Demethylase ALKBH5 protein liquid phase-solid phase-droplet phase conversion verification method
By dividing the ALKBH5 protein structure through the PONDR algorithm and constructing a transient overexpression system, the problem of unclear control of the ALKBH5 protein structure was solved, its functional threshold zoning analysis and intracellular distribution observation were realized, and the transformation characteristics of liquid phase, solid phase and droplet phase were revealed.
Patent Information
- Application Number
- CN202510689641.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-27
- Publication Date
- 2025-09-16
AI Technical Summary
The existing technology is unclear about the structural state of ALKBH5 protein and the control of different structures on the protein state, which hinders its functional research.
The PONDR algorithm was used to analyze the structural order of the ALKBH5 protein and the protein was divided into an N-terminal ordered region, an intermediate highly ordered region and a C-terminal disordered region. These regions were constructed into the pCDN3.1-EGFP vector for fusion protein expression and then transferred into HEK293T cells using liposome transient transfection technology for characterization and verification.
The analysis of the functional threshold zoning of ALKBH5 protein and the observation of its intracellular distribution were achieved, revealing its transformation characteristics between liquid phase, solid phase and droplet phase. A transient overexpression system with CMV as the promoter was constructed, which has higher expression level and observation advantages.
Smart Images
Figure CN120648747A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of protein modification, and in particular to a method for verifying liquid-solid-droplet phase transformation of a demethylase ALKBH5 protein. Background Art
[0002] The ALKBH5 protein is the primary m6A demethylase in mammals, involved in important physiological and pathological processes such as spermatogenesis and tumor development. Currently, ALKBH5 is primarily distributed in the cell nucleus, forming a droplet-like distribution associated with nucleosomes. However, its structural state and the control of different structures on protein status remain largely unknown, significantly hindering the study of its function. Summary of the Invention
[0003] The present invention provides a method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein, aiming to solve the technical problem that the prior art has unclear understanding of the structural state of the ALKBH5 protein and the control of the protein state by different structures.
[0004] To solve the above technical problems, the present invention provides a method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein, comprising the following steps: The structural order of ALKBH5 protein was analyzed and divided based on the PONDR algorithm, and the N-terminal ordered region, the middle highly ordered region and the C-terminal disordered region were obtained. After constructing the N-terminal ordered region, the intermediate highly ordered region, and the C-terminal disordered region into the pCDN3.1-EGFP vector for fusion protein expression, respectively, the protein phase transition results of the N-terminal ordered region, the intermediate highly ordered region, and the C-terminal disordered region are obtained; the protein phase transition results include the N-terminal ordered region being diffusely distributed in the cell nucleus, the intermediate highly ordered region being aggregated in the cytoplasm and presenting a solid state, and the C-terminal disordered region being distributed in a droplet-like manner in the cell nucleus; The NheI and KpnI enzyme-cleaved fragments were added to the DNA amplification primer sequences of the three corresponding protein fragments through primer synthesis, and then transiently transfected into HEK293T cells through liposomes. After transfection, characterization and verification were performed to obtain the verification results of ALKBH5 protein.
[0005] As some optional embodiments of the present invention, the N-terminal ordered region is 1aa-100aa, the middle highly ordered region is 101aa-270aa, and the C-terminal disordered region is 271aa-394aa.
[0006] As some optional embodiments of the present invention, the N-terminal ordered region is as follows: MAAASGYTDLREKLKSMTSRDNYKAGSREAAAAAAAAVAAAAAAAAAAEPYPVSGAKRKYQEDSDPERSDYEEQQLQKEEEARKVKSGIRQMRLFSQDEC.
[0007] As some optional embodiments of the present invention, the intermediate highly ordered region is as follows: AKIEARIDEVVSRAEKGLYNEHTVDRAPLRNKYFFGEGYTYGAQLQKRGPGQERLYPPGDVDEIPEWVHQLVIQKLVEHRVIPEGFVNSAVINDYQPGGCIVSHVDPIHIFERPIVSVSFFSDSALCFGCKFQFKPIRVSEPVLSLPVRRGSVTVLSGYAADEITHCIRP.
[0008] As some optional embodiments of the present invention, the C-terminal disordered region is as follows: QDIKERRAVIILRKTRLDAPRLETKSLSSSVLPPSYASDRLSGNNRDPALKPKRSHRKADPDAAHRPRILEMDKEENRRSVLLPTHRRRGSFSSENYWRKSYESSEDCSEAAGSPARKVKMRRH.
[0009] As some optional embodiments of the present invention, the characterization verification includes intracellular characterization verification, electron microscopy verification and FRAP experimental verification.
[0010] As some optional embodiments of the present invention, the intracellular characterization verification includes the following steps: The EGFP fusion overexpression vectors of different ALKBH5 protein regions were transiently transfected into HEK293T cells via liposomes, and the cell culture medium was changed 6 hours after transfection; 24 hours to 48 hours after transfection, the three forms of ALKBH5 protein, liquid phase, solid phase, and droplet phase, were observed by fluorescence microscopy. After purification, the protein was placed under a stereo microscope and observed using white light. The three forms of in vitro purified ALKBH5 protein, liquid phase, solid phase, and droplet phase, were observed.
[0011] As some optional embodiments of the present invention, the electron microscopy verification includes the following steps: Transmission electron microscopy scanning was performed on HEK293T cells transfected with EGFP fusion overexpression vectors of different ALKBH5 protein regions to observe the different states of the structures formed by EGFP fusion overexpression vectors of different ALKBH5 protein regions in the cells.
[0012] As some optional embodiments of the present invention, the FRAP experimental verification includes the following steps: Fluorescence photobleaching experiments were used to verify the characteristics of ALKBH5 protein in the solid phase within cells; If the ALKBH5 protein can recover fluorescence within a certain period of time after fluorescence bleaching, it means that the protein distribution presents a flowing liquid or droplet state; If the ALKBH5 protein cannot recover fluorescence within a certain period of time after fluorescence bleaching, it means that the protein distribution is in a non-flowing solid state.
[0013] As some optional embodiments of the present invention, the steps of adding NheI and KpnI enzyme-cleaved fragments to the DNA amplification primer sequences of the three corresponding protein fragments through primer synthesis, and then transiently transfecting them into HEK293T cells via liposomes, and performing characterization and verification after transfection to obtain verification results of the ALKBH5 protein include: The different fragments were synthesized by primers and the NheI and KpnI enzyme-digested fragments were added to the DNA amplification primer sequences of different protein fragments.
[0014] Extract genomic RNA from HEK293T cells and reverse transcribe to obtain cDNA. PCR is then performed to obtain DNA fragments of different protein regions. PCR product purification / gel purification is then performed to recover DNA fragments with single bands. The PCR products of different protein fragments and the pCDN3.1-EGFP vector were then digested with NheI and KpnI, respectively. After incubation at 37°C for 30 minutes, the PCR products were purified to obtain pure digestion products. A ligation reaction was then performed using T4 ligase, and the ligation products were transformed into DH5α competent cells. Single clones were picked and expanded for culture. Finally, sequencing was performed to verify the correct single clones, which were preserved and amplified, subjected to endotoxin extraction, and stored in the refrigerator for later use after concentration measurement. The EGFP fusion overexpression vectors of different ALKBH5 protein regions were transiently transfected into HEK293T cells via liposomes, and the cell culture medium was changed 6 hours after transfection. Characterization verification was performed 24 to 48 hours after transfection to obtain the verification results of ALKBH5 protein.
[0015] Compared with the prior art, the present invention uses the PONDR algorithm for the first time to analyze the structural orderliness of the ALKBH5 protein, and accordingly divides the N-terminal ordered region, the middle highly ordered region, and the C-terminal disordered region. Subsequently, these regions were constructed separately in the pCDN3.1-EGFP vector to achieve the expression of the fusion protein, and the protein phase transition results were further observed. Specifically, the N-terminal ordered region is diffusely distributed in the cell nucleus, the middle highly ordered region aggregates in the cytoplasm to form a solid structure, and the C-terminal disordered region is distributed in a droplet shape in the cell nucleus. In addition, by synthesizing primers and introducing NheI and KpnI enzyme fragments into the DNA amplification primer sequence of the corresponding protein fragment, the present invention uses liposome transient transfection technology to transfer the constructed vector into HEK293T cells, and performs characterization verification after transfection, thereby obtaining the verification results of the ALKBH5 protein. The present invention realizes the analysis of the functional threshold zoning of the ALKBH5 protein and the observation of its intracellular distribution for the first time, and reveals the transformation characteristics of different functional regions of the ALKBH5 protein between the liquid phase, solid phase and droplet phase for the first time. In addition, the present invention successfully constructed a transient overexpression system of different functional regions of the ALKBH5 protein using CMV as a promoter. Compared with the lentiviral system, this system has the advantages of higher expression levels and easier observation. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.
[0017] Figure 1 This is a diagram showing the orderliness of the protein sequence of ALKBH5 analyzed using the PONDR algorithm involved in the present invention; Figure 2 The figures show the expression of different fragments of ALKBH5-GFP protein in cells observed by microscope and the morphology of purified ALKBH5 protein in vitro; Figure 3 The transmission electron microscopy scans of the aggregation morphology of EGFP fusion overexpression vectors of different ALKBH5 protein regions in HEK293T cells involved in the present invention; Figure 4 The FRAP experiment involved in the present invention was performed to detect the solid distribution pattern of ALKBH5-M-GFP protein in cells; Figure 5 Schematic diagram of the process of liquid-solid-droplet phase transformation verification of the demethylase ALKBH5 protein involved in the present invention.
[0018] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION
[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.
[0020] As mentioned above, the ALKBH5 protein is the primary m6A demethylase in mammals, involved in important physiological and pathological processes such as spermatogenesis and tumor development. Currently, ALKBH5 is primarily distributed in the cell nucleus, forming a droplet-like distribution associated with nucleosomes. However, its structural state and the control of different structural states on the protein state remain largely unknown, significantly hindering the study of its function.
[0021] Based on this, the present invention used the PONDR algorithm to perform structural analysis on the protein. The results showed that according to the degree of order of the ALKBH5 protein sequence, it was divided into an N-terminal ordered region (1-100aa), an intermediate highly ordered region (101-270aa), and a C-terminal disordered region (271-394aa). In order to verify the function and distribution of different protein partitions, we constructed overexpression vectors respectively and found that the N-terminal ordered region (1-100aa) was diffusely distributed in the cell nucleus; the intermediate highly ordered region (101-270aa) aggregated in the cytoplasm and exhibited solid-state characteristics; and the C-terminal disordered region (271-394aa) exhibited droplet-like distribution characteristics in the cell nucleus. This discovery explains that the three protein partitions of the ALKBH5 protein control the characteristics of the protein's liquid-solid-droplet three-state transition, providing a certain reference for downstream functional research and a technical basis for downstream intracellular imaging.
[0022] Studies have shown that the disordered region (271-394aa) at the C-terminal end of the ALKBH5 protein promotes liquid-liquid phase separation of ALKBH5 and participates in the regulation of oxidative stress function. However, there have been no reports or studies on the diffuse distribution of the disordered region (1-100aa) at the N-terminal end of the ALKBH5 protein and the highly ordered region (101-270aa) in the middle to promote the solid-state formation of the protein. This study shows that the droplets produced by the disordered region (271-394aa) at the C-terminal end of the ALKBH5 protein are small and require oxidative stress stimulation. The reason is that the study uses a lentiviral transfection system, and its expression is inhibited by endogenous factors. The research of the present invention uses a transient transfection system with CMV as the promoter, which can quickly express protein sequences in large quantities. Therefore, in the expression system of the present invention, three forms of droplet phase, liquid dispersed phase and solid phase can be clearly seen.
[0023] The method of the present invention is mainly achieved by the following steps: The method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein comprises the following steps: The structural order of ALKBH5 protein was analyzed and divided based on the PONDR algorithm, and the N-terminal ordered region, the middle highly ordered region and the C-terminal disordered region were obtained. After constructing the N-terminal ordered region, the intermediate highly ordered region, and the C-terminal disordered region into the pCDN3.1-EGFP vector for fusion protein expression, respectively, the protein phase transition results of the N-terminal ordered region, the intermediate highly ordered region, and the C-terminal disordered region are obtained; the protein phase transition results include the N-terminal ordered region being diffusely distributed in the cell nucleus, the intermediate highly ordered region being aggregated in the cytoplasm and presenting a solid state, and the C-terminal disordered region being distributed in a droplet-like manner in the cell nucleus; The NheI and KpnI enzyme-cleaved fragments were added to the DNA amplification primer sequences of the three corresponding protein fragments through primer synthesis, and then transiently transfected into HEK293T cells through liposomes. After transfection, characterization and verification were performed to obtain the verification results of ALKBH5 protein.
[0024] Specifically, the N-terminal ordered region is 1aa-100aa, the middle highly ordered region is 101aa-270aa, and the C-terminal disordered region is 271aa-394aa.
[0025] Specifically, the N-terminal ordered region is as follows: MAAASGYTDLREKLKSMTSRDNYKAGSREAAAAAAAAVAAAAAAAAAAEPYPVSGAKRKYQEDSDPERSDYEEQQLQKEEEARKVKSGIRQMRLFSQDEC.
[0026] Specifically, the middle highly ordered region is as follows: AKIEARIDEVVSRAEKGLYNEHTVDRAPLRNKYFFGEGYTYGAQLQKRGPGQERLYPPGDVDEIPEWVHQLVIQKLVEHRVIPEGFVNSAVINDYQPGGCIVSHVDPIHIFERPIVSVSFFSDSALCFGCKFQFKPIRVSEPVLSLPVRRGSVTVLSGYAADEITHCIRP.
[0027] Specifically, the C-terminal disordered region is as follows: QDIKERRAVIILRKTRLDAPRLETKSLSSSVLPPSYASDRLSGNNRDPALKPKRSHRKADPDAAHRPRILEMDKEENRRSVLLPTHRRRGSFSSENYWRKSYESSEDCSEAAGSPARKVKMRRH.
[0028] Specifically, the characterization verification includes intracellular characterization verification, electron microscopy verification and FRAP experimental verification.
[0029] Specifically, the intracellular characterization verification includes the following steps: The EGFP fusion overexpression vectors of different ALKBH5 protein regions were transiently transfected into HEK293T cells via liposomes, and the cell culture medium was changed 6 hours after transfection; 24 hours to 48 hours after transfection, the three forms of ALKBH5 protein, liquid phase, solid phase, and droplet phase, were observed by fluorescence microscopy. After purification, the protein was placed under a stereo microscope and observed using white light. The three forms of in vitro purified ALKBH5 protein, liquid phase, solid phase, and droplet phase, were observed.
[0030] Specifically, the electron microscopy verification includes the following steps: Transmission electron microscopy scanning was performed on HEK293T cells transfected with EGFP fusion overexpression vectors of different ALKBH5 protein regions to observe the different states of the structures formed by EGFP fusion overexpression vectors of different ALKBH5 protein regions in the cells.
[0031] Specifically, the FRAP experimental verification includes the following steps: Fluorescence photobleaching experiments were used to verify the characteristics of ALKBH5 protein in the solid phase within cells; If the ALKBH5 protein can recover fluorescence within a certain period of time after fluorescence bleaching, it means that the protein distribution presents a flowing liquid or droplet state; If the ALKBH5 protein cannot recover fluorescence within a certain period of time after fluorescence bleaching, it means that the protein distribution is in a non-flowing solid state.
[0032] Specifically, the steps of adding NheI and KpnI enzyme-cleaved fragments to the DNA amplification primer sequences of the three corresponding protein fragments through primer synthesis, and then transiently transfecting them into HEK293T cells through liposomes, and performing characterization verification after transfection to obtain verification results of the ALKBH5 protein include: The different fragments were synthesized by primers and the NheI and KpnI enzyme-digested fragments were added to the DNA amplification primer sequences of different protein fragments.
[0033] Extract genomic RNA from HEK293T cells and reverse transcribe to obtain cDNA. PCR is then performed to obtain DNA fragments of different protein regions. PCR product purification / gel purification is then performed to recover DNA fragments with single bands. The PCR products of different protein fragments and the pCDN3.1-EGFP vector were then digested with NheI and KpnI, respectively. After incubation at 37°C for 30 minutes, the PCR products were purified to obtain pure digestion products. A ligation reaction was then performed using T4 ligase, and the ligation products were transformed into DH5α competent cells. Single clones were picked and expanded for culture. Finally, sequencing was performed to verify the correct single clones, which were preserved and amplified, subjected to endotoxin extraction, and stored in the refrigerator for later use after concentration measurement. The EGFP fusion overexpression vectors of different ALKBH5 protein regions were transiently transfected into HEK293T cells via liposomes, and the cell culture medium was changed 6 hours after transfection. Characterization verification was performed 24 to 48 hours after transfection to obtain the verification results of ALKBH5 protein.
[0034] The method of the present invention is further described in detail below with reference to specific embodiments: Step 1.1. Obtain the ALKBH5 protein sequence (NP_060228.3) and mRNA sequence (NM_017758.4) from the NCBI website. Obtain their FASTA formats as follows: >NP_060228.3 RNA demethylase ALKBH5 [Homo sapiens] MAAASGYTDLREKLKSMTSRDNYKAGSREAAAAAAAAVAAAAAAAAAAEPYPVSGAKRKYQEDSDPERSD YEEQQLQKEEEARKVKSGIRQMRLFSQDECAKIEARIDEVVSRAEKGLYNEHTVDRAPLRNKYFFGEGYT YGAQLQKRGPGQERLYPPGDVDEIPEWVHQLVIQKLVEHRVIPEGFVNSAVINDYQPGGCIVSHVDPIHI FERPIVSVSFFSDSALCFGCKFQFKPIRVSEPVLSLPVRRGSVTVLSGYAADEITHCIRPQDIKERRAVI ILRKTRLDAPRLETKSLSSSVLPPSYASDRLSGNNRDPALKPKRSHRKADPDAAHRPRILEMDKEENRRS VLLPTHRRRGSFSSENYWRKSYESSEDCSEAAGSPARKVKMRRH >NM_017758.4 Homo sapiens alkB homolog 5, RNA demethylase (ALKBH5),mRNA GAGGAGCCCGCTAAGGAGCGGCGCTGGCGGACGTCGGGCTGGCTGCCCGTGACGTCGTGCGGAGAGCTTT AAAGTGCGGGCCGGGCCGGGCGTCCGAGGGTCTGGTCGGGAGTCGGGCCGCGTCTCCGCAGCAGCCCTCC GCGGCATGAGGCGCTGCCGGCGCCCCTGCCCCGCGGGACGTGGAGAAGGTGGAGGAGGAAGAAGCCCCGT TGTCGCCACCGTTGCATGACCCGCCGCTCCTGAGGCCCTACCCCACGCCCGGACCCTCGACGCCCCCCGC CGGGTCCCCCACTCACGCATGGGGGTTCGGCGCTAAGGACCCCCCTCCCTCCGGGGGCCCCGGGGCGCGT CCCCTTAGAGCCATGCCCGGCTGCCCCGCCCGCCCCGGAGGACCCTAGAGCAGCGTCGTGGGGGCCATGG CGGCCGCCAGCGGCTACACGGACCTGCGTGAGAAGCTCAAGTCCATGACGTCCCGGGACAACTATAAGGC GGGCAGCCGGGAGGCCGCCGCCGCTGCCGCAGCCGCCGTAGCCGCCGCAGCCGCAGCCGCCGCTGCCGCC GAACCTTACCCTGTGTCCGGGGCCAAGCGCAAGTATCAGGAGGACTCGGACCCCGAGCGCAGCGACTATG AGGAGCAGCAGCTGCAGAAGGAGGAGGAGGCGCGCAAGGTGAAGAGCGGCATCCGCCAGATGCGCCTCTT CAGCCAGGACGAGTGCGCCAAGATCGAGGCCCGCATTGACGAGGTGGTGTCCCGCGCTGAGAAGGGCCTG TACAACGAGCACACGGTGGACCGGGCCCCACTGCGCAACAAGTACTTCTTCGGCGAAGGCTACACTTACG GCGCCCAGCTGCAGAAGCGCGGGCCCGGCCAGGAGCGCCTCTACCCGCCGGGCGACGTGGACGAGATCCC CGAGTGGGTGCACCAGCTGGTGATCCAAAAGCTGGTGGAGCACCGCGTCATCCCCGAGGGCTTCGTCAAC AGCGCCGTCATCAACGACTACCAGCCCGGCGGCTGCATCGTGTCTCACGTGGACCCCATCCACATCTTCG AGCGCCCCATCGTGTCCGTGTCCTTCTTTAGCGACTCTGCGCTGTGCTTCGGCTGCAAGTTCCAGTTCAA GCCTATTCGGGTGTCGGAACCAGTGCTTTCCCTGCCGGTGCGCAGGGGAAGCGTGACTGTGCTCAGTGGA TATGCTGCTGATGAAATCACTCACTGCATACGGCCTCAGGACATCAAGGAGCGCCGAGCAGTCATCATCC TCAGGAAGACAAGATTAGATGCACCCCGGTTGGAAACAAAGTCCCTGAGCAGCTCCGTGTTACCACCCAG CTATGCTTCAGATCGCCTGTCAGGAAACAACAGGGACCCTGCTCTGAAACCCAAGCGGTCCCACCGCAAG GCAGACCCTGATGCTGCCCACAGGCCACGGATCCTGGAGATGGACAAGGAAGAGAACCGGCGCTCGGTGC TGCTGCCCACACACCGGCGGAGGGGTAGCTTCAGCTCTGAGAACTACTGGCGCAAGTCATACGAGTCCTC AGAGGACTGCTCTGAGGCAGCAGGCAGCCCTGCCCGAAAGGTGAAGATGCGGCGGCACTGAGTCTACCCG CCGCCCTCCTGGGAACTCTGGCTCATCCTTACGTAGTTGCCCCTCCTTTTGTTTTGAGGGTTTTGTTTTT GTTCATTGGGGGGTTTTTGTTTTTTGTTTTTTGTTTTTTTTGATTCTATATATTTTTCCTTGGTTTTGTT GCCTGTTAGGGCTGAAGAATAGAATTGGCCAGGACCTAGGTTCTCATATTCTTGGTATTCCTCCTGGATG GAAAGGCTGTTGGCATCAATAGGGGACAGAGGCTGATGCTGGAGTGGCCAGTAGAGGTGGTGGAGCAGAG CAGCCATCTTTTAAGTGGGGCTGTATCAGGCTGGGTTTATTTAAAAGCAACAAAATGTTTTGGTTAAGAA AATTATTTTGCTTTCAGTGTAAATCTTCGCAGTGTTCTAAACAAAGTTCAGTCTTCTGCTCGCCCCTTTC CCTCACTGATGTCTGCACTTGGTTGAGGTCTCCTGGAGCCTCACAGGCTCTGCTGTTCTCCACTTCTCAC CTGCCATCCACGCCCTGCAAGCTCATGCAAACACCCTTTCTTCCTCCTGCGGCAGAGTTGTTCAGGTTGC CTGGGCAGGGGCTTAAACAGTGCCAGCCCCTGCCATCCCAAAGCTATTGTTAAGCCCCCCAGGCGTCCTC CACCCACGCCCACTAGCCTGCCATGTCCACAGTTCCTTGGGCTGCTGAGGGGCTAGTGCAGTGGTCCTGA CCTCTCTTATCAAGAGCACACTTCTTTGCTGGTTGCTCCTTTTGAGCATATGCGTGTGATTATTTGGAAC AGTTAGACTTGCCACGTTGGGTCAGTTTTAGAAATTGTTTCTAGCTAGAGGGACTGGTGTCCTTCCAAGT CTAGCATTTGGGGTATGGAAAATTGTTGTGGTGTGTGGTAGGGTTTTTGTTTTCTTTTTTGAGTTTTTTT TCCCCCTTTAGTCTCCTGGCTTTTTCCTTTCCCTTCCCTTCTCCACTGGCCAGCTTGGGCCTCATCCTCA TGTCATCCTTCTAGGAAGGCGCCTGCCCCATCTTGTCTGCCGGCAGCATGCATCCAAGGCCAGAGCTCAG GCCTGCAGACTGGGCTGGTGCCTCCTCCGCTTCAGGGTATGGGAGTTGGTGAAGGGGCTTTCAAAAAATA ATAAGGAAAAAAAGGTAAAGTCTTTGGTAGCTTCTATCCACTCAGATCCTGGAAGGCAGCAAGGTTTTGT GGATCTAGATTCATTAGGAATGTCTTCTTGTCAGCCAGGCCAGGACCCGGGCTTGCCAAGAGCAGAGGCC CTCCCAGCAACCAGGATACCACCACTTTGGGGGCTTTGTGTACAGAGGTCCGGGTCTGAGACCTCATAGG CTGCAGAAATCTGGGGCAGCCACCATCAAGAAGCCCCTCTCAGGGGCCAGAACTCCTTTGCCAGCGTGGA TTTCTCAAGTCGGGACTGCATAATTAAAGCAGTTGCAGTTTTATTTTTTTTACAGCTTTTTTCCCAAAAA TGATTTGTAGTTGTGTGTGCAGCACTTCGCCCTGATATGTGTGCTCTACAATAAAAACCAAATCTAATAT ATTTTGAAA Step 1.2: Copy the ALKBH5 protein sequence (NP_060228.3) in FASTA format to the Natural Disordered Regions (PONDR, www.pondr.com) website, select VSL2, VL3, and VL-XT parameters, and follow the default parameters for protein order analysis. The following results are obtained: Figure 1 The results of the PONDR algorithm analysis of the order of the ALKBH5 protein sequence are shown. The ALKBH5 protein is divided into an N-terminal ordered region (1-100aa), an intermediate highly ordered region (101-270aa), and a C-terminal disordered region (271-394aa). The sequences of the different protein regions are as follows: ALKBH5-N(1-100aa): MAAASGYTDLREKLKSMTSRDNYKAGSREAAAAAAAAVAAAAAAAAAAEPYPVSGAKRKYQEDSDPERSDYEEQQLQKEEEARKVKSGIRQMRLFSQDEC; ALKBH5-M(101-270aa): AKIEARIDEVVSRAEKGLYNEHTVDRAPLRNKYFFGEGYTYGAQLQKRGPGQERLYPPGDVDEIPEWVHQLVIQKLVEHRVIPEGFVNSAVINDYQPGGCIVSHVDPIHIFERPIVSVSFFSDSALCFGCKFQFKPIRVSEPVLSLPVRRGSVTVLSGYAADEITHCIRP; ALKBH5-C(271-394aa): QDIKERRAVIILRKTRLDAPRLETKSLSSSVLPPSYASDRLSGNNRDPALKPKRSHRKADPDAAHRPRILEMDKEENRRSVLLPTHRRRGSFSSENYWRKSYESSEDCSEAAGSPARKVKMRRH.
[0035] In step 1.3, the present invention divides the ALKBH5 protein sequence into an N-terminal ordered region (1-100 aa), a highly ordered intermediate region (101-270 aa), and a C-terminal disordered region (271-394 aa) based on the PONDR algorithm. Primers for amplifying different fragments are then designed based on the mRNA sequence (NM_017758.4). The NheI and KpnI digested fragments are then added to the DNA amplification primer sequences for the different protein fragments through primer synthesis. Genomic RNA from HEK293T cells is extracted and reverse transcribed to obtain cDNA. DNA fragments from the different protein regions are then obtained through PCR. PCR product purification / gel purification is then performed to recover DNA fragments with single bands. The PCR products of the different protein fragments and the pCDN3.1-EGFP vector are then digested using the NheI and KpnI double enzyme digestion system. After incubation at 37°C for 30 minutes, the PCR products are purified to obtain pure digested products. Then, the ligation reaction was carried out using T4 ligase, and the ligation product was transformed into DH5α competent cells. Single clones were picked and expanded. Finally, sequencing was performed to verify, and the correct single clone fragment was preserved and amplified. After endotoxin extraction, the concentration was measured and stored in the refrigerator for future use. The synthetic primer sequences are as follows: ALKBH5-N(1-100aa)-FP:GCTAGCatggcggccgccagcggcta; ALKBH5-N(1-100aa)-RP:gcactcgtcctggctgaagaCCATGG; ALKBH5-M(101-270aa)-FP:GCTAGCgccaagatcgaggcccgcat; ALKBH5-M(101-270aa)-FP:aggccgtatgcagtgagtgaCCATGG; ALKBH5-C(271-394aa)-FP:GCTAGCcaggacatcaaggagcgccg; ALKBH5-C(271-394aa)-RP:gtgccgccgcatcttcacctCCATGG.
[0036] Step 1.4, the EGFP fusion overexpression vectors of different ALKBH5 protein regions were transiently transfected into HEK293T cells via liposomes, and the cell medium was changed 6 hours after transfection. Between 24 hours and 48 hours after transfection, the three forms of ALKBH5 protein, liquid phase, solid phase, and droplet phase, could be clearly observed under a fluorescence microscope. After the protein was purified, it was placed under a stereo microscope and observed using white light, and the three forms of in vitro purified ALKBH5 protein, liquid phase, solid phase, and droplet phase, could also be seen, as shown in Figure 2. Figure 2 As shown, it was found that the N-terminal ordered region (1-100aa) was diffusely distributed in the cell nucleus; the middle highly ordered region (101-270aa) aggregated in the cytoplasm and showed solid characteristics; the C-terminal disordered region (271-394aa) showed droplet-like distribution characteristics in the cell nucleus.
[0037] Step 1.5: To further observe the different states of the intracellular structures formed by EGFP fusion overexpression vectors of different ALKBH5 protein regions, we used HEK293T cells transfected with EGFP fusion overexpression vectors of different ALKBH5 protein regions to perform transmission electron microscopy scanning to observe their intracellular structures. The results are as follows: Figure 3 shown.
[0038] Step 1.6. To further verify the solid phase characteristics of ALKBH5 protein in cells, we used fluorescence photobleaching experiment (FRAP experiment) to detect. If ALKBH5 protein can recover fluorescence within a certain period of time after fluorescence photobleaching, it means that the protein distribution is in a flowing liquid or droplet state; if ALKBH5 protein cannot recover fluorescence within a certain period of time after fluorescence photobleaching, it means that the protein distribution is in a non-flowing solid state. After verification, it was found that ALKBH5-WT-GFP (full-length ALKBH5 protein) formed a droplet phase in cells. In contrast, ALKBH5-M-GFP formed a solid phase in cells. The results are as follows: Figure 4 shown.
[0039] It can be seen that the present invention Figure 5The steps shown are the first use of the PONDR algorithm to analyze the structural orderliness of the ALKBH5 protein, and based on this, the N-terminal ordered region, the middle highly ordered region, and the C-terminal disordered region are divided. Subsequently, these regions are constructed separately in the pCDN3.1-EGFP vector to achieve the expression of the fusion protein, and the protein phase transition results are further observed. Specifically, the N-terminal ordered region is diffusely distributed in the cell nucleus, the middle highly ordered region aggregates in the cytoplasm to form a solid structure, and the C-terminal disordered region is distributed in a droplet shape in the cell nucleus. In addition, by synthesizing primers and introducing NheI and KpnI enzyme fragments into the DNA amplification primer sequence of the corresponding protein fragment, the present invention uses liposome transient transfection technology to transfer the constructed vector into HEK293T cells, and performs characterization verification after transfection, thereby obtaining the verification results of the ALKBH5 protein. The present invention realizes the analysis of the functional threshold zoning of the ALKBH5 protein and the observation of its intracellular distribution for the first time, and reveals the transformation characteristics of different functional regions of the ALKBH5 protein between the liquid phase, solid phase and droplet phase for the first time. In addition, the present invention successfully constructed a transient overexpression system of different functional regions of the ALKBH5 protein using CMV as a promoter. Compared with the lentiviral system, this system has the advantages of higher expression levels and easier observation.
[0040] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.
Claims
1. A method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein, characterized in that: The following steps are involved: The structural order of ALKBH5 protein was analyzed and divided based on the PONDR algorithm, and the N-terminal ordered region, the middle highly ordered region and the C-terminal disordered region were obtained. After constructing the N-terminal ordered region, the intermediate highly ordered region, and the C-terminal disordered region into the pCDN3.1-EGFP vector for fusion protein expression, respectively, the protein phase transition results of the N-terminal ordered region, the intermediate highly ordered region, and the C-terminal disordered region are obtained; the protein phase transition results include the N-terminal ordered region being diffusely distributed in the cell nucleus, the intermediate highly ordered region being aggregated in the cytoplasm and presenting a solid state, and the C-terminal disordered region being distributed in a droplet-like manner in the cell nucleus; The NheI and KpnI enzyme-cleaved fragments were added to the DNA amplification primer sequences of the three corresponding protein fragments through primer synthesis, and then transiently transfected into HEK293T cells through liposomes. After transfection, characterization and verification were performed to obtain the verification results of ALKBH5 protein.
2. The method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein according to claim 1, wherein: The N-terminal ordered region is 1aa-100aa, the middle highly ordered region is 101aa-270aa, and the C-terminal disordered region is 271aa-394aa.
3. The method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein according to claim 1, characterized in that: The N-terminal ordered region is as follows: MAAASGYTDLREKLKSMTSRDNYKAGSREAAAAAAAAVAAAAAAAAAAEPYPVSGAKRKYQEDSDPERSDYEEQQLQKEEEARKVKSGIRQMRLFSQDEC.
4. The method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein according to claim 1, characterized in that: The intermediate highly ordered region is shown below: AKIEARIDEVVSRAEKGLYNEHTVDRAPLRNKYFFGEGYTYGAQLQKRGPGQERLYPPGDVDEIPEWVHQLVIQKLVEHRVIPEGFVNSAVINDYQPGGCIVSHVDPIHIFERPIVSVSFFSDSALCFGCKFQFKPIRVSEPVLSLPVRRGSVTVLSGYAADEITHCIRP.
5. The method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein according to claim 1, characterized in that: The C-terminal disordered region is as follows: QDIKERRAVIILRKTRLDAPRLETKSLSSSVLPPSYASDRLSGNNRDPALKPKRSHRKADPDAAHRPRILEMDKEENRRSVLLPTHRRRGSFSSENYWRKSYESSEDCSEAAGSPARKVKMRRH.
6. The method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein according to claim 1, characterized in that: The characterization verification includes intracellular characterization verification, electron microscopy verification and FRAP experimental verification.
7. The method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein according to claim 6, characterized in that: The intracellular characterization validation includes the following steps: The EGFP fusion overexpression vectors of different ALKBH5 protein regions were transiently transfected into HEK293T cells via liposomes, and the cell culture medium was changed 6 hours after transfection; 24 hours to 48 hours after transfection, the three forms of ALKBH5 protein, liquid phase, solid phase, and droplet phase, were observed by fluorescence microscopy. After purification, the protein was placed under a stereo microscope and observed using white light. The three forms of in vitro purified ALKBH5 protein, liquid phase, solid phase, and droplet phase, were observed.
8. The method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein according to claim 6, characterized in that: The electron microscopy verification comprises the following steps: Transmission electron microscopy scanning was performed on HEK293T cells transfected with EGFP fusion overexpression vectors of different ALKBH5 protein regions to observe the different states of the structures formed by EGFP fusion overexpression vectors of different ALKBH5 protein regions in the cells.
9. The method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein according to claim 6, characterized in that: The FRAP experimental verification includes the following steps: Fluorescence photobleaching experiments were used to verify the characteristics of ALKBH5 protein in the solid phase within cells; If the ALKBH5 protein can recover fluorescence within a certain period of time after fluorescence bleaching, it means that the protein distribution presents a flowing liquid or droplet state; If the ALKBH5 protein cannot recover fluorescence within a certain period of time after fluorescence bleaching, it means that the protein distribution is in a non-flowing solid state.
10. The method for verifying the liquid-solid-droplet phase transformation of the demethylase ALKBH5 protein according to claim 1, characterized in that: The steps of adding the NheI and KpnI enzyme-cleaved fragments to the DNA amplification primer sequences of the three corresponding protein fragments through primer synthesis, and then transiently transfecting them into HEK293T cells through liposomes, and performing characterization and verification after transfection to obtain verification results of the ALKBH5 protein include: The different fragments were synthesized by primers. The NheI and KpnI enzyme-digested fragments were added to the DNA amplification primer sequences of different protein fragments. Extract genomic RNA from HEK293T cells and reverse transcribe to obtain cDNA. PCR is then performed to obtain DNA fragments of different protein regions. PCR product purification / gel purification is then performed to recover DNA fragments with single bands. The PCR products of different protein fragments and the pCDN3.1-EGFP vector were then digested with NheI and KpnI, respectively. After incubation at 37°C for 30 minutes, the PCR products were purified to obtain pure digestion products. A ligation reaction was then performed using T4 ligase, and the ligation products were transformed into DH5α competent cells. Single clones were picked and expanded for culture. Finally, sequencing was performed to verify the correct single clones, which were preserved and amplified, subjected to endotoxin extraction, and stored in the refrigerator for later use after concentration measurement. The EGFP fusion overexpression vectors of different ALKBH5 protein regions were transiently transfected into HEK293T cells via liposomes, and the cell culture medium was changed 6 hours after transfection. Characterization verification was performed 24 to 48 hours after transfection to obtain the verification results of ALKBH5 protein.