A method for regulating the liquid-liquid phase separation ability and function of HBB

Abstract

The present invention provides a method for regulating the liquid-liquid phase separation ability and function of HBB. By mutating the intrinsically disordered region of HBB, its liquid-liquid phase separation ability and function can be regulated; specifically, by deleting the amino acids at positions 139 to 147 of HBB or mutating alanine at position 139 to proline. The present invention discovers for the first time that the β subunit of hemoglobin can undergo LLPS, and further verifies through experiments that mutating the intrinsically disordered region at its carboxyl terminus can effectively down-regulate its LLPS ability, and it is found that this mutation significantly increases the ability of HBB to down-regulate reactive oxygen species (ROS). The method provided by the present invention for regulating the liquid-liquid phase separation ability and function of HBB by mutating the intrinsically disordered region of HBB helps in the development of related drugs, and thus is expected to intervene in and treat human diseases by regulating the polymerization ability and function of hemoglobin.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for regulating the liquid-liquid phase separation ability and function of HBB. Background Art

[0002] The lipid bilayer membrane of membrane-bound organelles creates a space isolated from the outside world, enclosing specific proteins, nucleic acids, and other substances, thus enabling the normal progress of life activities within the organelles. In contrast, membrane-less organelles do not have any substantial membrane to establish a specific compartment. Liquid–liquid phase separation (LLPS), as a molecular mechanism for the formation and function of membrane-less organelles, has attracted increasing attention. During the LLPS process, the interaction forces between multivalent macromolecules can change the physical state of these macromolecules themselves, resulting in a phase change, thereby promoting the formation of membrane-less organelles or cell structures. After the phase change, the local protein concentration increases relative to the surrounding medium, and the movement of molecules weakens, thus improving the efficiency of corresponding biochemical reactions. In previous studies, the structures formed by these aggregations exhibit the characteristics of a liquid phase and are defined as granules, droplets, puncta, bodies, and condensates according to their morphology. LLPS of macromolecules, especially protein molecules, plays an important role in various cellular processes.

[0003] The basic principle of LLPS formation is the interaction of multivalent molecules. In the study of protein LLPS, intrinsically disordered regions (IDRs) often but not uniquely promote the interaction between multivalent macromolecules. This region lacks a stable 3D structure, and its structural heterogeneity allows interacting molecules to have interaction forces in multiple directions, leading to unstable binding and separation between molecules, and ultimately mediating the occurrence of protein LLPS.

[0004] Hemoglobin itself, as the main component in red blood cells, is responsible for transporting oxygen. In the minds of most people and even some life science workers, it is generally believed that adult hemoglobin only exists in red blood cells. However, according to research findings, hemoglobin is also expressed in non-erythroid cells, and it has been detected in various cells and tissues such as airway epithelial cells, macrophages, mesangial cells, midbrain dopaminergic neurons and glial cells, breast cancer cells, and liver cancer cells. Moreover, in addition to its oxygen-carrying and oxygen-releasing functions, research results show that its expression level may be related to the cellular oxidative stress state.

[0005] As one of the most important effector molecules in the process of cellular oxidative stress, reactive oxygen species (ROS) have positive effects on physiological cell death, cell proliferation, signal transduction, cell metabolism, cell differentiation, tissue development, and immune function when at physiological levels. However, increased production of ROS due to genetic mutations, tissue damage, chronic inflammation, and other inducing factors can cause DNA damage, leading to potential carcinogenic mutations and promoting the transformation of normal cells. ROS can also act as signaling molecules to promote tumor metastasis, the survival of tumor cells, and increased malignancy, including epithelial-mesenchymal transition, migration, invasion, and adhesion to endothelial cells, thereby promoting tumorigenesis and development. However, excessive ROS accumulation caused by oncogene activation or a high ROS environment in circulation can also lead to tumor cell death. Therefore, ROS play an important role in various physiological and pathological processes, and the regulatory mechanism of ROS also has an important impact on life activities.

[0006] Hemoglobin exists in the cytoplasm without a clear organelle or cellular structure localization. Does liquid-liquid phase separation (LLPS) occur? Does the intrinsically disordered region (IDR) affect the formation of LLPS of hemoglobin? And does the influence on its LLPS have an impact on its ROS regulatory function? There is a lack of corresponding research and model establishment for the above questions. Summary of the Invention

[0007] The object of the present invention is to provide a method for regulating the liquid-liquid phase separation ability and function of HBB by mutating the intrinsically disordered region of HBB, thereby regulating its liquid-liquid phase separation ability and function.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a method for regulating the liquid-liquid phase separation ability and function of HBB by mutating the intrinsically disordered region of HBB, thereby regulating its liquid-liquid phase separation ability and function;

[0010] The method for regulating the liquid-liquid phase separation ability and function of HBB can specifically reduce the polymerization ability of HBB and enhance its ability to downregulate ROS by deleting amino acids 139 to 147 of HBB or mutating alanine at position 139 to proline. Further, by mutating the intrinsically disordered region of HBB, the inhibition of liquid-liquid phase separation of HBB in cell and in vitro models can be achieved; the enhancement of the ability of HBB to downregulate ROS is verified in cell models.

[0011] Further, the regulation method is divided into the following Method A or Method B:

[0012] Method A: Delete amino acids 139 to 147 of HBB;

[0013] Method B: Mutate alanine at position 139 of HBB to proline.

[0014] Furthermore, in Methods A and B, molecular cloning technology is used to construct expression vectors for wild-type and mutants. Taking the wild-type coding sequence (CDS sequence) of HBB as a control, two mutants with amino acids 139 to 147 deleted or alanine at position 139 mutated to proline are constructed on this basis.

[0015] Furthermore, in Methods A and B, the HBB coding sequence is derived from the NCBI database, with the CDS sequence number: CCDS7753.1, Gene ID: 3043, Protein ID: NP_000509.1.

[0016] Furthermore, in Methods A and B, the C-terminal amino acid sequences affecting the liquid-liquid phase separation and ROS regulation ability of HBB are obtained: ANALAHKYH and alanine at position 139.

[0017] Furthermore, in Methods A and B, molecular cloning technology is used to construct expression vectors for wild-type and mutants. Taking the coding sequence (CDS sequence) of HBB as the wild-type control, two mutants with amino acids 139 to 147 deleted or alanine at position 139 mutated to proline are constructed on this basis. Among them, the eukaryotic expression vector is based on the retroviral vector pQCXIP-EGFP-N1, and the inserted sequences are the protein coding regions (CDS) of gene HBB and its mutants respectively, and overexpression vectors for HBB and its mutants are constructed respectively. The CDS region is inserted between the restriction enzyme sites XhoI and BamHI of the viral vector pQCXIP-EGFP-N1 to obtain the respective eukaryotic expression vectors.

[0018] Furthermore, in Methods A and B, an intracellular LLPS model of HBB and its mutants is established by using overexpression technology to express intracellular proteins of wild-type and mutants. The specific operation is to transfer equal amounts of the above-mentioned expression vectors of HBB and its mutants into cells by liposome transfection method, so as to express their protein products in target cells. In cells, the protein products undergo LLPS, and at the same time, the LLPS formation ability of HBB and mutants is compared according to the ratio of positive cells with fluorescent dots / overexpressed successfully cells.

[0019] Furthermore, the ability of HBB to regulate ROS was verified in a cell model by using overexpression technology to express wild-type and mutant intracellular proteins, followed by normoxic and hypoxic treatments of the cells, and finally detecting the intracellular ROS level by DCFH-DA staining and subsequent flow cytometry analysis.

[0020] Further, in the method, an in vitro liquid-liquid phase separation model of HBB and its mutants was established by using prokaryotic expression and protein purification techniques to obtain purified proteins of wild-type and mutants, and then adding them to a specific LLPS buffer.

[0021] Furthermore, the ability of the mutant to enhance the downregulation of ROS by HBB was verified in a cell model by using overexpression technology to express wild-type and mutant intracellular proteins, and then detecting the intracellular ROS level by DCFH-DA staining and subsequent flow cytometry analysis.

[0022] The second aspect of the present invention provides an in vitro model of HBB forming LLPS, which is obtained by the method of regulating the liquid-liquid phase separation ability and function of HBB. The present invention also claims the in vitro model of HBB forming LLPS obtained by the method described above.

[0023] The in vitro model of HBB forming LLPS obtained in the embodiments of the present invention is mainly achieved through the following techniques and conditions. First, construct prokaryotic expression vectors of HBB and its mutants, and use protein purification technology to obtain single and highly pure HBB and its mutant proteins; dissolve the proteins in LLPS buffer, add the solution to a confocal dish, and observe the solution using a confocal microscope; through the simulation of the in vivo environment and the optimization of conditions, finally confirm that the components of the LLPS buffer are: 150 mM KH2PO4 / K2HPO4, pH = 7.35, 10% PEG2000; use a microscope to observe and compare the formation of bodies by HBB and its mutants in the buffer to compare the LLPS formation ability among the three.

[0024] In a specific implementation of the present invention, molecular cloning technology was used to construct prokaryotic expression vectors for wild-type and mutants. Among them, the CDS sequence of HBB was used as the wild-type control, and on this basis, two mutants were constructed by deleting amino acids at positions 139 to 147 or mutating alanine at position 139 to proline. Among them, the prokaryotic expression vector pET-30a(+) was used as the backbone, and the inserted sequences were the protein-coding regions (CDS) of the gene HBB and its mutants respectively, and the prokaryotic expression vectors of HBB and its mutants were constructed respectively. The CDS region was inserted between the restriction enzyme sites NdeI and HindIII of the viral vector pET-30a(+) to obtain their respective prokaryotic expression vectors. For wild-type HBB, the HBB-EGFP prokaryotic expression vector was prepared according to the above method. For the purified proteins of HBB and its mutants, the confocal DIC channel was used for observation, and for the purified HBB-EGFP, the confocal FITC channel was used for observation.

[0025] The third aspect of the present invention provides an amino acid that affects the liquid-liquid phase separation and ROS regulation ability of HBB, obtained by the above method. The amino acid that affects the liquid-liquid phase separation and ROS regulation ability of HBB is: the C-terminal amino acid or alanine at position 139;

[0026] The C-terminal amino acid sequence is: ANALAHKYH.

[0027] The fourth aspect of the present invention provides the application of the C-terminal amino acid sequence or single amino acid site (alanine at position 139) that affects the liquid-liquid phase separation and ROS regulation ability of HBB in any of the following:

[0028] 1) Design of artificial hemoglobin through the amino acid sequence or single amino acid site;

[0029] 2) Design of novel application proteins or polypeptides using the amino acid sequence or single amino acid site;

[0030] 3) Establishment of related mutant animal models using the amino acid sequence or single amino acid site;

[0031] 4) Research and development of related drugs by targeting the amino acid sequence or single amino acid site;

[0032] The above related drugs can be: compounds, polypeptides, proteins, gene therapy vectors, etc.

[0033] The beneficial effects of the present invention:

[0034] 1. The present invention provides a method for regulating the liquid-liquid phase separation ability and function of HBB by mutating the intrinsically disordered region of HBB, thereby regulating its liquid-liquid phase separation ability and function. Specifically, by deleting and mutating the intrinsically disordered region at the carboxyl terminus of hemoglobin beta subunit (HBB), its liquid-liquid phase separation ability and function are regulated.

[0035] 2. The object of the present invention is to provide a method for regulating the liquid-liquid phase separation ability and function of hemoglobin beta subunit by deleting and mutating the intrinsically disordered region at its carboxyl terminus. To achieve the above object, the present invention provides the following technical solutions: The present invention first overexpresses HBB intracellularly to construct an intracellular liquid-liquid phase separation model. Further, the inventors explored the formula of the in vitro liquid-liquid phase separation buffer, thereby establishing an in vitro liquid-liquid phase separation model. The present invention discovers for the first time that the beta subunit of hemoglobin has liquid-liquid phase separation characteristics, and further verifies through experiments that mutating the intrinsically disordered region at its carboxyl terminus can effectively down-regulate its liquid-liquid phase separation ability, and it is found that this mutation significantly increases the ability of HBB to down-regulate reactive oxygen species (ROS).

[0036] 3. The present invention provides a method for regulating its LLPS ability and function by deleting and mutating the intrinsically disordered region at the carboxyl terminus of hemoglobin beta subunit (HBB).

[0037] The present invention discovers for the first time that the beta subunit of hemoglobin has liquid-liquid phase separation characteristics, and further verifies through experiments that mutating the intrinsically disordered region at its carboxyl terminus can effectively down-regulate its liquid-liquid phase separation ability, and it is found that this mutation significantly increases the ROS regulation ability of HBB. The method provided by the present invention for regulating the liquid-liquid phase separation ability and function of HBB by mutating the intrinsically disordered region helps in the development of related drugs, and thus is expected to intervene in and treat human diseases by regulating the polymerization ability and function of hemoglobin.

[0038] 4. The present invention first overexpresses HBB intracellularly to construct an intracellular liquid-liquid phase separation model. Further, the inventors explored the formula of the in vitro liquid-liquid phase separation buffer, thereby establishing an extracellular liquid-liquid phase separation model. It provides a research basis and model for the subsequent study of hemoglobin LLPS, and provides ideas and mechanism explanations for the functional regulation and related research of hemoglobin.

[0039] 5. The present invention regulates the liquid-liquid phase separation (LLPS) ability and function by deleting and mutating the intrinsically disordered region at the carboxyl terminus of hemoglobin beta subunit (HBB). First, the present invention constructs an intracellular liquid-liquid phase separation model by overexpressing HBB intracellularly. Further, the inventors have explored an in vitro liquid-liquid phase separation buffer formulation, thereby achieving the establishment of an extracellular liquid-liquid phase separation model. The present invention discovers for the first time that the beta subunit of hemoglobin can undergo LLPS, and further verifies through experiments that mutating the intrinsically disordered region at its carboxyl terminus can effectively downregulate its LLPS ability, and it is found that this mutation significantly increases the ability of HBB to downregulate reactive oxygen species (ROS). The method provided by the present invention for regulating the liquid-liquid phase separation ability and function by mutating the intrinsically disordered region of HBB helps in the development of related drugs, and thus is expected to intervene in and treat human diseases by regulating the polymerization ability and function of hemoglobin. Description of the Drawings

[0040] Figure 1 Intracellular verification of the liquid-liquid phase separation characteristics of hemoglobin beta subunit. Panel A: After 48 hours of hypoxia in Huh7 cells, immunofluorescence staining shows a punctate distribution of HBB aggregated in the cytoplasm. The blue Hoechst (Hoch.) marks the cell nucleus, the green marks the HIF-1α protein that accumulates in the nucleus after hypoxia, and the red marks the HBB protein. Magnification: 20×10, large field scale bar: 10 μm, enlarged view scale bar: 5 μm. Panel B: Representative images of EGFP and HBB-EGFP expressed in 293T cells. No aggregated punctate distribution was observed for the EGFP protein, while HBB-EGFP formed punctate aggregates at a high proportion. The red CAAX marks the cell membrane. The blue Hoechst (Hoch.) marks the cell nucleus. The arrows indicate the aggregates formed by HBB-EGFP. Magnification: 60×10, scale bar: 20 μm. Panel C: The proportion of aggregate formation in different cell lines (number of cells forming aggregates / number of cells positive for fluorescent protein). Data are the mean ± standard deviation of 5 fields of view, and the number of fluorescent protein-positive cells analyzed for each cell line is greater than or equal to 300. Panels D and E: Panel E shows time-lapse images of the fluorescence recovery after photobleaching. After the highly fluorescent aggregates marked by the white dashed circles were photobleached by a laser, the fluorescence disappeared and then recovered within a few minutes. Magnification: 100×10, left original image scale bar: 10 μm; right enlarged view scale bar: 5 μm. Panel D shows the change curve of the relative fluorescence intensity (real-time fluorescence intensity / initial fluorescence intensity) recorded in the photobleaching experiment of 10 aggregates (n = 10).

[0041] Figure 2In vitro verification of the liquid-liquid phase separation characteristics of hemoglobin β subunit. Panel A: Droplet-like distribution formed by the liquid-liquid phase separation of HBB-EGFP in LLPS buffer (150 mM KH2PO4 / K2HPO4, pH = 7.35, 10% (w / v) PEG2000), magnification: 60×10. Panel B: Time-lapse imaging of the fusion between HBB-EGFP protein droplets. The white arrow and the red arrow respectively indicate the two droplets before fusion, and the yellow arrow indicates the droplet after fusion. Magnification: 60×10, scale bar: 5 μm. Panel C: Protein droplets formed in the solution after adding purified unlabeled HBB protein at the same protein concentration in the phase separation buffer (150 mM KH2PO4 / K2HPO4, pH = 7.35, 10% (w / v) PEG 2000) under optimal conditions. Magnification: 60×10, scale bar: 30 μm. Panel D: Time-lapse imaging of the fusion between in vitro purified unlabeled HBB droplets. Magnification: 60×10, scale bar: 5 μm..

[0042] Figure 3 Effects of mutations in intrinsically disordered regions on the liquid-liquid phase separation and function of HBB. Panel A: Schematic diagram of the constructed HBB mutants according to the predicted intrinsically disordered regions, with single or combined disordered regions (gray boxes) deleted (ΔN: deletion of the amino-terminal IDR; ΔC: deletion of the carboxyl-terminal IDR; ΔN&ΔC: simultaneous deletion of the amino-terminal and carboxyl-terminal) or point mutation A139P (red bar). Panels B and C: Quantitative chart (B) and representative image (C) of the proportion of aggregate formation of HBB-EGFP and its mutants in 293T cells. Magnification: 20×10, scale bar: 200 μm. Panel D: After purifying HBB, HBB-ΔC, HBB-A139P, two mutants that inhibit LLPS, and wild-type HBB protein, the droplet formation in the corresponding protein solutions under the phase separation buffer (150 mM KH2PO4 / K2HPO4, pH = 7.35, 10% (w / v) PEG 2000) under optimal conditions. Panels E and F: After transfecting HBB, HBB-ΔC, HBB-A139P, two mutants that inhibit LLPS, and control plasmids into Hep3B cells respectively, flow cytometry detection chart (E) and quantification chart (F) of ROS levels after hypoxia treatment. The proportion of cells within the gate represents the proportion of cells with low ROS. Detailed implementation methods

[0043] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0044] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods.

[0045] Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels.

[0046] PLC / PRF / 5 cells: ATCC cell bank, #CRL-8024.

[0047] HEK 293T cells: Beina Biological Cell Bank, #BNCC353535.

[0048] HepG2 cells: ATCC cell bank, #HB-8065

[0049] HCT 116 cells: Procell Life Science & Technology Co., Ltd., #CL-0096

[0050] KH2PO4: Solarbio, #7778-77-0

[0051] K2HPO4: Solarbio, 7758-11-4

[0052] PEG 2000: Solarbio, 25322-68-3

[0053] Reactive oxygen species staining kit: Yeasen Biotech Co., Ltd., 50101ES01

[0054] The pET30a(+) prokaryotic expression plasmid was provided by Nanjing Detai Biology, and the construction of its wild-type and mutant prokaryotic expression vectors and protein purification were also commissioned to Nanjing Detai Biology to complete.

[0055] The retroviral vector pQCXIP-EGFP-N1: is described in the article "Wang M, Ning X, Chen A, Huang H, Ni C, Zhou C, et al. Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression. Scientific reports. 2015; 5: 12223", and the public can obtain it from the applicant and can only be used for repeating the experiments of the present invention.

[0056] Example 1: Intracellular Verification of the Liquid-Liquid Phase Separation Characteristics of Hemoglobin β Subunit

[0057] The target cells in this example are HEK 293T human renal epithelial cells, HepG2 liver cancer cells, PLC / PRF / 5 liver cancer cells, and HCT 116 colon cancer cells. The transferred plasmid pQCXIP-HBB-EGFP was constructed and preserved by the inventors. The main verification methods are as follows: 1. Formation of HBB aggregates in cells; 2. Photobleaching recovery of HBB aggregates; 3. Fusion between HBB aggregates. The above methods are important criteria for whether a protein molecule can undergo LLPS.

[0058] I. Construction of Eukaryotic Expression Plasmids Overexpressing HBB and HBB-EGFP

[0059] 1) PCR amplification of the HBB fragment: Using cDNA as a template, PCR amplify the homologous sequence required for homologous recombination, and purify it using a gel DNA fragment recovery kit (Tiangen, #DP208-02).

[0060] 2) Enzyme digestion: Mix the pQCXIP-EGFP-N1 plasmid with the restriction endonucleases XhoI and BamHI (New England Biolabs) and reaction buffer, and place it at 37 °C for 1 h for double enzyme digestion; then perform gel recovery and purification of the DNA fragment again.

[0061] 3) DNA fragment recombination: Mix the above double-digested backbone plasmid with the HBB insert fragment and homologous recombination enzyme, and place it at room temperature for 30 minutes.

[0062] 4) Transformation: Transform the above ligated prokaryotic expression plasmid into Escherichia coli competent Trans10 using the method of water bath heat shock and ice bath cooling, and screen with ampicillin;

[0063] 5) Sequencing: Pick monoclonal colonies for sequencing, and select the plasmid with correct sequencing for preservation.

[0064] II. Aggregate Formation and Statistics of HBB-EGFP in Cells

[0065] 1) Cell seeding: The day before transfection, inoculate 3×105 HEK 293T, HepG2, HCT116, and PLC / PR / F5 cells per well into 1 mL of medium (6-well glass bottom plates).

[0066] 2) Plasmid transfection: The cell usage per well in the transfection system is as follows. Use 250 μL / well of Opti-MEM TM(Thermofiher, #31985070) Dilute the plasmid (2 μg) with serum-free medium, shake gently and flick to mix well, and let it stand at room temperature for 5 min. Take out Lipofectamine TM 2000 (Thermofiher, #L3000075) transfection reagent, flick to mix well, and dilute it according to the ratio of adding 5 μL to 250 μL of Opti-MEM TM serum-free medium, mix gently, and incubate at room temperature for 5 min. Mix the plasmid and Lipofectamine TM 2000 transfection reagent diluted in the previous two steps, gently shake well, and let it stand at room temperature for 20 min. Continue to add fresh normal DMEM complete medium to the above mixed solution until the volume is 1 mL, gently shake well, and replace the original medium of the cells with the transfection solution in turn. Dosage of plasmid (pQCXIP-HBB-EGFP): 2 μg / well, 5 μL / well of transfection reagent. After 6 h of transfection, change to the respective fresh medium suitable for the cells, and place the cells in a normoxic incubator for 24 h.

[0067] 3) Photograph and observe: Take out the cells at the above time points, place them under a wide-field fluorescence microscope, and use the FITC fluorescence channel and DIC channel for photographing and counting. Using the cells with green fluorescence positive as the base value, the number of cells forming aggregates is divided by the base value to obtain the relative value, and then a proportional graph is made.

[0068] III. Photobleaching verification of aggregates of HBB-EGFP in cells

[0069] 1) Cell seeding: Seed 3×105 HepG2 cells per well in 2 mL of medium (in confocal dishes) one day before transfection.

[0070] 2) Plasmid transfection: The specific operation is the same as the above plasmid transfection operation. Dosage of plasmid (pQCXIP-HBB-EGFP): 1 μg / dish, 2.5 μL / dish of transfection reagent. After 6 h of transfection, change to fresh medium, and place the cells in a normoxic incubator for 16 h. To prevent further aggregation of aggregates to form solid agglomerates, 24 h is not selected.

[0071] 3) Laser photobleaching: Cells were taken out at the above time points and placed under a confocal fluorescence microscope. Cells that had formed small-volume aggregates were selected for the photobleaching experiment. The FRAP module of the Nikon confocal microscope system was used to perform FRAP measurements on the aggregates inside the cells. The Hbb-EGFP aggregates were bleached using a 488 nm laser beam. The bleaching laser beam was focused on the area where the aggregates formed using 100% laser power for 10 seconds, and then images were taken every 5 seconds to collect time-lapse images. The Nikon confocal microscope system was used to measure the fluorescence intensity. The fluorescence values at each time point were calculated as relative values to the fluorescence at the time point before bleaching. GraphPad Prism was used to plot and analyze the FRAP results.

[0072] 4) IV. Aggregate fusion experiment of HBB-EGFP

[0073] 5) Cell seeding: 3×105 HepG2 cells were seeded per well in 2 mL of medium (25 mm glass-bottomed dish) the afternoon before transfection.

[0074] 6) Plasmid transfection: The specific operation was as described above for plasmid overexpression. Dosage of plasmid (pQCXIP-HBB-EGFP):

[0075] 1 μg / dish, and 2.5 μL / dish of transfection reagent. After 6 h of transfection, fresh medium was used, and the cells were cultured in a normoxic incubator for 12 h. 24 h was not chosen to prevent further aggregation of the aggregates into solid coagulants.

[0076] 7) Photography and observation: Cells were taken out at the above time points and placed under a confocal fluorescence microscope. Cells that had formed small-volume aggregates were selected for photography and observation. Note to use a low exposure time to avoid damaging the cells. The laser channel was: 488 nm excitation.

[0077] Example 2. In vitro verification of the liquid-liquid phase separation characteristics of hemoglobin β subunit

[0078] Both HBB and HBB-EGFP in this example are proteins after protein purification. The composition of the LLPS buffer used is: 150 mM KH2PO4 / K2HPO4, pH = 7.35, 10% (w / v) PEG 2000. This composition ratio is the result of screening by the inventor.

[0079] I. Construction of prokaryotic expression plasmids overexpressing HBB and HBB-EGFP

[0080] 1) Synthesis of HBB fragment: Using the HBB or HBB-EGFP fragment as the source sequence, after prokaryotic expression optimization, gene synthesis and purification were carried out.

[0081] 2) Enzyme digestion: Mix the pET-30a(+) plasmid with the restriction endonucleases NdeI and HindIII (New England Biolabs) and the reaction buffer, and incubate at 37 °C for 1 h for double enzyme digestion; then perform gel extraction and purification of the DNA fragment again.

[0082] 3) DNA fragment recombination: Mix the above double-digested backbone plasmid with the HBB or HBB-EGFP insertion fragment and the homologous recombination enzyme, and incubate at room temperature for 30 minutes.

[0083] 4) Transformation: Transform the above ligated prokaryotic expression plasmid into Escherichia coli competent Trans10 by the method of water bath heat shock and ice bath cooling, and screen with kanamycin;

[0084] 5) Sequencing: Pick monoclonal colonies for sequencing, and select the plasmids with correct sequencing for preservation.

[0085] 6) After transferring the plasmid into Escherichia coli, perform protein purification.

[0086] II. In vitro liquid-liquid phase separation experiment of HBB and HBB-EGFP

[0087] Use an in vitro standard liquid-liquid phase separation buffer (150 mM KH2PO4 / K2HPO4, pH = 7.35, 10% (w / v) PEG2000) to dissolve HBB to form liquid-liquid phase separation. Briefly, first dissolve the HBB protein in H2O at a concentration of 1 mg / μL. To achieve the working concentration of each component, use a high-concentration LLPS buffer (300 mM KH2PO4 / K2HPO4, pH = 7.35, 20% PEG2000) for equal ratio mixing, and then dilute the protein to the final concentration: 500 ng / μL. Finally, drop the mixture onto a confocal dish and image the solution with a confocal microscope. Collect images using the DIC channel or FITC channel of a 60× Apo objective lens.

[0088] Example 3. Effect of mutants in the disordered region of HBB on the ability to form LLPS

[0089] I. Intracellular liquid-liquid phase separation experiment of HBB-EGFP and its mutants

[0090] 1) In this study, the IDR sequences of Hbb were predicted by the online server: https: / / d2p2.pro / , and two IDR sequences were predicted, one located at the amino terminus (1-9 amino acids) and one located at the carboxyl terminus (140-147 amino acids).

[0091] 2) Expression vectors of truncated mutants with N-terminal and C-terminal deleted as well as both deleted simultaneously and A139P mutant with alanine at position 139 mutated to proline were constructed by homologous recombination.

[0092] 3) Cell seeding: One day before transfection, 3×10⁵ 293T cells per well were seeded in 2 mL of medium (six-well plate).

[0093] 4) Plasmid transfection: The specific operation was the same as that in 2.2.2.8 Plasmid overexpression. Plasmid dosage: 1 μg / dish, transfection reagent 2.5 μL / dish.

[0094] 5) After 6 h of transfection, the cells were changed to fresh medium and cultured in a normoxic incubator for 24 h.

[0095] 6) Using the cells with positive green fluorescence as the base value, the number of cells forming aggregates was divided by the base value to obtain the relative value, and then a proportional graph was made.

[0096] II. In vitro liquid-liquid phase separation experiment of HBB and its mutants

[0097] 1. Construction of prokaryotic expression plasmid overexpressing HBB mutants

[0098] 1) PCR amplification of HBB fragment: Using the HBB fragment as the source sequence, PCR amplification was carried out, and purification was performed using a gel DNA fragment recovery kit (Tiangen, #DP208-02).

[0099] 2) Enzyme digestion: The pET-30a(+) plasmid was mixed with restriction endonucleases NdeI and HindIII (New England Biolabs) and reaction buffer, and double enzyme digestion was carried out at 37 °C for 1 h; DNA fragment was recovered and purified by gel electrophoresis again.

[0100] 3) DNA fragment recombination: The above double-digested backbone plasmid was mixed with the inserted fragment of the mutated HBB fragment and homologous recombinase, and incubated at room temperature for 30 minutes.

[0101] 4) Transformation: The above ligated prokaryotic expression plasmid was transformed into Escherichia coli competent Trans10 by the method of water bath heat shock and ice bath cooling, and screened with kanamycin;

[0102] 5) Sequencing: Single colonies were selected for sequencing, and the plasmids with correct sequencing were selected for preservation.

[0103] 6) After the plasmid was transferred into Escherichia coli, protein purification was carried out.

[0104] 2. Construction of in vitro LLPS model and the effect of disordered region on the LLPS ability of HBB

[0105] Dissolve HBB in an in vitro standard liquid-liquid phase separation buffer (150 mM KH2PO4 / K2HPO4, pH = 7.35, 10% (w / v) PEG2000) to form a liquid-liquid phase separation. Briefly, first dissolve HBB and mutant proteins in H2O at a concentration of 1 mg / μL. To achieve the working concentration of each component, use a high-concentration LLPS buffer (300 mM KH2PO4 / K2HPO4, pH = 7.35, 20% PEG2000) for proportional mixing, and then dilute the protein to the final concentration: 500 ng / μL. Finally, drop the mixture onto a confocal dish and image the solution with a confocal microscope. Acquire images using the DIC channel of a 60× Apo objective lens.

[0106] III. Influence of the disordered region on the ROS regulation ability of HBB

[0107] For ROS indicator staining, the Reactive Oxygen Species Assay Kit from Yeasen Biotech Co., Ltd. is used, and the active ingredient is DCFH-DA. This staining agent can pass through the cell membrane and enter the cell. DCFH itself has no fluorescence and is oxidized by reactive oxygen species in the cell to generate DCF, which has green fluorescence activity. The intensity of the green fluorescence is linearly related to the level of reactive oxygen species in the cell. Its maximum excitation wavelength is 480 nm, and its maximum emission wavelength is 525 nm. Its fluorescence signal can be detected by instruments such as flow cytometry, fluorescence microscopy, or laser confocal microscopy. The specific operation procedure is as follows:

[0108] 1) Cell seeding: One day before transfection, seed 3×10 5 HepG2 cells per well into 2 mL of medium (six-well plate).

[0109] 2) Preparation of transfection solution: The amount of cells per well is as follows. Dilute the HBB and its mutant plasmids (2 μg) with 250 μL / well of Opti-MEM TM serum-free medium, shake gently and flick to mix evenly, and let it stand at room temperature for 5 min. Take out the Lipofectamine TM 2000 transfection reagent, flick to mix evenly, and dilute it according to the ratio of adding 5 μL to 250 μL of Opti-MEM TM serum-free medium, gently mix evenly, and incubate at room temperature for 5 min.

[0110] 3) Mix the plasmids and Lipofectamine TM 2000 transfection reagent diluted in the previous two steps, gently shake well, and place at room temperature for 20 min.

[0111] 4) Add fresh normal DMEM complete medium to the above-mentioned mixed solution until the volume reaches 1 mL, gently shake well, and sequentially replace the original culture medium of the cells with the transfection solution.

[0112] 5) After 6 hours of transfection, replace it with 2 mL of fresh DMEM complete medium. After 24 hours, place the cells in a normal oxygen (20%) and a low oxygen (1%) incubator for 24 hours, and then perform ROS indicator staining and flow cytometry detection.

[0113] 6) Dye preparation: Dilute DCFH-DA at a ratio of 1:1000 with serum-free culture medium to make the working concentration of the dye 10 μM.

[0114] 7) In-situ staining: After hypoxia treatment or drug treatment, aspirate the original culture medium, and slowly add the DCFH-DA working solution into the cell culture vessel. The staining solution should completely cover the cells to be detected. Incubate at 37°C in the dark for 1 hour, in a cell culture incubator or a hypoxia incubator.

[0115] 8) Washing after staining: Aspirate all the original staining solution. If observing directly, wash the cells 2 times with PBS. If further detection is required after digestion, first wash 1 time with PBS, after digestion, neutralize and centrifuge, aspirate the neutralization culture medium, wash 1 time with PBS, centrifuge again, resuspend with PBS, and then detect. Note that do not detect the cells in the culture medium, as the background value is relatively high.

[0116] 9) Fluorescence signal detection: The samples in the confocal culture well plates can be directly observed and photographed using a laser confocal microscope. The cells can also be digested, centrifuged, and resuspended, and then detected using a fluorescence microplate reader, a flow cytometer, or a fluorescence spectrophotometer.

[0117] It should be noted that in this article, terms such as "including", "comprising", or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device including a series of elements not only includes those elements but also includes other elements not explicitly listed, or elements inherent to such a process, method, article, or device. Without further limitations, an element defined by the statement "including one..." does not exclude the existence of additional identical elements in the process, method, article, or device including the said element.

[0118] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for regulating the liquid-liquid phase separation ability and function of HBB, characterized in that, Regulate its liquid-liquid phase separation ability and function by mutating the intrinsically disordered region of HBB; Specifically, delete the amino acids at positions 139 to 147 of HBB or mutate alanine at position 139 to proline; The coding sequence of the said HBB is from the NCBI database, CDS sequence number: CCDS7753.1, Gene ID: 3043, Protein ID: NP_000509.

1.

2. The method for regulating the liquid-liquid phase separation ability and function of HBB according to claim 1, wherein Inhibit the liquid-liquid phase separation of HBB in cell and in vitro models by mutating the intrinsically disordered region of HBB; The enhanced ability of HBB to down-regulate ROS was verified in the cell model.

3. A method for regulating the liquid-liquid phase separation ability and function of HBB, according to claim 1, characterized in that, Use molecular cloning technology to construct expression vectors of wild-type and mutants, using the wild-type of the coding sequence (CDS sequence) of HBB as a control, and on this basis, construct two mutants with deletion of amino acids at positions 139 to 147 or mutation of alanine at position 139 to proline.

4. A method for regulating the liquid-liquid phase separation ability and function of HBB, according to claim 2, characterized in that The intracellular LLPS model of HBB and its mutants was established by using overexpression technology to express wild-type and mutant intracellular proteins; The enhanced ability of the mutant to down-regulate ROS in the cell model was verified by using overexpression technology to express wild-type and mutant intracellular proteins, and then detecting the intracellular ROS level by DCFH-DA staining and subsequent flow cytometry analysis.

5. A method for regulating the liquid-liquid phase separation ability and function of HBB, according to claim 2, wherein In the said method, the in vitro liquid-liquid phase separation model of HBB and its mutants was established by using prokaryotic expression and protein purification technology to obtain purified proteins of wild-type and mutants, and then adding them to the LLPS buffer; The components of the LLPS buffer are: 150 + mM KH2PO4 / K2HPO4, pH = 7.35, 10% PEG2000.

6. An HBB mutant that inhibits liquid-liquid phase separation and improves ROS regulation ability, obtained by any one of the methods for regulating the liquid-liquid phase separation ability and function of HBB as claimed in claims 1-5, characterized in that, Contain one of the following mutations: (a) Deletion of amino acids at positions 139 to 147; (b) Mutation of alanine at position 139 to proline (A139P).

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