under physiological conditions, the activity of the intracellular γ-tubulin ring complex is complexly regulated in vivo to strictly control the nucleation and dynamic properties of microtubules. Microtubule dysfunction has been shown to be highly associated with a variety of neurological diseases and cancers. For example, in gliomas, breast cancer and oral cancer cells, the expression of γ-tubulin is significantly increased, resulting in changes in microtubule nucleation activity and microtubule dysfunction. However, the current research on this microtubule regulatory mechanism is still incomplete, and there is also a lack of a tool that can directly indicate microtubule nucleation activity. Summary of the invention

[0003] In view of the above technical problems, the present invention provides a biomarker, a therapeutic target and a detection tool thereof.

[0004] The first aspect of the present invention aims to provide a use of a substance for detecting phosphorylation of γ-tubulin.

[0005] The second aspect of the present invention aims to provide the use of a γ-tubulin phosphorylation inhibitor.

[0006] The third aspect of the present invention aims to provide an antibody for detecting phosphorylation of γ-tubulin.

[0007] The fourth aspect of the present invention aims to provide a method for preparing an antibody for detecting phosphorylation of γ-tubulin.

[0008] The fifth aspect of the present invention aims to provide a method for detecting γ-tubulin phosphorylation in vitro.

[0009] The sixth application of the present invention is to provide the application of γ-tubulin phosphorylated at the Ser364 site as a target in the development of prevention and treatment of diseases related to microtubule disorders.

[0010] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides the use of a substance for detecting phosphorylation of γ-tubulin in (a1) to (a3):

[0012] (a1) preparing a product indicative of microtubule nucleation defects;

[0013] (a2) preparing products indicative of mitotic elongation;

[0014] (a3) preparing a product indicative of spindle defects;

[0015] The phosphorylation site of the γ-tubulin is Ser364.

[0016] The amino acid sequence of the γ-tubulin is shown in SEQ ID NO.1.

[0017] In some embodiments of the present invention, the substance for detecting phosphorylation of γ-tubulin is a substance for detecting phosphorylation of γ-tubulin at the protein level.

[0018] In some embodiments of the present invention, the substance for detecting phosphorylation of γ-tubulin includes a substance for detecting phosphorylation of γ-tubulin by enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, co-immunoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescence immunochromatography, surface plasmon resonance, or biotin-avidin technology.

[0019] In some embodiments of the present invention, the substance for detecting phosphorylation of γ-tubulin comprises an antibody.

[0020] In some embodiments of the present invention, the antibody comprises at least one of a polyclonal antibody, a monoclonal antibody, a single-chain antibody, a functional antibody fragment, an antibody Fab region, a nanobody, a chimeric antibody, and a multispecific antibody.

[0021] In some embodiments of the present invention, the product includes at least one of a reagent, a kit, a system and a chip.

[0022] The second aspect of the present invention provides the use of a γ-tubulin phosphorylation inhibitor in (b1) to (b3):

[0023] (b1) preparing a product that inhibits microtubule nucleation defects;

[0024] (b2) preparing a product that inhibits mitotic elongation;

[0025] (b3) Preparation of a product that suppresses spindle defects.

[0026] In some embodiments of the present invention, the γ-tubulin phosphorylation inhibitor includes a Cdk1 specific inhibitor; preferably, the Cdk1 specific inhibitor includes RO-3306.

[0027] The third aspect of the present invention provides an antibody for detecting phosphorylation of γ-tubulin, wherein the antibody is produced by immunizing an animal with the polypeptide VALSRK(pS)PYLPS (SEQ ID NO: 2).

[0028] In some embodiments of the present invention, the animal comprises at least one of a rabbit, a mouse, and a rat.

[0029] The fourth aspect of the present invention provides a method for preparing the antibody described in the third aspect of the present invention, using the polypeptide VALSRK(pS)PYLPS to immunize a rabbit, passing the serum through an affinity column coupled to non-phosphorylated γ-tubulin at Ser364 and an affinity column coupled to phosphorylated γ-tubulin at Ser364, and finally collecting the eluate to purify the antibody.

[0030] In some embodiments of the present invention, the preparation method comprises:

[0031] Preparation of immunization antigens: Antigenic peptides (VALSRK(pS)PYLPS) and control peptides (VALSRKSPYLPS) were synthesized and then coupled to hemocyanin (KLH).

[0032] Immunization of animals: Serum is collected after immunization of animals.

[0033] Affinity purification: The antigen peptide and the control peptide are coupled to the affinity column respectively, and balanced with phosphate buffer (PBS, the formula is 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4·2H2O, 2mM KH2PO4, pH7.4); the immunized rabbit serum is filtered through a filter membrane, first passed through the control peptide column, the non-phosphorylation-specific antibodies are removed, and the flow-through is collected; the flow-through liquid is passed through the antigen peptide column, balanced with PBS, and the solution is exhausted. Add antibody eluent to the column, collect the eluent, and dialyze. The fifth aspect of the present invention provides an in vitro method for detecting γ-tubulin phosphorylation:

[0034] Construct the γ-tubulin gene expression vector, transfect and express, separate and purify the protein, and detect the phosphorylation status.

[0035] The gene expression vector comprises: at least one of a pET28a vector, a pRetroX-Puro-tight vector, a pLIB vector, a pBIG1a vector, a pBIG1b vector, a pBIG1c vector, and a pBIG2abc vector.

[0036] The sixth aspect of the present invention provides the use of γ-tubulin phosphorylated at Ser364 as a target in the development of prevention and treatment of diseases related to microtubule disorders.

[0037] The microtubule disorder-related diseases include oral cancer.

[0038] The beneficial effects of the present invention are:

[0039] The present invention utilizes the property that phosphorylation at Ser364 of γ-tubulin is crucial for microtubule nucleation, mitosis and spindle formation, and uses γ-tubulin phosphorylated at Ser364 as a biomarker for indicating microtubule nucleation defects and related diseases. At the same time, antibodies to phosphorylated γ-tubulin at Ser364 are used as detection tools. The Ser364 site of γ-tubulin can be used as a therapeutic target for diseases related to microtubule disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Shown are human γ-tubulin ring complexes purified from HEK293T cells.

[0041] Figure 2 The mass spectrum of the peptide containing phosphorylated γ-tubulin Ser364 in the purified human γ-tubulin ring complex sample is shown; the red arrow indicates the phosphorylation site.

[0042] Figure 3 Shown is an amino acid sequence alignment of γ-tubulin among different species.

[0043] Figure 4 Shown are immunoblots of cell lysates of hTERT RPE-1 (hereafter referred to as RPE-1) stable cell lines transfected with or without doxycycline to induce γ-tubulin-myc wild-type or mutant expression and siRNA.

[0044] Figure 5 Immunofluorescence images of microtubule regeneration experiments are shown. Arrows point to centrosomes. Scale bar is 5 μm.

[0045] Figure 6 Statistical results of microtubule regeneration experiments are shown. Data are presented by the mean ± SEM from three independent experiments (for centrosomal microtubule regeneration experiments of γ-tubulin wild type S364A, S364D, respectively n = 108, 144, 138; for non-centrosomal microtubule regeneration experiments of γ-tubulin wild type and S364D, respectively; n = 80, 83) and unpaired t-tests are used to compare the differences with γ-tubulin wild type, ****, p < 0.0001, ns, not significant.

[0046] Figure 7 Shown are protein gel staining images and immunoblotting of recombinant γ-tubulin ring complex purified from Sf9 cells; the components of the purified complex were examined by Sypro Ruby staining (A) and immunoblotting for MZT1 and MZT2 (B).

[0047] Figure 8 Shown is a gel image of sucrose density gradient centrifugation of recombinant γ-tubulin ring complex / γ-tubulin wild type and S364D mutant; samples were analyzed by SDS-PAGE and Sypro Ruby staining.

[0048] Fig. 9 Negatively stained electron micrographs of recombinant γ-tubulin ring complex / γ-tubulin wild type are shown; scale bar, 200 nm.

[0049] Fig.10 An image of a microtubule nucleation experiment in solution is shown. Immunostaining was performed with anti-α-tubulin antibody 647 conjugate (Merck); scale bar, 10 μm.

[0050] Fig.11 Statistical results of the in-solution microtubule nucleation assay are shown; data are presented as mean ± SEM from three independent experiments (n = 30 for all groups); unpaired t-test was used to compare differences from γ-tubulin ring complex / γ-tubulin wild type. ****, p < 0.0001; ns, not significant.

[0051] Fig.12 Mitotic index of RPE-1 stable cell lines expressing γ-tubulin wild type and S364D mutant is shown; data are presented as mean ± SEM from three independent experiments (n = 40 for all groups); unpaired t-test was used to compare differences from γ-tubulin wild type; ***, p < 0.001.

[0052] Fig.13 Statistical results of mitotic duration of RPE-1 stable cell lines expressing γ-tubulin wild type and Ser364 mutant are shown; data are presented as mean ± SEM from three independent experiments (γ-tubulin wild type, S364A, S364D, n = 60, 89, 79, respectively); unpaired t-test was used to compare the difference with γ-tubulin wild type; **, p < 0.01. ns, not significant.

[0053] Fig.14Statistical results of β-tubulin intensity in mitotic spindles of RPE-1 stable cell lines expressing γ-tubulin wild type and S364D mutant are shown; data are presented by mean ± SEM from three independent experiments (γ-tubulin wild type and S364D, n = 69, 82, respectively); unpaired t-test was used to compare the difference with γ-tubulin wild type. ****, p < 0.0001.

[0054] Fig.15 Immunoblotting and statistical results of Ser364 phosphorylated γ-tubulin in the γ-tubulin ring complex of HEK293T cells in interphase and mitosis are shown; quantification was normalized using the intensity of γ-tubulin; data are presented as mean ± SEM from three independent experiments. Unpaired t-test was used. ****, p<0.0001.

[0055] Fig.16 Shown are immunoblots of in vitro kinase assays using recombinant γ-tubulin ring complex / γ-tubulin wild type and S364A mutant and Cdk1 / cyclin B.

[0056] Fig.17 Shown is an immunoblot of γ-tubulin phosphorylated at Ser364 in HEK293T cells treated with DMSO or 9 μM RO-3306 for 16 hours.

[0057] Fig.18 The statistics of centrosome microtubule intensity in the microtubule regeneration experiment are shown; RPE-1 cells were treated with DMSO or 9 μM RO-3306 for 16 hours before the microtubule regeneration experiment; data are presented by the mean ± SEM from three independent experiments (DMSO and RO-3306 treatment groups of random cells were n=33 and 40, respectively; DMSO and RO-3306 treatment groups of late G2 cells were n=30 and 39, respectively); unpaired t-test was used to compare the differences with the γ-tubulin wild type. ****, p<0.0001. *, p<0.1 (P=0.0315 in this experiment).

[0058] Fig.19 The statistical results of γ-tubulin expression levels in oral cancer tumor tissues and normal tissues in TCGA are shown; the expression levels are expressed as median and minimum to maximum range; the difference in expression between tumor tissues and normal tissues was tested using unpaired t-test. ****, p<0.0001.

[0059] Fig. 20Immunoblotting of γ-tubulin phosphorylated at Ser364 in the γ-tubulin ring complex of human normal oral keratinocytes (HOK) oral cancer cells (HSC3 and HN6) is shown. DETAILED DESCRIPTION

[0060] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0061] The technical idea proposed by the present invention is: using the property that phosphorylation of Ser364 of γ-tubulin is crucial for microtubule nucleation, mitosis and spindle formation, using γ-tubulin phosphorylated at Ser364 as a diagnostic biomarker for indicating microtubule nucleation defects. At the same time, an antibody to phosphorylated γ-tubulin at Ser364 is used as a detection tool. Using the Cdk1-mediated phosphorylation of Ser364 of γ-tubulin discovered by the present invention, using RO-3306, a specific inhibitor of Cdk1, as a method for downregulating the phosphorylation of Ser364 of γ-tubulin, it is used as a therapeutic target for diseases related to microtubule disorders.

[0062] Protein phosphorylation is a common way to regulate cell functions. The present invention separates and purifies the γ-tubulin ring complex from cells, performs mass spectrometry on it, and finds the phosphorylation of the Ser364 site of γ-tubulin. Through microtubule regeneration experiments, the present invention finds that the phosphorylation of the Ser364 site of γ-tubulin has a significant inhibitory effect on the nucleation of centrosomal microtubules and non-centrosomal microtubules. The present invention also uses the in vitro recombinant γ-tubulin ring complex to introduce relevant point mutations of γ-tubulin, and verifies through in vitro microtubule nucleation experiments that the phosphorylation of the Ser364 site of γ-tubulin is necessary for the microtubule nucleation activity of the γ-tubulin ring complex.

[0063] In addition, the present invention also found that cells expressing the γ-tubulin simulated phosphorylation mutant S364D have a higher mitotic index. Further studies have found that the γ-tubulin S364D mutant leads to the elongation of the prometaphase-metaphase in mitosis and the reduction of the microtubule density of the spindle.

[0064] These findings reveal the key role of phosphorylation of Ser364 of γ-tubulin in microtubule nucleation and cell mitosis, suggesting that Ser364 of γ-tubulin may serve as a potential diagnostic biomarker for diseases associated with microtubule disorders.

[0065] The following antibodies were used in the present invention: rabbit polyclonal anti-β-tubulin (Abcam), mouse monoclonal anti-Myc (Cell Signaling), sheep polyclonal anti-TGN46 (Bio-Rad), Hoechst 33258 (Sigma-Aldrich), Anti-α-tubulin antibody 647 conjugate (Merck), 488 Donkey anti-rabbit IgG (H+L), 568 Donkey anti-mouse IgG (H+L), 647 Donkey anti-Sheep IgG (H+L) (Thermo).

[0066] The following antibody vectors were used in the examples of the present invention: pET28a vector was purchased from Novagen #69864, and pRetroX-Puro-tight vector was purchased from Clontech #631128. The following vectors were all from Addgene: pLIB (#80610), pBIG1a (#80611), pBIG1b (#80612), pBIG1c (#80613), and pBIG2abc (#80617). The relevant literature is: Weissmann F, Petzold G, VanderLinden R, et al. biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes. Proc Natl Acad Sci U SA. 2016; 113 (19): E2564-E2569. doi: 10.1073 / pnas.1604935113.

[0067] The inventors generated a γ-tubulin antibody phosphorylated at Ser364, which was generated by immunizing rabbits with the synthetic peptide VALSRK(pS)PYLPS (γ-tubulin 358-369) and purified from rabbit serum. The effectiveness of the antibody was verified by an in vitro kinase experiment. Using this antibody, the present invention found that the phosphorylation level of the γ-tubulin Ser364 site was significantly increased during the cell mitosis phase and was inhibited by the Cdk1-specific inhibitor RO-3306. Treatment with the Cdk1 inhibitor also significantly enhanced the nucleation activity of the cell centrosome microtubules. Thus, the present invention found that Cdk1 is a kinase that can specifically phosphorylate the γ-tubulin Ser364 site.

[0068] The specific phosphorylated antibody of the Ser364 site of γ-tubulin generated by the present invention becomes a detection tool for cell microtubule nucleation and mitosis defects. Subsequent discoveries revealed that Cdk1, as a kinase that specifically phosphorylates the Ser364 site of γ-tubulin, and Cdk1-specific inhibitors have an inhibitory effect on the phosphorylation of Ser364 of γ-tubulin, and can become a therapeutic target for microtubule disorder-related diseases (such as cancers such as oral cancer and neurodegenerative diseases). Among them, oral cancer has a clear connection with microtubule disorder-related diseases. Oral squamous cell carcinoma is a malignant tumor of squamous epithelial origin that occurs in the lips, buccal mucosa, gums, tongue, palate and maxillofacial skin. According to tumor staging and pathological diagnosis, oral squamous cell carcinoma is mainly treated with surgery, radiotherapy, chemotherapy or combined treatment. Among them, chemotherapy is progressing rapidly. Paclitaxel is an important chemotherapeutic drug, and its pharmacological mechanism is to inhibit the movement of microtubule spindles, prevent cell proliferation and self-repair of DNA. [1] At present, more and more studies have shown that microtubule-associated proteins can be used as biomarkers for oral squamous cell carcinoma. Among them, CDK5RAP2 is highly expressed in oral cancer as a γTuRC regulatory protein, affecting the mitotic spindle angle and increasing the number of tumor stem cells. [2] The expression of microtubule-associated protein 1 light chain 3 increased significantly with the increase of malignancy of oral mucosal tissue [3] .

[0069] [1] Ji Zhibo, Chen Ruiguo, Yang Hao, Ma Kun, Hou Jun [review], Sun Lei [review]. Current status and research progress of chemotherapy drugs for oral squamous cell carcinoma. Journal of Anhui Medical University. 2024Apr; 59(4).

[0070] [2]Shen Y, Chen Y, Lin Y, et al. CDK5RAP2 is a Wnt target gene and promotes stemness and progression of oral squamous cell carcinoma. Cell DeathDis. 2023; 14(2):107. Published 2023Feb11.doi:10.1038 / s41419-023-05652-z.

[0071] [3] Dong Xiaolin, Liu Zhiwen. Expression and clinical significance of microtubule-associated protein 1 light chain 3 and target of rapamycin in oral leukoplakia and oral squamous cell carcinoma. West China Journal of Stomatology. 2018;36(6):613-618.doi:10.7518 / hxkq.2018.06.006.

[0072] Example 1 Identification of phosphorylation sites of human γ-tubulin

[0073] 1. Experimental Methods

[0074] 1) Construction of gene expression vector

[0075] In order to clone the recombinant γ-tubulin ring complex, this example uses an improved version of the biGBac cloning system (disclosed in Weissmann F, Peters JM. Expressing Multi-subunit Complexes UsingbiGBac. Methods Mol Biol. 2018; 1764: 329-343. doi: 10.1007 / 978-1-4939-7759-8_21). First, a single gene is cloned into the pLIB vector by Gibson assembly. Next, up to 4 genes (GECs) in the constructed pLIB are combined and cloned into pBIG1, and Gibson assembly is performed using DNA Linker to form a multi-gene cassette (PGC). The PGC is then further transferred to the pBIG2 vector by PmeI (Thermo) digestion and Gibson assembly. Finally, up to 25 genes can be expressed simultaneously using this system. The flowchart for cloning the recombinant γ-tubulin ring complex is shown below. Fig.19 All plasmids constructed and used in this study are listed in Table 1.

[0076] For other plasmids, site mutations were introduced by primer design. This example uses PCR to amplify each strand of the plasmid and fuses them together by denaturation. All vectors were amplified by transformation into E. coli DH5α. MiniPlus TM Plasmid DNA extraction system (Viogene) was used to extract plasmid. The plasmid vectors involved in the present invention are shown in Table 1.

[0077] Table 1

[0078]

[0079]

[0080] Among them, the inserted fragments shown are all full-length genes, and the order is from N-terminus to C-terminus, such as GCP6-TEV-Twinstrep, which means that there are TEV and Twinstrep sequences in the downstream of GCP6 in the order of NC-terminus. γ-tubulin WT / S364A / S364D represents γ-tubulin WT, γ-tubulin S364A and γ-tubulin S364D, and the same applies to others. . The mutants shown are marked with the mutation sites (such as γ-tubulin S364D represents that the S364 site of γ-tubulin has mutated to D).

[0081] The sequence of γ-tubulin is as follows: MPREIITLQLGQCGNQIGFEFWKQLCAEHGISPEG IVEEFATEGTDRKDVFFYQADDEHYIPRAVLLDLEPRVIHSILNSPYAKLYNPENIYLSEHGGGAGNNWASGFSQGEKIHEDIFDIIDREADGSDSLEGFVLCHS IAGGTGSLGSYLLERLNDRYPKKLVQTYSVFPNQDEMSDVVVQPYNSLLTLKRLTQNADCVVVLDNTALNRIATDRLHIQNPSFSQINQLVSTIMSASTTTLRY PGYMNNDLIGLIASLIPTPRLHFLMTGYTPLTTDQSVASVRKTTVLDVMRRLLQPKNVMVSTGRDRQTNHCYIAILNIIQGEVDPTQVHKSLQRIRERKLANFIP WGPASIQVALSRKSPYLPSAHRVSGLMMANHTSISSLFERTCRQYDKLRKREAFLEQFRKEDMFKDNFDEMDTSREIVQQLIDEYHAATRPDYISWGTQEQ(SEQ ID NO: 1).

[0082] 2) Cell culture, synchronization and transfection

[0083] HEK293T and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco), and RPE-1 cells were cultured in DMEM / Ham's F12 medium containing 10 v / v% fetal bovine serum (Gibco) and 1 v / v% penicillin-streptomycin (Gibco). The cells were cultured at 37°C in a humidified environment with 5% carbon dioxide.

[0084] To synchronize HEK293T cells, cells were treated with 1.6 μg / mL aphidicolin (Sigma) or 100 ng / mL nocodazole (Sigma) to arrest them in interphase or mitosis, respectively.

[0085] HEK293T or HeLa cells were transfected with polyethyleneimine (PEI). After transfection, cells were kept in culture for 48 hours for protein expression.

[0086] 3) Bacterial expression of FLAG-2TEV-SBP-CDK5RAP2 1-100 recombinant protein

[0087] The pET28a FLAG-2TEV-SBP-CDK5RAP2 1-100 plasmid was transformed into the BL21 strain, and a single clone of the transformed BL21 cells was cultured with shaking overnight at 37°C. The cells were inoculated at 1:100 and expanded with shaking at 37°C until the OD value reached 0.6, and then 0.2 mM IPTG was added to induce protein expression at 18°C ​​overnight.

[0088] Cells were collected and sonicated in lysis buffer (50 mM sodium hydrogen phosphate, pH 8.0, 500 mM sodium chloride, 10 mM imidazole, 5 mM 2-mercaptoethanol, 1 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride). 1% Triton X-100 was added to the lysate and incubated for 30 minutes. The lysate was centrifuged and clarified and incubated with Ni-NTA agarose gel beads for 2 hours. The beads were washed with wash buffer (50 mM sodium hydrogen phosphate, pH 8.0, 500 mM sodium chloride, 20 mM imidazole, 5 mM 2-mercaptoethanol, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride and 0.1% Triton X-100). Finally, the protein was eluted with a wash buffer containing 500 mM imidazole.

[0089] 4) Purification of γ-tubulin ring complex from mammalian cells

[0090] Streptavidin magnetic beads (Invitrogen) were incubated with bacterially expressed FLAG-2TEV-SBP-CDK5RAP2 1-100 recombinant protein at 4°C overnight in advance. HEK293T cells were collected and washed with phosphate-buffered saline (PBS), and then lysed in cell lysis buffer (50mM HEPES, pH 7.4, 150mM sodium chloride, 1mM magnesium chloride, 1mM ethylene glycol bisaminoethyl ether tetraacetic acid (EGTA), 0.5% Triton X-100, 1mM dithiothreitol (DTT), 1× complete protease inhibitor cocktail (Roche), 10mM sodium fluoride, 10mM β-glycerophosphate and 0.2mM guanosine triphosphate). The cell lysate was centrifuged at 16,100g for 20 minutes at 4°C. The supernatant was collected for the next step.

[0091] The beads were washed with wash buffer (50 mM HEPES, pH 7.4, 150 mM sodium chloride, 1 mM magnesium chloride, 1 mM EGTA, 0.1% Triton X-100, 1 mM DTT, 1× complete protease inhibitor cocktail (Roche), 10 mM sodium fluoride, 10 mM β-glycerophosphate, and 0.2 mM guanosine triphosphate) and incubated with cell lysate for 2 hours at 4° C. The beads were further washed 4 times, and then the proteins were eluted with 20 mM biotin.

[0092] 5) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and protein blotting

[0093] The proteins obtained in the above steps were loaded as samples onto sodium dodecyl sulfate polyacrylamide gels of different concentrations and electrophoresed in SDS-PAGE electrophoresis buffer (25mM Tris, pH 8.3, 192mM glycine, 0.1% sodium dodecyl sulfate). After electrophoresis, the proteins were separated according to molecular weight. The proteins were then electrotransferred from the gel to a nitrocellulose membrane (Pall Corporation) in a protein transfer buffer (25mM Tris, pH 8.3, 192mM glycine, 20% methanol) at 4°C. After protein transfer, the membrane was blocked in a blocking buffer (5% skim milk in tris-buffered saline and Tween 20 (TTBS); the formula of TTBS is: 25mM Tris, pH 7.5, 150mM sodium chloride, 0.1% Tween 20) at room temperature for 1 hour to avoid nonspecific binding of the membrane. The membrane was incubated with the primary antibody in TTBS at 4°C overnight, and then washed 3 times with TTBS at room temperature for 10 minutes each time. The membrane was then incubated with the secondary antibody for 2 hours at room temperature. After washing, chemiluminescent substrate (ECL) (PerkinElmer) was added to the membrane. Protein bands were observed using the Chemidoc imaging system (Bio-Rad), and then the intensity of protein bands was quantified using Image Lab.

[0094] 6) Mass spectrometry analysis

[0095] Protein samples were trypsin digested in solution or after SDS-PAGE and gel staining, the γ-tubulin band was cut out for in-gel digestion. The digested peptides were desalted by Pierce C18 Spin Tips (Thermo Scientific) and analyzed by timsTOF Pro (Bruker) mass spectrometer. The mass spectrometry data were analyzed using PEAKS Studio software.

[0096] 2. Experimental results

[0097] In order to study the phosphorylation of γ-tubulin in human cells, the present invention first purified the γ-tubulin ring complex from the HEK293T cell line. Figure 1 As shown in Figure 2, after protein gel electrophoresis and Coomassie brilliant blue staining, bands of all core components of the γ-tubulin ring complex can be seen in the purified sample. Next, the sample was analyzed using a timsTOFPro mass spectrometer (Bruker), as shown in Figure 2. Figure 2 As shown, phosphorylation of Ser364 of γ-tubulin was detected.

[0098] Example 2 Phosphorylation of γ-tubulin Ser364 inhibits microtubule nucleation activity

[0099] 1. Experimental Methods

[0100] 1) Preparation of RPE-1Tet-on stable cell line

[0101] The pRetroX-Puro-tight vector containing γ-tubulin-myc-His (γ-tubulin wild type) or S364A / S364D mutations was transfected into Phoenix-AMPHO cells to produce lentivirus. Parental RPE-1Tet-on cells were infected with the corresponding viruses and selected with puromycin to select single clones of surviving cells.

[0102] hTERT RPE-1 cells were purchased from ATCC CRL-4000. hTERT RPE-1 cells were infected with pRetroX-Tet-On Advanced (Clontech) virus and introduced with tetracycline transcriptional regulatory system (Tet-on), thereby obtaining RPE-1Tet-on parental cells.

[0103] By adding doxycycline, the RPE-1Tet-on stable cell line was induced to express the corresponding protein. TM RNAiMAX transfection reagent (Invitrogen) was used to transfect γ-tubulin siRNA (5′-CGCAUCUCUUUCUCAUAUA-3′, SEQ ID NO: 3) to knock down endogenous γ-tubulin.

[0104] 2) Cell microtubule regeneration experiment

[0105] Cells grown on coverslips of cell culture plates were placed on an ice-water mixture for 1 hour to depolymerize microtubules, and then the plates were immediately transferred to 37° C. to regenerate their microtubules for 85 seconds. After regeneration, cells were fixed with Paraformaldehyde (PFA) fixation buffer (60 mM PIPES-potassium, 25 mM HEPES, 4 wt% PFA, 2 wt% sucrose, 1 mM magnesium chloride, 10 mM EGTA, 0.5 v / v% Triton X-100) at room temperature for 15 minutes, and then the cells were immunostained.

[0106] 3) Immunostaining and microscopy

[0107] Cells grown on coverslips were fixed with methanol at –20°C or PFA fixation buffer at room temperature. After fixation and rinsing, immunostaining was performed using rabbit polyclonal anti-β-tubulin (Abcam), mouse monoclonal anti-Myc (Cell Signaling), sheep polyclonal anti-TGN46 (Bio-Rad), Hoechst 33258 (Sigma-Aldrich) and corresponding secondary antibodies; coverslips were stained. Immunofluorescence images were acquired using a microscope (Axio Observer Z1, Carl Zeiss) equipped with an ORCA-Flash 4.0 camera (Hamamatsu Photonics) and processed using ZEN microscope software (2011 version, Carl Zeiss).

[0108] Live cell imaging was performed on a spinning disk microscope (Axio Observer ZI, Carl Zeiss; laser-based spinning disk system, Andor). Live cell DNA was stained using the SiR-DNA kit (Spirochrome).

[0109] 4) Preparation of baculovirus

[0110] First, the pBIG2 recombinant vector containing the wild-type γ-TuRC or the γ-TuRC containing the S364A and S364D mutations of γ-tubulin was transformed into DH10EMBacY Escherichia coli and cultured on blue-white screening plates (containing 50 μg / mL kanamycin, 10 μg / mL tetracycline, 7 μg / mL gentamicin, 50 μg / mL Bluo-Gal LB agar, 0.1 mM IPTG) to generate baculovirus shuttle vectors. The recombinant shuttle vector was isolated by isopropanol precipitation and PEI was used to (Polysciences) was transfected into Sf9 cells. The transfected cells were cultured for 96 hours for virus amplification, and the first generation virus (V0) was harvested from the culture supernatant. Sf9 cells were infected again and cultured for 96 hours to collect V1 to further amplify V0. 500 mL of 2×10 6 Sf9 cells at 10 cells / mL were infected with V1 and cultured for 96 hours, and finally the cell pellets were collected and washed with PBS.

[0111] 5) Purification of recombinant γ-tubulin ring complex from Sf9 cells

[0112] The cells in step 4) were suspended in a lysis buffer (50 mM HEPES, pH 7.4; 150 mM KCl; 5 mM MgCl; 1 mM EGTA; 1 mM DTT; 0.1 mM GTP; 0.2% IGEPAL CA 360; 1× cOmplete TM EDTA-free protease inhibitor cocktail (Roche)) and ground with a tissue grinder. The cell lysate was clarified by centrifugation at 18,000 rpm for 30 min at 4°C, and the supernatant was filtered through a 0.45 μm PES syringe filter and Agarose beads (IBA Lifesciences) were incubated for 2 hours at 4°C. The beads were washed with wash buffer (50 mM HEPES; 150 mM KCl; 5 mM MgCl; 1 mM EGTA; 1 mM DTT; 0.1 mM GTP), and finally the proteins were eluted twice with elution buffer (wash buffer containing 2.5 mM desthiobiotin), each elution at 4°C for 1 hour.

[0113] 6) Microtubule nucleation assay of recombinant γ-tubulin ring complex

[0114] Different concentrations of clarified recombinant γ-tubulin ring complexes were mixed with 15 μM α / β-tubulin and 2 mM GTP in nucleation buffer (80 mM potassium-PIPES, pH 6.8; 1 mM magnesium chloride; 1 mM EGTA) containing 150 mM potassium chloride. The reaction solution was placed on ice for 10 minutes and transferred to 37°C for 5 minutes. Fixative (1% glutaraldehyde in potassium chloride nucleation buffer) was added and incubated at room temperature for 2 minutes to terminate the reaction, and then the reaction solution was diluted with cold potassium chloride nucleation buffer.

[0115] Prepare 15% glycerol buffer in a 2.2 mL centrifuge tube. Place a coverslip on the polytetrafluoroethylene pad of the centrifuge tube, carefully add the reaction solution on top of the glycerol buffer, and centrifuge at 100,000 g and 4 °C for 15 minutes. Then use anti-α-tubulin antibody Coverslips were stained with 647 conjugate (Millipore).

[0116] 2. Experimental results

[0117] like Figure 3As shown, the Ser364 site of γ-tubulin is highly conserved in evolution, suggesting that phosphorylation of this site may have important functions. In order to further study the function of phosphorylation at the Ser364 site of γ-tubulin, this example constructed an RPE-1 stable cell line of the tet-on system, which can induce the expression of wild-type, non-phosphorylated mutant (S364A) and pseudo-phosphorylated mutant (S364D) of γ-tubulin with a Myc tag. By adding different doses of doxycycline, the expression level of γ-tubulin can be adjusted to the endogenous level. At the same time, as Figure 4 As shown in Figure 2, endogenous γ-tubulin was depleted by RNA interference. After replacing endogenous γ-tubulin with different mutants, microtubule regeneration experiments were performed: microtubules in cells were depolymerized by low temperature treatment and then transferred to 37°C to allow microtubules to regenerate for a certain period of time, which was used to measure the ability of microtubule nucleation and growth. Figure 5 As shown, the Ser364 phosphomimetic mutant of γ-tubulin inhibited centrosomal and non-centrosomal microtubule nucleation, whereas no inhibitory effect was observed with the non-phosphorylatable mutant. Figure 6 As shown, the centrosomal microtubule intensity of the γ-tubulin phosphomimetic mutant decreased by about 80% compared with the wild type.

[0118] In order to verify the microtubule nucleation defect caused by phosphorylation of Ser364 of γ-tubulin, this example uses the insect cell biGBac expression system to reconstruct the human γ-tubulin ring complex in vitro. A total of 11 proteins were co-expressed in Sf9 insect cells: γ-tubulin (or mutant), GCP2, GCP3, GCP4, GCP5, GCP6-TEV-Twinstrep, MZT1, MZT2, β-actin, RUVBL1 and RUVBL2. Protein purification was performed by the affinity tag on GCP6. Finally, a total of 3 recombinant γ-tubulin ring complexes containing γ-tubulin WT, S364A and S364D mutants were obtained.

[0119] like Figure 7 As shown, the composition of the purified complex has been verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining, and all core components of the γ-tubulin ring complex are present. The integrity of the recombinant γ-tubulin ring complex was further verified by 5-40% sucrose gradient centrifugation. Figure 8 As shown, all core components of the recombinant γ-tubulin ring complex / γ-tubulin WT and S364D mutant were deposited to a peak similar to that of the endogenous γ-tubulin ring complex, indicating that the γ-tubulin S364D phosphomimetic mutation does not affect the integrity of the γ-tubulin ring complex. Fig. 9As shown, the structure of the wild-type recombinant γ-tubulin ring complex was observed by negative staining electron microscopy, which showed a uniform ring structure. The results showed that the recombinant γ-tubulin ring complex retained the same structure as the endogenous mammalian γ-tubulin ring complex.

[0120] Next, this example uses the recombinant γ-tubulin ring complex to conduct a microtubule nucleation experiment in solution. Fig.10 and 11 As shown, the nucleated microtubules of the γ-tubulin ring complex / γ-tubulin S364D pseudophosphorylated mutant were reduced by about 85%. No significant difference was observed for the non-phosphorylated mutant S364A of the recombinant γ-tubulin ring complex / γ-tubulin. This result is consistent with the in vivo microtubule regeneration experiment, verifying that phosphorylation of the Ser364 site of γ-tubulin inhibits the microtubule nucleation activity of the γ-tubulin ring complex.

[0121] Example 3 Phosphorylation of γ-tubulin Ser364 affects cell mitosis

[0122] It is noteworthy that after knockdown of endogenous γ-tubulin, the RPE-1 stable cell line expressing the γ-tubulin S364D pseudophosphopeptide mutant grew more slowly than the wild type. This example detected the mitotic index (number of mitotic cells / total number of cells) of these cells. Fig.12 As shown, the mitotic index of cells expressing the γ-tubulin S364D phosphomimetic mutant was increased by 30% relative to that of cells expressing the γ-tubulin wild type.

[0123] Determination of mitotic index: After knocking down endogenous γ-tubulin in the RPE-1 stable cell line and inducing the expression of wild-type or S364D mutant γ-tubulin, the cell DNA was immunostained with Hoechst 33342 and observed under a microscope. The cells in interphase or division were distinguished by nuclear or chromosome morphology and counted separately. Finally, the mitotic index of each cell line was calculated according to the formula (mitotic index = number of mitotic cells / total number of cells).

[0124] In order to find out the cause of the increase in mitotic index, this example uses live cell imaging technology to measure the duration of cell mitosis. Fig.13 As shown, expression of the γ-tubulin S364D phosphomimetic mutant prolonged the duration of prometaphase-metaphase by approximately 20% compared with cells expressing wild-type γ-tubulin, but had no effect on anaphase. These data suggest that phosphorylation of γ-tubulin at Ser364 prolongs mitosis, leading to an elevated mitotic index.

[0125] Example 4 Phosphorylation of γ-tubulin Ser364 reduces spindle microtubule strength

[0126] In order to study the mechanism of mitotic defects in γ-tubulin S364D phosphomimetic mutant cells, this example used immunofluorescence staining to detect the phenotype of mitotic spindles. Fig.14 As shown, the spindle microtubule intensity of γ-tubulin S364D phosphomimetic mutant cells was reduced by 45%. However, the spindle length, spindle area, chromosome alignment and arrangement of phosphomimetic mutant cells were not affected. Therefore, the reduction of spindle microtubule density in γ-tubulin S364D phosphomimetic mutant cells may be the cause of their mitotic defects.

[0127] Example 5 Preparation and effectiveness verification of polyclonal antibody against γ-tubulin phosphorylated at Ser364

[0128] The polyclonal antibody was generated by immunizing rabbits with the synthetic peptide VALSRK(pS)PYLPS (γ-tubulin 358-369). The peptides without phosphorylated Ser364 and the peptides with phosphorylated Ser364 were coupled to affinity columns respectively, and then the immune rabbit serum was passed through the non-phosphorylated and phosphorylated peptide affinity columns, and the eluate was finally collected to obtain the purified phosphorylated antibody. The specific method is as follows.

[0129] 1. Antigen immunization:

[0130] 1.1 Synthetic antigen peptide (VALSRK(pS)PYLPS) and control peptide (VALSRKSPYLPS).

[0131] 1.2 Use Sulfo-SMCC coupling agent to couple the antigen peptide with KLH as the immune antigen.

[0132] 1.3 Dilute the antigen to 1 mg / mL with PBS.

[0133] 1.4 Four New Zealand white rabbits were selected and immunized 4 times in total. On days 1, 15, 29 and 43, 2 mL of the immunizing antigen was mixed with 2 ml of Freund's complete adjuvant and emulsified, and injected subcutaneously at the back of the rabbit's neck, with at least 8 injection sites per rabbit.

[0134] 1.5 On the 53rd day, blood was collected from the carotid artery of the rabbits and the rabbits were killed.

[0135] 1.6 The collected rabbit blood was placed at 4°C overnight, then centrifuged at 4°C, 10,000 rpm for 30 minutes, and the supernatant was collected.

[0136] 2. Antibody purification:

[0137] 2.1 Connect the antigen peptide and control peptide to the activated SulfoLink TM On the Coupling Resin, an antigen peptide affinity column and a control peptide affinity column were prepared.

[0138] 2.2 Equilibrate the above affinity column with 10 column volumes of PBS.

[0139] 2.3 Filter the rabbit serum collected above using a 0.45 μm filter, then pass it through a control peptide affinity column to remove antibodies without phosphorylation specificity, and collect the flow-through.

[0140] 2.4 Pass the flow-through solution through the antigen peptide affinity column, and then balance it with 10 times the column volume of PBS to allow the solution to flow out.

[0141] 2.5 Add 5 mL of antibody eluate (1 M glycine solution, pH 2.7) to the antigen peptide affinity column and collect the eluate.

[0142] 2.6 The collected eluate was dialyzed with PBS and stored at -20°C.

[0143] In order to verify the sensitivity and specificity of the γ-tubulin Ser364 phosphorylation antibody, this example performed an in vitro kinase assay using recombinant γ-tubulin ring complex purified from Sf9 cells and Cdk1-cyclin B (14-450, Millipore) recombinant kinase.

[0144] In vitro kinase assay: 1 μg of recombinant γ-tubulin ring complex was incubated with 1.5 μg of Cdk1 / cyclin B (Millipore) in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM magnesium chloride, 10 mM sodium fluoride, 1 mM DTT, 0.1 mM ATP) at 30°C for 1 hour, and then SDS sample buffer was added to terminate the reaction. Samples were analyzed by SDS-PAGE and Coomassie blue staining.

[0145] As shown in Table 2, Fig.15 As shown, after the addition of kinase, the wild-type γ-tubulin showed a strong band in the anti-phospho-Ser364 antibody blot. However, if Cdk1 was not added, no significant band was observed. To demonstrate that the antibody is site-specific, the γ-tubulin ring complex / γ-tubulin S364A non-phosphorylated mutant was added to the in vitro kinase assay. The γ-tubulin S364A mutant showed no bands with or without the kinase Cdk1. These results indicate that the antibody is able to detect γ-tubulin phosphorylated at Ser364 in a site-specific manner. Therefore, the antibody also becomes a tool for detecting defects in microtubule nucleation and mitosis in cells.

[0146] Example 6 Cdk1 kinase can phosphorylate Ser364 of γ-tubulin both in vitro and in vivo

[0147] Since the kinase activity of Cdk1 is cell cycle-dependent, it is speculated that the phosphorylation of γ-tubulin Ser364 also has a cell cycle-dependent pattern. In this example, HEK293T cells were synchronized to interphase or mitosis using aphidicolin and nocodazole, and then the γ-tubulin ring complex was purified. Fig.16 As shown, Western blotting using an anti-Ser364 phosphorylated γ-tubulin antibody showed that the phosphorylation level of γ-tubulin Ser364 in mitotic cells increased by about 60% compared with interphase cells. This experimental data is consistent with the higher kinase activity of Cdk1 in mitosis. This result shows that Cdk1 is a candidate kinase for phosphorylating γ-tubulin Ser364.

[0148] In the above-mentioned in vitro kinase experiment, the Ser364 phosphorylated γ-tubulin antibody has been used to prove that Cdk1 can phosphorylate the Ser364 site of γ-tubulin in vitro. For further verification, this example also performed mass spectrometry analysis on the samples of the in vitro kinase experiment, and detected phosphorylation of the Ser364 site in both the control group and the kinase group samples. However, as shown in Table 2, the proportion of phosphorylated peptide matching (PSM) ions in the kinase group samples was 7%, much higher than 0.36% of the control group samples. This shows that the addition of Cdk1 / cyclin B catalyzes the phosphorylation of the Ser364 site of γ-tubulin. Therefore, this example proves that the Ser364 site of γ-tubulin is a substrate of Cdk1 kinase in vitro.

[0149] Example 7 Cdk1 inhibitor RO-3306 can reduce the phosphorylation level of γ-tubulin Ser364 in vivo and promote microtubule nucleation

[0150] In order to further study the role of Cdk1 in the phosphorylation of γ-tubulin Ser364 in vivo, this example used a Cdk1 specific inhibitor RO-3306 to treat cells and detected the phosphorylation level of γ-tubulin Ser364 in the γ-tubulin ring complex. Fig.17 As shown, after cells were treated with 9 μM RO-3306 overnight, the phosphorylation level of γ-tubulin Ser364 was significantly reduced. This result verified that Cdk1 can phosphorylate the Ser364 site of γ-tubulin in vivo.

[0151] like Fig.18As shown, after treatment with the Cdk1 inhibitor RO-3306, the centrosomal microtubule nucleation activity was significantly promoted. However, inhibition of Cdk1 kinase activity will cause cells to stagnate in the G2 / M phase. In order to exclude the possibility that different cell stages lead to differences in microtubule nucleation, this example selected late G2 cells for quantification, and centrosomal microtubule nucleation still increased slightly. This result shows that the inhibition of Cdk1 activity caused by RO-3306 avoids the microtubule nucleation inhibition caused by phosphorylation of the Ser364 site of γ-tubulin, suggesting that it may become a therapeutic target for diseases related to microtubule disorders (such as cancers such as oral cancer and neurodegenerative diseases).

[0152] Example 8 The phosphorylation level of γ-tubulin Ser364 site increases in oral cancer cells

[0153] To further verify the expression and phosphorylation of γ-tubulin in tumors, the TCGA database was used for analysis, such as Fig.19 As shown, this example found that the expression of γ-tubulin was increased in oral cancer. In order to further verify the phosphorylation level of γ-tubulin Ser364 in oral cancer cells, this example selected human normal oral keratinocytes HOK and oral cancer cells HSC3 and HN6, and used bacterial vectors to express and purify FLAG-2TEV-SBP-CDK5RAP2 1-100 recombinant protein co-immunoprecipitation to pull down the γ-tubulin ring complex, and performed immunoblotting experiments. Fig. 20 As shown, the overall γ-tubulin content of immunoprecipitation was basically the same in the three cell lines, while the phosphorylation level of γ-tubulin Ser364 was significantly increased in oral cancer cells, suggesting that phosphorylation of γ-tubulin Ser364 is expected to be a therapeutic target.

Claims

1. Application of substances for detecting phosphorylation of γ-tubulin in (a1) to (a3): (a1) preparing a product indicative of microtubule nucleation defects; (a2) preparing products indicative of mitotic elongation; (a3) preparing a product indicative of spindle defects; The phosphorylation site of the γ-tubulin is Ser364.

2. The use according to claim 1, characterized in that: The substance for detecting phosphorylation of γ-tubulin is a substance for detecting phosphorylation of γ-tubulin at the protein level.

3. The use according to claim 2, characterized in that: The product includes at least one of a reagent, a kit, a system and a chip.

4. Application of γ-tubulin phosphorylation inhibitors in (b1) to (b3): (b1) preparing a product that inhibits microtubule nucleation defects; (b2) preparing a product that inhibits mitotic elongation; (b3) Preparation of a product that suppresses spindle defects.

5. The use according to claim 4, characterized in that: The γ-tubulin phosphorylation inhibitor includes a Cdk1 specific inhibitor.

6. An antibody for detecting phosphorylation of γ-tubulin, characterized in that: The immunogenic site of the antibody is VALSRK(pS)PYLPS.

7. A method for preparing the antibody according to any one of claims 6 to 7, comprising the following steps: Animals were immunized with the polypeptide VALSRK(pS)PYLPS, and the serum was passed through an affinity column coupled to non-phosphorylated γ-tubulin at Ser364 and an affinity column coupled to phosphorylated γ-tubulin at Ser364, and the eluate was finally collected to purify the antibody.

8. A method for detecting γ-tubulin phosphorylation in vitro: Construct the γ-tubulin gene expression vector, transfect and express, separate and purify the protein, and detect the phosphorylation status.

9. Application of γ-tubulin phosphorylated at Ser364 as a target in the development of prevention and treatment of diseases related to microtubule disorders.

10. The use according to claim 9, characterized in that: The microtubule disorder-related diseases include oral cancer.

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