CN112481232B

CN112481232B - Bacterial protein lysine deacetylation modification enzyme and application thereof

Info

Publication number: CN112481232B
Application number: CN202011467147.3A
Authority: CN
Inventors: 林向民; 汪玉倩; 张丽珊
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2022-12-20
Anticipated expiration: 2040-12-14
Also published as: CN112481232A

Abstract

The invention discloses a bacterial protein lysine deacetylation modification enzyme and application thereof, wherein the amino acid sequence of the bacterial protein lysine deacetylation modification enzyme is shown as SEQ ID NO.1, and the acetylation modification of the bacterial protein lysine plays an important role in a plurality of key biological processes, such as transcription regulation, quorum sensing, metabolism and the like. Bacteria can acetylate and modify the lysine residues of proteins by a plurality of acetylases or non-enzymatic chemical reactions, but only a few kinds of lysine deacetylation modifying enzymes have been found so far. The invention provides a novel bacterial protein lysine deacetylation modification enzyme, which further improves the understanding of people on protein posttranslational modification, plays a promoting role in the research of the complex physiological functions of bacteria and is beneficial to the prevention and treatment of the bacteria.

Description

Bacterial protein lysine deacetylation modification enzyme and application thereof

Technical Field

The invention relates to a bacterial protein lysine deacetylation modification enzyme and application thereof, belonging to the technical field of biology.

Background

Protein posttranslational modification (PTM)) is widely present in natural life bodies, greatly expands and extends the limited functional range of proteins formed by direct coding and transcription of DNA, and plays an important role in cell differentiation, signal recognition, and metabolic physiological processes. Since the first discovery of lysine acetylation modification in histones by Allfrey et al in 1964, lysine acetylation modification has been intensively studied. At present, the modification plays a significant role in various organisms, and can be involved in various important biological regulation processes such as chromatin structure, transcription and replication, metabolism, signal transduction and the like.

On the other hand, the protein needs a specific lysine deacetylation modification enzyme to perform reversible regulation on the function of the PTMs protein. The function and species of lysine deacetylation modification enzyme are most deeply studied in eukaryotes at present. There are four classes of lysine deacetylation modifying enzymes (KDACs) in eukaryotic cells, of which types I, II and IV are Zn2+ -dependent KDACs, and type III KDACs, also known as Sirtuins, require NAD + cofactors. Sirtuins can be divided into 7 classes (SIRT 1-7) which are distributed in different spaces of the cell and may also have other deacylase activities. While KDACs in bacteria tend to have homology with KDACs from eukaryotic cells, mainly including Zn2+ -dependent RpLda (homologous to type II), zn2+ -dependent AcuC (homologous to type I), and NAD + -dependent Sirtuins (homologous to type III) family proteins. The Sirtuins represented by escherichia coli CobB and family proteins thereof are distributed most widely, the research on substrate proteins of the escherichia coli CobB and the family proteins thereof is also most widely, and the Sirtuins are found in various bacterial species and relate to the regulation and control of various important physiological functions such as bacterial virulence, chemotaxis, growth metabolism and the like. Coli YcgC is also reported in eLife journal in 2015 to be a novel deacetylation modifying enzyme independent of Zn2+ and NAD +, and 2016, nature Reviews Microbiology journal recommends the discovery as a research hotspot, which indicates the importance of discovering the novel deacetylation modifying enzyme, but the protein function of the enzyme is yet to be further confirmed. In addition, no new lysine deacetylation modification enzyme has been found for bacterial proteins.

However, the number of lysine acetylation-modified proteins in different species varies from hundreds to thousands, involving thousands of modification sites, participating in numerous physiological functions and complex environmental responses, and theoretically is unlikely to be regulated by only one or a few deacetylation-modified enzymes, but the currently known types of bacterial deacetylation-modified enzymes are obviously far different from the scale of acetylation-modified proteins, and it is likely that some unknown KDACs in bacteria act on specific protein substrates to maintain normal physiological functions of cells. Therefore, the search for new bacterial protein lysine deacetylation modification enzyme and its function is one of the important research contents in the current biomacromolecule modification field.

Disclosure of Invention

The invention aims to provide a bacterial protein lysine deacetylation modified enzyme and application thereof. The protein does not need to depend on Zn ²⁺ With NAD ⁺ The auxiliary can obviously remove BSA acetylation modification, has no obvious homology with the currently known deacetylation modification enzyme, and is a novel deacetylation enzyme family protein family.

A bacterial protein lysine deacetylation modifying enzyme is A0KI27, and the amino acid sequence thereof is shown in SEQ ID NO. 1. Found in Aeromonas hydrophila ATCC 7966, the gene of which is encoded asAHA_1389。

SEQ ID NO.1：MIVWTVANQKGGVGKTTTVVSLAGILAQRGQRVLLIDTDPHASLTSYLDFDSDRLDGTLYELFQAVKPTAELVNKLTLRTKFDNIHLLPASITLATLDRVMGNREGMGLVLKRALLRIQDQYDYVLIDCPPVLGVMMVNALAACDRILVPVQTEFLALKGLERMMKTFEIMQRSKREKFRYTVIPTMFDKRTRASLMTLQSIKEQHGNAVWNAVIPIDTKFRDASLLHIPPSIYSPSSRGTYAYETLLNYLDAQERQRAHEVTS。

The gene of the bacterial protein lysine deacetylation modifying enzyme is coded, and the gene sequence is shown as SEQ ID NO. 2.

SEQ ID NO.2：gtgattgtttggacggttgccaaccaaaagggtggggtcggtaaaaccaccacagtggtctcgctggccggtattctggcccagcgcggccagcgggtgctgttgattgacaccgaccctcatgcctccctgacctcttatctcgattttgattccgaccggctggatggcactctttatgagctgtttcaggcggtcaaaccaacggcagagctcgtcaataaactgacgctgcgcaccaagtttgacaacattcatctgctgcccgcgtcaatcaccctggcgaccctggatcgggtaatgggcaatcgggaagggatgggacttgtgctcaagcgagcgttgctgcgcattcaggatcagtacgattatgtgctgatcgactgcccgcccgtcttgggggtcatgatggtcaatgcgctggcggcgtgtgaccggatcctggtgccggtacagaccgagtttctggcgctcaaagggctggagcggatgatgaagacgtttgaaatcatgcagcgctccaagcgggagaagttccgttataccgtcattcccaccatgtttgacaagcgaacccgtgcctcgctgatgactctgcagtccatcaaggagcagcacggcaatgcggtatggaatgccgtcatacccatagataccaaattccgggatgccagtttgcttcacattcccccctccatctattcgccgagcagccgtggcacctatgcctatgagaccctgctcaactatcttgatgcgcaagagcgtcagcgtgctcatgaggtaacatcatga。

The bacterial protein lysine deacetylation modification enzyme is applied to lysine deacetylation modification.

The invention has the advantages that:

the protein does not need to depend on Zn ²⁺ With NAD ⁺ The auxiliary can obviously remove BSA acetylation modification, has no obvious homology with the currently known deacetylation modification enzyme, and is a novel deacetylation enzyme family protein family. Further improves the understanding of people on protein modification, plays a promoting role in the research on the complex physiological function of bacteria and is beneficial to the prevention and treatment of the bacteria.

Drawings

FIG. 1 is a view showing an embodiment of the present inventionAHA_1389Constructing a gene deletion strain; (A) Is composed ofcobB(771 bp) andAHA_1389PCR validation of the (792 bp) deletion strain.

Lanes

1 and 2, respectivelyWith WT andΔcobBas a template, amplificationcobBAbout 500bp fragments of the upstream and the downstream of the gene respectively;

lanes

3 and 4 are WT and 4, respectivelyΔahcobBAs a template, amplificationcobBA gene fragment; lanes 5-8 are amplified as beforeAHA_1389A fragment of interest. The PCR products in

lanes

2 and 6 were sent to the company for sequence verification to confirm that the target gene was successfully knocked out.

FIG. 2 the resulting Aeromonas hydrophila CobB and A0KI27 proteins were purified. A and B are 6 XHis-tagged CobB and A0KI27 proteins purified by nickel column, respectively.

FIG. 3 dot blotting validation of Aeromonas hydrophilacobBAndAHA_1389deletion results in an increased level of acetyl modification of the protein. Is shown inΔcobB、ΔAHA_1389And the upper part of the acetyl modification condition is the protein loading amount shown by the dyeing of PVDF membrane G350 under the condition of different protein dilution gradients of wild type aeromonas hydrophila, and the lower part is the Dolt blotting result of the anti-specificity PTM antibody.

FIG. 4 Aeromonas hydrophilacobBAndAHA_1389deletion strain acetylation modified immunoblot analysis. Are respectively asΔ cobB、ΔAHA_1389And SDS-PAGE Coomassie blue staining of wild type Aeromonas hydrophila holoprotein (A) and Western blotting against acetylation (B). In the case where the amount of the sample is substantially uniform,ΔcobBandΔAHA_1389the level of this acetylation modification was significantly increased.

FIG. 5 Western blotting validated in vitro acetylated BSA; 2 lanes are immunoblot experiments of acetylated anti-experience treated acetylated BSA versus untreated BSA in vitro, respectively.

FIG. 6 is a graph showing the results of experiments on deacetylation modification in a specific embodiment; western blotting experiment shows that Aeromonas hydrophila A0KI27, ahCobB and acetylated BSA are in NAD ⁺ After incubation, the BSA acetylation of the cells was changed. The upper part of the graph, showing the level of acetylation modification of the various samples under different treatments, and the lower part of the graph, showing the G350 staining of their PVDF membranes, shows that the samples are loaded in substantially uniform amounts, and the above results are repeated at least 3 times.

FIG. 7 is a graph of domain comparison analysis showing that Aeromonas hydrophila CobB and A0KI27 proteins belong to different protein families; aeromonas hydrophila CobB belongs to the Sirtuin protein family, and A0KI27 is homologous with proteins of chromosome division ATPase, vitamin B12 synthetic protein, plasmid partition protein and the like of other species, and all have AAA-31 Pfam belonging to P-loop _ NTPase.

Detailed Description

Example 1

1. Material method

Experimental materials the vectors pET-32a (+), pRE112 and Aeromonas hydrophila ATCC 7966 required for the experiment were stored in the laboratory, the enzyme was purchased from Thermo, and the Escherichia coli BL21 competent cells were purchased from holo gold biotechnology, inc.; the gel recovery kit and the plasmid extraction kit are purchased from Omega company; antibodies were purchased from PTM Biolabs, other relevant reagents from Sigma, and primers synthesized by foryowa bon.

2. Experimental procedure

2.1 construction of Gene-deleted Strain

According to the whole genome sequence of Aeromonas hydrophila ATCC 7966, fragments of about 500bp of the upstream and downstream target genes A0KI27 and CobB are respectively taken, primers at two ends of the upstream fragment are respectively corresponding P1 and P2, primers at two ends of the downstream fragment are respectively corresponding P3 and P4, and primers at two ends of the target gene are respectively corresponding P5 and P6. In addition, setting a primer P7 on the fragment about 100bp upstream of P1, and similarly setting a primer P8 on the fragment about 100bp downstream of P4; then, respectively amplifying fragments of 500bp at the upstream and downstream of the target gene, connecting the fragments to the digested pRE112 plasmid by using a seamless ligase (C113-01/02, vazyme Biotech Co. Ltd.), transferring the fragments into MC1061 competence, selecting a single clone, shaking bacteria at 37 ℃,200rpm, verifying by PCR (P1P 4), sequencing quality-improved particles, and preserving the bacteria at-20 ℃.

Transferring the positive plasmid in the step into escherichia coli S17-1 competence, selecting a monoclonal, shaking the bacteria at 37 ℃,200rpm, verifying by PCR (P1P 4), and preserving the bacteria at-20 ℃ for later use; next, the correct S17-1 bacterium was mixed with the waterRespectively shaking aeromonas ATCC 7966 to OD 1.0, uniformly mixing according to the proportion of 4; next, the filter paper containing the culture in the previous step was taken out and placed in an EP tube, 500. Mu.l of fresh LB was repeatedly washed, the culture was completely washed off, the filter paper was discarded, centrifuged, and the supernatant was discarded. Washing repeatedly for 2-3 times, re-suspending the precipitate with 500 μ l fresh LB, applying 100 μ l bacterial liquid to LB plate containing ampicillin (100 μ g/ml) and chloramphenicol (30 μ g/ml) resistance, picking single clone, shaking at 30 deg.C and 200rpm, PCR (P1P 4) bacterial liquid, sequencing, and preserving at-20 deg.C; finally, the correct overnight strain verified in the previous step is taken, diluted by 1000 times, 100 mul of diluted bacterial liquid is taken and coated on an LB plate containing 20% sucrose, a monoclonal is selected, shaken at 30 ℃ and 200rpm for 4h, then, 2 mul of bacterial liquid is taken and spotted on a chlorine mycin plate drawn with lattices, bacteria which do not grow on the chlorine mycin plate are selected for PCR verification, sequencing is carried out, heredity is stabilized, and bacteria are preserved at-80 ℃ for later use. Is finally obtainedcobBDeletion strains andAHA_1389a gene-deleted strain.

The results are shown in FIG. 1,

lanes

1 and 2 are WT and WT, respectivelyΔcobB（cobBDeletion strain) (2.1 is the gene deletion method) as a template, and amplifying fragments of about 500bp respectively at the upstream and the downstream of the cobB gene;

lanes

3 and 4 are WT and 4, respectivelyΔcobBAs a template, cobB gene fragments were amplified; lanes 5-8 the AHA _1389 target fragment was amplified as before. The PCR products in

lanes

2.2 Purification of A0KI27 protein

Using Aeromonas hydrophila ATCC 7966 total genome as a templateAHA_1389AndcobBthe gene primers were subjected to PCR amplification, respectively. The obtained specific fragment is connected with pET-32a plasmid which is respectively subjected to double enzyme digestion by HindIII and EcoRI to obtain recombinant plasmid, and the recombinant plasmid is transformed intoE.coliBL21 competent cells were plated on LB (100. Mu.g/mL AMP.) plates and incubated overnight at 37 ℃. Picked positive single clones were cultured overnight. Then transferred to 200 mL of fresh LB culture broth (containing 100. Mu.g/mL AMP) was cultured at 37 ℃ and 200rpm until OD600=0.3-0.6, and 1 mmol/L IPTG was added to induce the strain at 16 ℃ for 12 h. Then, the cells were centrifuged at 10 000 Xg at 4 ℃ and the supernatant was discarded and washed twice with Phosphate Buffered Saline (PBS), and the cells were resuspended in 5 mmol/L imidazole solution and sonicated. After centrifugation, the supernatant was purified by passing through a Ni-NTA resin column, the purified band was detected by SDS-PAGE (FIG. 2), and the obtained protein was stored at-20 ℃. As can be seen from FIG. 2, the band of the target protein obtained by purification is single and the size is as expected.

The target protein obtained by sequencing is A0KI27, and the amino acid sequence of the target protein is shown in SEQ ID NO. 1.

2.3 Dot-spot

Approximately 10 μ g of protein was spotted onto PVDF membrane. After air drying the samples, the membranes were blocked with 5% BSA in TBST for 4h and then incubated overnight with 10. Mu.l of an anti-lysine acetyl modified antibody (purchased from Chongzhou Jingjie, (PTM Biolabs Inc., hangzhou, china)). Subsequently, after five washes in TBS, the samples were incubated for 1h with horseradish peroxidase-conjugated goat anti-rabbit antibody (Comwin, beijing, china) (secondary antibody) containing a mouse antibody with 3% BSA. Membranes from dot blots were detected using a Clarity Western ECL substrate and visualized by Image Lab software (BioRad) on a ChemiDoc MP imaging system. Finally, the PVDF membrane was stained with Coomassie R-350 to verify that the loadings were equal. The results are shown in figure 3 of the drawings,ΔcobB、ΔAHA_1389the level of acetyl modification is significantly greater than that of wild type Aeromonas hydrophila, due to the lack of deacetylation modifying enzymes, consistent with protein function.

2.4 Western blotting

Approximately 20 μ g protein samples were separated by SDS-PAGE and then transferred to PVDF membrane using a Trans-Blot Turbo Transfer System (BioRad). Then, after blocking with 5% skim milk in PBS buffer, the membrane was attached toAnti-lysine acetyl modified antibody (purchased from huntington) was incubated at room temperature for 2 hours, then washed 3 times with PBST buffer, and then the membrane was incubated with secondary antibody at room temperature for 2 hours. Finally, the Western blotted membranes were detected using a clay Western ECL substrate (BioRad) and visualized by Image Lab software (BioRad) on a ChemiDoc MP imaging system (BioRad, hercules, CA, usa). Finally, the PVDF membrane was stained with Coomassie R-350 to verify that the loading was equal. As can be seen from fig. 4, in the case where the sample amounts are substantially uniform,ΔcobBandΔAHA_1389the acetylation modification level is obviously improved. The protein has the function of deacetylation modification.

2.5 preparation of acetylated modified BSA

Weighing 1mg BSA dissolved in 1mL 0.1M Na ₂ Co ₃ In (1), 0.04g of acetic anhydride (4% of the total reaction volume) was added while slowly shaking. Subsequently, the above solution was incubated at room temperature (37 ℃) for 3 hours. Finally, 20 μ l 1M Tris was added to the reaction mixture to terminate the reaction, after WB validation (fig. 5), placed at 4 ℃ for future use. As shown in the figure, the treated BSA showed a clear band compared to the unmodified BSA, indicating successful modification.

2.6 A0KI27 deacetylation modification function verification

Mu.g of BSA modified in 2.5 above with 0.2. Mu.g of A0KI27 at 150mM Tris-HCl (pH 8.0), 10 mM MgCl ₂ 、0.5 mM ATP、0.25 mM NAD ⁺ And 10% glycerol, left to stand at 37 ℃ for 4 hours, and then subjected to Western blotting (FIG. 6). The experiment shows that in the case of consistent loading, aeromonas hydrophila AhCobb is in the presence of NAD ⁺ In the case of (2), the effect of removing the acetylation modification of BSA was significant, whereas A0KI27 was observed regardless of the presence or absence of NAD ⁺ The acetylation modification could be significantly removed, showing that it does not need to rely on Zn ²⁺ With NAD ⁺ Assisted, deacetylation modification is possible.

2.7 bioinformatics analysis

The protein family sequences with high similarity between CobB and A0KI27 are obtained through BLAST of NCBI, an evolutionary tree is constructed by using MEGA _7.0.14 software, a protein domain is predicted by using a pfam database (https:// pfam. Xfam. Org /), and a graph is generated by using online software iTOL (https:// iTOL. Embl. De /) (FIG. 7). As shown in FIG. 7, aeromonas hydrophila CobB belongs to the Sirtuin protein family, and A0KI27 is homologous with proteins such as chromosome division ATPase, vitamin B12 synthetic protein, plasmid partition protein and the like of other species, and all have AAA-31 Pfam belonging to P-loop _ NTPase. This indicates that the A0KI27 found in the invention has no obvious homology with the deacetylation modified enzymes known at present, and is a novel protein family of deacetylation enzymes.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> Fujian agriculture and forestry university

<120> bacterial protein lysine deacetylation modification enzyme and application thereof

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atgggcaatc gggaagggat gggacttgtg ctcaagcgag cgttgctgcg cattcaggat 360

cagtacgatt atgtgctgat cgactgcccg cccgtcttgg gggtcatgat ggtcaatgcg 420

ctggcggcgt gtgaccggat cctggtgccg gtacagaccg agtttctggc gctcaaaggg 480

ctggagcgga tgatgaagac gtttgaaatc atgcagcgct ccaagcggga gaagttccgt 540

tataccgtca ttcccaccat gtttgacaag cgaacccgtg cctcgctgat gactctgcag 600

tccatcaagg agcagcacgg caatgcggta tggaatgccg tcatacccat agataccaaa 660

ttccgggatg ccagtttgct tcacattccc ccctccatct attcgccgag cagccgtggc 720

acctatgcct atgagaccct gctcaactat cttgatgcgc aagagcgtca gcgtgctcat 780

gaggtaacat catga 795

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gcaaccaaca gcagtttgt 19

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gccctgccgc tga 13

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catgaattcc cgggagagct ccgagctgag ctcaggtctg ttg 43

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ctctgtatca atcctgcctc gttgtt 26

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gaggcaggat tgatacagag ataccaccca agttcggg 38

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cgatcccaag cttcttctag actcggtcaa cggcaccgc 39

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gtgattgttt ggacggttgc c 21

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gatgttacct catgagcacg ctg 23

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tgcagccacg cagcgacggt ttgct 25

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ggcgccattt gacctggtca cgatg 25

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Claims

1. Bacterial protein lysineThe application of acid deacetylation modification enzyme in lysine deacetylation modification; the bacterial protein lysine deacetylation modification enzyme is A0KI27, and the amino acid sequence of the bacterial protein lysine deacetylation modification enzyme is shown in SEQ ID NO. 1; the gene sequence of the lysine deacetylation modified enzyme for encoding the bacterial protein is shown as SEQ ID NO. 2; the bacterial protein lysine deacetylation modification enzyme does not need to depend on Zn in lysine deacetylation modification ²⁺ With NAD ⁺ And (4) assisting.