DPS protein composition for protecting organisms under heat stress

Abstract

The present invention relates to a composition comprising a DNA-binding protein (DPS) within a poor cell that protects an organism under heat stress. More specifically, the invention relates to a composition that protects a prokaryotic organism under heat stress, comprising a DPS protein that acts as a molecular chaperone to protect intracellular proteins under heat stress.

Description

Technology Field

[0001] The present invention relates to a composition comprising a DNA-binding protein (DPS) within a poor cell that protects an organism under heat stress. More specifically, the invention relates to a composition that protects a prokaryotic organism under heat stress, comprising a DPS protein that acts as a molecular chaperone to protect intracellular proteins under heat stress. Background Technology

[0003] DNA-binding protein within impoverished cells, known as DPS protein (sequence number: 1), was observed to have the function of protecting E. coli from external heat stress.

[0005] All organisms are subjected to various external stresses throughout their lives and possess defense mechanisms to protect themselves from environmental stress and survive. Environmental stress often causes DNA mutations or deletions, leading to damage to genetic information. Consequently, each organism has its own unique DNA protection mechanism.

[0007] For example, eukaryotes use histone proteins that bind strongly to DNA to compress this complex into nucleosomes to protect it from external stress within the nucleus. Unlike eukaryotic cells, prokaryotic cells lack a nucleus, and their DNA is distributed in the cytoplasm. However, they possess various histone-like proteins that bind to and stabilize DNA without nucleosome compression. The DPS protein, named the DNA-binding protein of the impoverished cell, is a novel DNA-binding protein that performs DNA regulatory and protective functions; it was discovered in impoverished E. coli cultures under nutrient-deficient conditions while investigating DNA-binding proteins in prokaryotes.

[0009] DPS oligomers form a shell-like structure composed of 12 monomeric units assembled in tetrahedral symmetry around a pore approximately 45° in diameter. This forms a highly stable DNA-protein complex that binds to DNA without distinguishing specific sequences, thereby defending against DNase-mediated DNA degradation. These DNA-protein complexes polymerize to form complex structures of varying sizes that stably protect DNA from environmental stress. In particular, DPS increases resistance to cytotoxic attacks by regulating the expression of stress-tolerance genes during starvation. The protein structure of DPS and the model by which this protein binds to and stabilizes DNA have been reported in numerous studies and are well-established.

[0011] DPS protein plays an essential role in protecting E. coli during various stresses such as oxidative stress, gamma and UV irradiation, high pressure, heat shock, and iron toxicity. E. coli DPS is the Fe of Listeria innocua 2+ It has a structure similar to ferritin, a protein that binds and stores, and is composed of 12 subunits. It binds to DNA to provide protection against reactive oxygen species and performs functions similar to ferritin.

[0013] Ferritin protects DNA during oxidative stress by reducing iron and hydrogen peroxide, which can generate harmful hydroxyl radicals, through peroxidase activity, namely the Fenton reaction. In particular, two iron ions oxidize one reduced H2O2 molecule, preventing the generation of hydroxyl radicals through Fenton chemistry. Fe 2+ + H2O2→ Fe 3+ + OH - + 'OH. Therefore, to protect E. coli from external stress, not only the DNA binding activity of DPS but also the action of peroxidase is essential.

[0015] Despite numerous reports on the structural characteristics and DNA binding activity of DPS proteins, studies on their in vitro biochemical activity and function under environmental stresses such as heat shock have been limited. DPS mutants have been shown to have increased sensitivity to heat shock compared to the wild type. Since DPS proteins, composed of 12 monomers in an oligomeric structure, protect E. coli from heat and oxidative stress, we hypothesized that these DPS oligomers are heat-stable and potentially function as molecular chaperones.

[0017] Accordingly, the inventors purified the recombinant GST-DPS protein and confirmed that the DPS protein exists in the form of a high molecular weight oligomeric complex and exhibits high heat stress stability. They also confirmed that the hydrophobic region of the GST-DPS protein prevents the aggregation of substrate proteins and exhibits molecular chaperone activity, thereby conferring heat resistance to prokaryotes such as E. coli, and thus completed the present invention. The problem to be solved

[0019] Therefore, the problem that the present invention aims to solve is to purify the recombinant GST-DPS protein, confirm that the DPS protein exists in the form of a high molecular weight oligomeric complex and exhibits high heat stress stability, and to measure that the hydrophobic domain of the GST-DPS protein prevents the aggregation of substrate proteins and exhibits molecular chaperone activity, thereby conferring heat resistance to prokaryotes such as E. coli. means of solving the problem

[0021] The object of the present invention is to provide a composition comprising a DNA binding protein (DPS) in a poor cell that protects prokaryotes under heat stress, wherein the DPS protein having the amino acid sequence of SEQ ID NO: 1 is converted from a basic oligomer composed of 12 monomers under heat stress into a high molecular weight oligomer complex comprising multiple monomers, and exhibits molecular chaperone activity capable of preventing denaturation or aggregation of substrate proteins from heat shock.

[0023] At this time, the above DPS protein is characterized as being a GST-DPS protein obtained by lysing wild-type E. coli, amplifying DNA by performing 35 cycles of PCR cycling of denaturation, annealing, and extension using a forward primer having the nucleotide sequence of SEQ ID NO: 2 and a reverse primer having the nucleotide sequence of SEQ ID NO: 3, and electrophoresis on an agarose gel.

[0025] In addition, the above DPS protein is characterized by having chaperone activity of a Holdase molecule, which prevents protein denaturation and restores the original protein tertiary structure when proteins in prokaryotes are exposed to stress, as the basic oligomer is converted into a high molecular weight oligomer complex under heat stress.

[0027] Meanwhile, the above-mentioned DPS protein is characterized by exhibiting Holdase molecular chaperone activity by exposing the hydrophobic domain of the DPS protein within a prokaryotic organism to bind to a substrate protein and prevent the aggregation of the substrate protein. Effects of the invention

[0029] The effect of the present invention is to provide a composition comprising a DPS protein that protects organisms under heat stress, by confirming that after purifying the recombinant GST-DPS protein, the DPS protein exists in the form of a high molecular weight oligomeric complex and exhibits high heat stress stability, and confirming that the hydrophobic region of the GST-DPS protein prevents the aggregation of substrate proteins and exhibits molecular chaperone activity, thereby conferring heat resistance to prokaryotes such as E. coli. Brief explanation of the drawing

[0031] Figure 1 shows the thermal stability of the GST-DPS recombinant protein. (A) shows the nucleotide and amino acid sequences of DPS. Asterisks indicate stop codons, and the numbers on the right indicate the amino acid positions of DPS. (B) shows a schematic diagram of DPS. Ferritin-like domains were indicated using black boxes. (C) shows the purity of the GST-DPS recombinant protein analyzed using SDS-PAGE. (D, E) show the protein stability of MDH and GST-DPS. Each protein was exposed to temperatures of 25°C, 40°C, and 50°C for 30 minutes, after which the supernatant and pellet fractions were separated by centrifugation. The proteins were loaded onto a reduction gel and detected by silver staining. The solubility ratios of the samples are shown in (E). Data are expressed as mean ± standard deviation (n=3). Results were repeated at least three times using independent samples. Figure 2 shows the protein structure of heat-shock treated GST-DPS. (A) shows the structural changes in GST-DPS induced by heat shock. Recombinant proteins were heat-treated at 25°C, 40°C, and 50°C for 30 minutes, then analyzed using Native-PAGE (top) or SDS-PAGE (bottom) followed by silver staining. (B) shows the SEC analysis of the protein samples described in (A). In (C), the hydrophobicity score of DPS was analyzed using the ProtScale program. (D) shows the bis-ANS analysis to measure the heat stress-induced exposure of the hydrophobic regions of GST and GST-DPS. The fluorescence of bis-ANS was measured at excitation 380 nm and emission scan 400–600 nm. The samples used were bis-ANS (■), bis-ANS + GST ​​treated at 25°C (■) or 50°C (△), and bis-ANS + GST-DPS treated at 25°C (○) or 50°C (●). The results were repeated at least 3 times using independent samples. Figure 3 shows the molecular chaperone activity of GST-DPS. (A) to (C) show the chaperone activity assays. Light scattering was measured at 340 nm for 20 minutes. Concentration-dependent chaperone activity was analyzed using MDH (A) and CS (B) as substrates. The substrates were incubated with various concentrations of GST-DPS in the reaction mixture. The concentration ratios used for MDH:GST-DPS were 1:1 (●), 1:2 (▲), and 1:3 (◆). GST (■) was used as a negative control. (C) shows the effect of thermal shock on the chaperone activity of GST-DPS. The ratio of MDH to GST-DPS used at 25°C (●) or 50°C (○) was 1:1, and at 25°C (◆) or 50°C (◇) was 1:3. MDH alone (??) was used as a control. The results were repeated at least three times using independent samples. Figure 4 shows a schematic model illustrating the activity of DPS under various stresses. Under oxidative stress, oligomeric DPS protects DNA through DNA binding activity, DNA-protein complex formation, and peroxidase activity, and reduces harmful hydroxyl radicals via the Fenton reaction. Additionally, under heat stress, DPS acts as a molecular chaperone to protect intracellular proteins. These activities demonstrate the important role of DPS proteins in protecting prokaryotic cells from damage caused by oxidative and heat stress. Specific details for implementing the invention

[0032] The present invention is to provide a composition comprising a DNA-binding protein (DPS) within a poor cell that protects an organism under heat stress. More specifically, the invention is to provide a composition that protects a prokaryotic organism under heat stress, comprising a DPS protein that acts as a molecular chaperone to protect intracellular proteins under heat stress.

[0034] The present invention will be described in detail below.

[0036] 1. Purification and Thermal Stability of GST-DPS Protein

[0038] The inventors identified structural and functional domains in the protein sequence of DPS using the web-based tool NCBI Conserved Domain Search (CD-Search; http: / / www.ncbi.nlm.nih.gov / stcture / cdd / wrpsb.cgi). DPS is known to be encoded by 167 amino acids consisting mainly of a ferritin-like domain (Figs. 1A and B) and to form a cage oligomer composed of 12 monomers.

[0040] Various proteins exhibit molecular chaperone functions through oligomer formation to prevent substrate aggregation during heat shock. To confirm whether DPS oligomers also exhibit molecular chaperone activity, GST-fusion DPS recombinant protein (GST-DPS) was purified, and its purity was verified by SDS-PAGE (Fig. 1C).

[0042] Purified GST-DPS protein was used to characterize biochemical functions. To evaluate potential chaperone activity, the stability of GST-DPS under heat shock was assessed. After heat-treating GST-DPS protein at 40°C and 50°C, the soluble fraction and pellet fraction were separated for further analysis. Maleate dehydrogenase (MDH) was used as a control protein. While common proteins such as MDH are unstable and prone to aggregation under heat shock conditions, GST-DPS protein exhibits high stability (Fig. 1D). Solubility measurements showed that both MDH and GST-DPS were soluble under normal conditions.

[0044] However, when exposed to thermal shock, the solubility of MDH decreases sharply, whereas the solubility of GST-DPS does not change significantly (Fig. 1E). These results indicate that GST-DPS exhibits significant stability during thermal shock and has the potential to act as a molecular chaperone under such conditions.

[0046] 2. Protein structure of GST-DPS under heat shock

[0048] We confirmed that the DPS protein forms oligomers composed of 12 monomers and verified the heat shock stability of the GST-DPS protein (Figs. 1D and E). Subsequently, we investigated whether the structure of GST-DPS changes in response to heat shock. Native-PAGE analysis of the heat-treated GST-DPS protein revealed that, similar to previous reports, GST-DPS exists primarily as oligomers under normal conditions, with larger oligomers forming at 50°C (Fig. 2A). These findings were further confirmed using SEC. Notably, GST-DPS exists in an oligomer fraction greater than 440 kDa under normal conditions, and the results were similar to those under heat shock (Fig. 2B).

[0050] Molecular chaperones protect proteins from stress by binding to substrates through hydrophobic sites. To confirm the potential of GST-DPS as a molecular chaperone, the hydrophobicity of GST-DPS was evaluated using bioinformatics tools such as ProtScale (https: / / web.expasy.org / protscale / ). The hydrophobicity was approximately 40% (Fig. 2C). Subsequently, the hydrophobicity of GST-DPS was measured using bis-ANS [1,1-bi(4-anilinonaphthalene-5-sulfonic acid)], which binds to the hydrophobic site.

[0052] The reaction between GST-DPS and bis-ANS increased fluorescence intensity compared to the GST control. The fluorescence intensity of heat-treated GST-DPS increased further (Fig. 2D). These results imply that the hydrophobic regions of GST-DPS are exposed during thermal shock, potentially increasing binding affinity to the substrate.

[0054] 3. Holdase chaperone activity of GST-DPS

[0056] To verify the chaperone activity of GST-DPS, the holdase chaperone activity was evaluated using MDH and citrate synthase (CS) as substrates. Thermal shock induces the aggregation of MDH and CS, thereby increasing absorbance. Therefore, the inventors measured whether GST-DPS protects against substrate aggregation. The results showed that as the concentration of GST-DPS increased, it effectively protected against the aggregation of MDH and CS, demonstrating its role as a molecular chaperone (Figs. 3A and B).

[0058] In addition, the inventors investigated the effect of heat shock on the chaperone activity of GST-DPS, specifically whether heat-stressed GST-DPS exhibits higher chaperone activity than normal GST-DPS due to potentially larger oligomer sizes. In particular, the inventors' findings showed that heat-stressed GST-DPS exhibited significantly higher chaperone activity compared to normal GST-DPS at the same concentration (Fig. 3C). These results provide new mechanistic insights into the role of heat-induced, highly oligomeric DPS as a potent molecular chaperone protecting intracellular proteins under heat shock.

[0060] DPS protects E. coli under oxidative stress through DNA binding and peroxidase activity. In this study, the inventors demonstrated a novel function of DPS as a molecular chaperone during heat shock (Fig. 4). In particular, when subjected to heat shock, the hydrophobic regions of DPS are exposed to bind to the substrate and act as a molecular chaperone that prevents aggregation.

[0062] While multifunctional proteins with multiple domains are common, this finding is noteworthy because DPS proteins possess only a single ferritin-like domain. PTM modifications induce structural changes that enable a single protein to recognize various signals and perform diverse functions. For example, proteins that switch functions in response to external stresses, such as heat or cold stress, are well-documented. In yeast, cPrxI switches from a peroxidase to a superchaperone during oxidative stress.

[0064] AtUSP acts as a molecular chaperone under heat shock and as an RNA chaperone under cold shock. DPS confers resistance to heat shock by acting as a chaperone that protects DNA through DNA binding and peroxidase activity under oxidative stress and protects substrate proteins under heat stress. Therefore, further experiments are required regarding the signaling mechanisms used by DPS to distinguish between the two conditions.

[0066] DPS uses Fe through a peroxidase center 2+ It reduces the generation of hydroxyl radicals that bind to ions and are promoted by the Fenton reaction. Streptococcus suis Dpr, belonging to the DPS family, binds to and removes intracellular zinc ions. Therefore, ions such as iron or zinc are likely to assist DPS in regulating mechanisms such as distinguishing between DNA binding and chaperones.

[0068] The present invention will be explained in more detail through the following examples.

[0070] (Example 1) DPS protein replication

[0072] Wild-type E. coli cells were lysed and DNA was released by treating them with a boiling step for 5 minutes. PCR primers were specially designed based on the DPS gene sequence for gene amplification using polymerase chain reaction (PCR). The primers used for PCR are as follows: DPS forward (BamHI), 5'-GGATCCATGAGCACCGCGAAACTG-3' (Sequence No. 2) and DPS reverse (HindIII), 5'-AAGCCTTTATTCAATGTTGCTTTCAATAAACCACAG-3' (Sequence No. 3). PCR was performed after incubation at 95°C for 5 minutes, followed by 35 cycles of 30 seconds at 95°C, 30 seconds at 58°C, 1 minute at 72°C, and 10 minutes at 72°C. The size and purity of the amplified genes were confirmed by electrophoresis using a 0.8% agarose gel stained with ethidium bromide.

[0074] (Example 2) Purification of DPS recombinant protein in E. coli

[0076] As previously described, the cDNA of the DPS gene was inserted into a pGEX expression vector, and the plasmid was introduced into E. coli BL21(DE3) cells. Transformed colonies were cultured overnight in LB medium at 37°C, followed by subculture at 30°C until the absorbance at 600 nm reached 0.5–0.6. Expression of the DPS recombinant protein was induced by adding 0.5 mM isopropyl-bD-thiogalactopyranoside (IPTG), and the cells were cultured at 30°C for 5 hours. Cells were harvested by centrifugation and resuspended in a buffer [1.8 mM KH2PO4 (pH 8.0), 140 mM NaCl, 2.7 mM KCl, and 10 mM Na2HPO4]. After freezing and thawing, the cells were lysed by sonication, and the supernatant was loaded into a glutathione-cephalosporose matrix. GST-fusion DPS recombinant protein (GST-DPS) was eluted using 10 mM reduced glutathione and dialyzed against 10 mM HEPES-NaOH (pH 8.0) buffer.

[0078] (Example 3) Thermal stability of MDH and GST-DPS

[0080] The thermal stability of malate dehydrogenase (MDH) and GST-DPS was evaluated using recombinant MDH and GST-DPS proteins according to the previously described modified protocol. The recombinant proteins were subjected to heat shock at 40°C and 50°C for 30 minutes. After centrifugation, the soluble fraction and the pellet fraction were separated. Subsequently, each protein was loaded onto a 12% reducing gel and detected using silver staining.

[0082] (Example 4) Fluorescence measurement

[0084] The hydrophobicity of GST-DPS was evaluated by measuring the fluorescence of bis-ANS [1,1-bi(4-anilinonaphthalene-5-sulfonic acid)] using an SFM 25 spectrofluorometer excited at 380 nm, and the emission was scanned between 400 and 600 nm according to the protocol described above. A reaction mixture containing 10 mM DPS and bis-ANS was incubated at 25°C for 30 minutes.

[0086] (Example 5) Analysis of Structural Changes in GST-DPS Due to Thermal Shock

[0088] Structural changes in GST-DPS due to heat shock were observed using recombinant GST-DPS proteins treated with heat shock at 40°C and 50°C. The proteins were separated using basic PAGE and detected using silver staining by modifying the previously described protocol.

[0090] (Example 6) Size exclusion chromatography (SEC)

[0092] The structure of GST-DPS was analyzed using a GE Healthcare Superdex 200 HR 10 / 30 column equilibrated with 20 mM HEPES-KOH (pH 8.0) buffer. The previously described protocol was followed with modifications.

[0094] (Example 7) Analysis of Holdase Chaperone Activity of GST-DPS

[0096] The holdase chaperone activity of GST-DPS was evaluated using MDH and citrate synthase (CS) as substrates. The substrates were mixed with GST-DPS or heat-shocked GST-DPS at various concentrations. Turbidity caused by thermal aggregation of MDH or CS at 45°C was monitored using a DU800 spectrophotometer at A340 nm according to the previously described method. A chaperone is a protein involved in protein folding; for example, when a protein is subjected to stress such as heat shock, its properly folded three-dimensional structure unfolds, preventing it from performing its role normally. A chaperone is a protein that recognizes and binds to these unfolded proteins, creating an appropriate environment for them to fold properly again.

[0098] Molecular chaperone activity is distinguished into holdase and foldase activities. Holdase refers to the function of creating an environment where, for example, when a protein is exposed to stress—such as oxidative stress or heat stress—it becomes denatured, causing its folded tertiary structure to partially unravel and hydrophobic amino acid residues to be exposed, and when this process intensifies, the denatured proteins irregularly clump together to form aggregates that are then degraded and eliminated by proteolytic enzymes. In this case, chaperone proteins (SHSPs, DnaJ) bind to the hydrophobic amino acids of the protein that has partially unraveled due to stress, preventing the protein from forming aggregates and allowing it to return to its original 3D structure.

Claims

Claim 1 A composition comprising a DPS protein having the amino acid sequence of SEQ ID NO: 1, which is converted from a basic oligomer composed of 12 monomers into a high molecular weight oligomer complex containing multiple monomers under heat stress to prevent denaturation or aggregation of substrate proteins from heat shock, wherein the DPS protein possesses Holdase molecular chaperone activity such that the basic oligomer is converted into a high molecular weight oligomer complex under heat stress to prevent protein denaturation and restore the original protein tertiary structure when the protein in the prokaryotic organism is exposed to stress, and the DPS protein exhibits Holdase molecular chaperone activity by exposing the hydrophobic region of the DPS protein within the prokaryotic organism to bind to the substrate protein and prevent the aggregation of the substrate protein. Claim 2 delete Claim 3 delete Claim 4 delete

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