CN112481232A

CN112481232A - Bacterial protein lysine deacetylation modification enzyme and application thereof

Info

Publication number: CN112481232A
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: 2021-03-12
Anticipated expiration: 2040-12-14
Also published as: CN112481232B

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, further perfects 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 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, more intensive studies on lysine acetylation modification were carried out. 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 types (SIRT 1-7) which are distributed in different spaces of cells and may also have other deacylase activities. While KDACs in bacteria tend to have homology with KDACs from eukaryotic cells, including mainly 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 bacterial protein lysine deacetylation modification enzyme has been found.

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 species of bacterial deacetylation-modified enzymes are obviously too 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 modification enzyme and application thereof. The protein does not need to depend on Zn²⁺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 modification enzyme is coded, and the gene sequence is shown in 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 Zn²⁺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 knowledge of protein modification of people, plays a promoting role in the research of 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(771bp) andAHA_1389PCR validation of the (792bp) deletion strain.

Lanes

1 and 2 are WT and 2, respectivelyΔcobBAs 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 6XHis tag CobB and A0KI27 proteins purified from 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 under different protein dilution gradients of wild aeromonas hydrophila, the upper part of the protein is the PVDF membrane G350 staining showing the protein loading amount, and the lower part of the protein is the Dolt staining result of the anti-specificity PTM antibody under the acetyl modification condition.

FIG. 4 Aeromonas hydrophilacobBAndAHA_1389acetylation of deletion strainsModified 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 samples 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 in vitro treated acetylated BSA versus untreated BSA, respectively.

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

FIG. 7 is a graph of a 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 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.

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 competent cell of Escherichia coli BL21 was 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 were synthesized by ford bonbonbon.

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 P1 and P2 corresponding to the upstream fragment, primers at two ends of the downstream fragment are respectively P3 and P4, and primers at two ends of the target gene are respectively P5 and P6. In addition, a primer P7 is arranged on a fragment about 100bp upstream of P1, and similarly, a primer P8 is arranged on a fragment about 100bp downstream of P4; then, 500bp fragments of the upstream and downstream of the target gene are respectively amplified, the fragments are connected to the digested pRE112 plasmid by utilizing seamless ligase (C113-01/02, Vazyme Biotech Co. Ltd), the plasmid is transferred into MC1061 to be competent, a single clone is selected, bacteria is shaken at 37 ℃ and 200rpm, PCR (P1P4) verification is carried out, and sequencing of the quality-improved particles is carried out, and bacteria are preserved at-20 ℃.

Transferring the positive plasmid in the step into escherichia coli S17-1 competence, selecting a monoclonal, shaking the bacteria at 37 ℃, 200rpm, and verifying by PCR (P1P4) at-20 ℃ for later use; next, the correct S17-1 strain and Aeromonas hydrophila ATCC 7966 were shaken to OD 1.0, mixed uniformly at a ratio of 4:1, centrifuged at 4000rpm for 5min, the supernatant was discarded, 50. mu.l of fresh LB medium was added for resuspension, all dropped on filter paper (sterilized) placed on blank LB medium, and left to stand at 37 ℃ for 18 h; 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 (P1P4) bacterial liquid, sequencing, and preserving at-20 deg.C; finally, the correct overnight strain is diluted 1000 times, 100. mu.l of diluted bacterial liquid is applied to LB plate containing 20% sucrose, monoclonal is selected, shaken at 30 ℃ and 200rpm for 4h, then 2. mu.l of bacterial liquid is spotted on a chlorine mycin plate drawn with lattices, and bacteria not growing on the chlorine mycin plate are selected for PCR verification, and are subjected to sequencing and stable inheritance, 8Preserving bacteria at 0 ℃ 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 a gene deletion method) is taken as a template, and fragments of about 500bp respectively at the upstream and the downstream of the cobB gene are amplified;

lanes

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

lanes

2.2A 0KI27 protein purification

Using Aeromonas hydrophila ATCC 7966 total genome as templateAHA_1389AndcobBthe gene primers were subjected to PCR amplification. 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 medium (containing 100. mu.g/mL AMP), cultured at 37 ℃ and 200rpm to OD600=0.3-0.6, and the strain was induced at 16 ℃ for 12 h with the addition of 1 mmol/L IPTG. Then, the cells were centrifuged at 10000 Xg at 4 ℃ to discard the supernatant, washed twice with Phosphate Buffered Saline (PBS), and then suspended in 5 mmol/L imidazole solution to be sonicated. After centrifugation, the supernatant was purified by 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 purified target protein has a single band and the size is as expected.

The target protein is A0KI27 obtained by sequencing, 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 the sample was air dried, the film was driedBlocking was performed with TBST containing 5% BSA for 4h, followed by overnight incubation with 10 μ l of anti-lysine acetyl modified antibody (purchased from huntington kingdom, (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), a mouse antibody containing 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 loading was equal. The results are shown in figure 3 of the drawings,ΔcobB、ΔAHA_1389the level of acetyl modification is significantly enhanced compared to wild-type Aeromonas hydrophila, due to the lack of deacetylation-modified enzymes, which is 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). Next, after blocking with 5% skim milk in PBS buffer, the membrane was incubated with anti-lysine acetyl modified antibody (purchased from huntington) for 2 hours at room temperature, then washed 3 times with PBST buffer, and then incubated with secondary antibody for 2 hours at room temperature. Finally, the Western blotted membranes were detected using a Clarity Western ECL substrate (BioRad) and visualized by Image Lab software (BioRad) on a ChemiDoc MP imaging system (BioRad, Hercules, Calif., 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

1mg BSA was weighed out and dissolved in 1mL 0.1M Na₂Co₃In (5), 0.04g of acetic anhydride (4% of the total volume of the reaction) was added while slowly shaking. Subsequently, the above solution was incubated at room temperature (37 ℃) for 3 hours. Finally, 20 was added to the reaction mixtureμ l 1M Tris to stop the reaction, after WB validation (FIG. 5), placed at 4 ℃ for future use. As shown in the figure, the modified BSA had a clear band compared to the unmodified BSA, indicating successful modification.

2.6A 0KI27 deacetylation modification function verification

Mu.g of the 2.5 modified BSA described above were mixed with 0.2 mu g A0KI27 in 150mM Tris-HCl (pH 8.0), 10 mM MgCl₂、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). Experiments show that in the case of consistent loading, Aeromonas hydrophila AhCobB is in the presence of NAD⁺In the case of (2), there was a clear effect of removing the acetylation modification of BSA, whereas A0KI27 was present regardless of the presence or absence of NAD⁺The acetylation modification could be significantly removed, showing that it does not need to rely on Zn²⁺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, the MEGA _7.0.14 software is used for constructing an evolutionary tree, meanwhile, a pfam database (https:// pfam. xfam. org /) is used for predicting protein domains, and then online software ITOL (https:// iTOL. embl. de /) is used for generating graphs (figure 7). As shown in FIG. 7, Aeromonas hydrophila CobB belongs to the Sirtuin protein family, and A0KI27 has homology 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 enzyme known at present, and is a novel protein family of deacetylation enzyme family.

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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aagtttgaca acattcatct gctgcccgcg tcaatcaccc tggcgaccct ggatcgggta 300

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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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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gctgatatcg gatccgaatt cgtgattgtt tggacggttg cc 42

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ctcgagtgcg gccgcaagct ttcatgatgt tacctcatga gcacg 45

Claims

1. A bacterial protein lysine deacetylation modification enzyme, which is characterized in that: the enzyme is A0KI27, and the amino acid sequence of the enzyme is shown in SEQ ID NO. 1.

2. A gene encoding the bacterial protein lysine deacetylation modifying enzyme as claimed in claim 1, wherein: the gene sequence is shown in SEQ ID NO. 2.

3. The use of a bacterial protein lysine deacetylation modifying enzyme as defined in claim 1 for lysine deacetylation modification.