AtPFN2 protein composition acting as a molecular chaperone under thermal shock stress

Abstract

The present invention relates to a protein composition of Arabidopsis thaliana propilin 2 (AtPFN2) that acts as a molecular chaperone under heat shock stress. More specifically, the invention relates to a protein composition of Arabidopsis thaliana propilin 2 (AtPFN2) that protects a plant from heat shock by acting as a molecular chaperone under heat shock stress and converting into a high molecular weight complex.

Description

Technology Field

[0001] The present invention relates to a protein composition of Arabidopsis thaliana propilin 2 (AtPFN2) that acts as a molecular chaperone under heat shock stress. More specifically, the invention relates to a protein composition of Arabidopsis thaliana propilin 2 (AtPFN2) that protects a plant from heat shock by acting as a molecular chaperone under heat shock stress and converting into a high molecular weight complex. Background Technology

[0003] Phloem is the living tissue of vascular plants that transports nutrients to all parts of the plant. It is a transport system that acts as a conduit for viruses, RNA, proteins, lipids, and other small molecules. For example, plant viruses target phloem to rapidly establish a generalized infection. Through this, plant viruses must overcome the host's defense response. Phloem-associated proteins effective in preventing viral transport play various roles in plant defense. Therefore, the proper functioning of plant phloem is essential for growth and development, as well as serving as a defense mechanism against biological and abiotic stresses.

[0005] Much research has been conducted on immune defense mechanisms against pathogen invasion and external stress. When pathogens penetrate plant tissues, powerful defense molecules in the phloem induce apoptosis by destroying the pathogen's plasma membrane. The phloem produces various compounds, such as phytoalexins, antifungal peptides, and small proteins, that inhibit the growth of pathogens.

[0007] Propylin was originally identified as being involved in actin polymerization, but it is also known to play an important regulatory role in F-actin dynamics. Previous studies confirmed that propylin-1 and propylin-2 (AtPFN1 and AtPFN2, respectively), extracted from the phloem sap of *Arabidopsis*, exhibit antifungal functions through apoptosis by inducing membrane potential disruption and cytochrome C release in fungal cells. Additionally, it was confirmed that AtPFN1 and AtPFN2 can inhibit fungal growth by generating cellular reactive oxygen species and mitochondrial peroxides.

[0009] In addition, propylin ligands containing phosphatidylinositol polyphosphate and proline-rich domain-containing proteins were identified. The different affinities of propylin for these ligands are due to sequence differences between isoforms. Previous studies proposed interesting results from size exclusion chromatography (SEC) analysis to confirm the purity of AtPFN1 and AtPFN2 proteins. This study suggested that AtPFN1 and AtPFN2 proteins exhibit various oligomeric structures. These results are similar to those reported in previous studies. In 1996, Babich et al. confirmed the existence of propylin dimers and tetramers, and in 2002, Wopfner et al. reported the spontaneous oligomerization of propylin.

[0011] Accordingly, the inventors analyzed the secondary structures of AtPFN1 and AtPFN2 proteins using CD spectroscopy. As predicted from their amino acid sequences, the α-helical content of AtPFN2 should be higher than that of AtPFN1; however, AtPFN2 exhibited a lower helical content and a higher strand content than AtPFN1, thereby demonstrating strong antifungal activity in low pH buffer. The antifungal activity of AtPFN1 and AtPFN2 proteins was measured using microtiter plate assay. It was confirmed through MIC values ​​that AtPFN2 was 2 to 4 times higher than AtPFN1, and this was disclosed in Korean Patent Publication No. 10-2021-004045, "Antifungal protein composition having propylene as the main component of Arabidopsis thaliana."

[0013] Furthermore, through continuous research on the proteins, the inventors confirmed that the difference in minimum inhibitory concentration (MIC) values ​​between the AtPFN1 and AtPFN2 proteins is due to structural differences between the two proteins. By performing structural analysis of the AtPFN1 and AtPFN2 proteins and measuring potential chaperone activity, they confirmed structural changes in the low molecular weight (LMW) and high molecular weight (HMW) of AtPFN1 and AtPFN2 in vitro.

[0015] Furthermore, the inventors determined that the AtPFN2 protein predominantly functions as a holdase chaperone in the HMW complex, whereas no chaperone function is observed in the LMW form of the AtPFN1 protein; and completed the present invention by measuring the resistance to external heat shock stress through the fact that AtPFN2 acts as a holdase chaperone in the HMW form in a structure-dependent manner. The problem to be solved

[0017] The problem that this invention aims to solve is to confirm that the difference in minimum inhibitory concentration (MIC) values ​​between AtPFN1 and AtPFN2 proteins is due to structural differences between the two proteins, and to measure their potential chaperone activity by performing structural analysis. Furthermore, by confirming structural changes in AtPFN1 and AtPFN2 at low molecular weight (LMW) and high molecular weight (HMW) in vitro, and by measuring that AtPFN2 functions as a holdadase chaperone in the HMW complex while AtPFN1 lacks chaperone function in the LMW form, the invention aims to determine that AtPFN2 acts as a holdadase chaperone in the HMW form in a structure-dependent manner to confer resistance to external heat shock stress. means of solving the problem

[0019] The present invention provides an AtPFN protein composition comprising an AtPFN1 protein having the amino acid sequence of SEQ ID NO: 1 and an AtPFN2 protein having the amino acid sequence of SEQ ID NO: 2, wherein the AtPFN2 protein acts as a molecular chaperone under heat shock stress and is converted into a high molecular weight complex to protect the plant body from heat shock.

[0021] In addition, at this time, the AtPFN2 protein acts as a holdase chaperone in the high molecular weight (HMW) complex, while the AtPFN1 protein does not act as a chaperone in the low molecular weight (LMW) form.

[0023] In this case, the AtPFN2 protein complex is characterized by increased exposure of hydrophobic residues during heat shock stress, which converts it into a high molecular weight complex.

[0025] In addition, the AtPFN2 protein is characterized by having antifungal activity against Candida crusae (accession number: CCARM-14017), Candida tropicalis (accession number: KCTC-7221), Fusarium graminarum (accession number: KCTC-16656), and Cryptococcus (accession number: KCTC-17072) in its low molecular weight structure. Effects of the invention

[0027] The effect of the present invention is to confirm structural changes in AtPFN1 and AtPFN2 in the low molecular weight (LMW) and high molecular weight (HMW) forms in vitro, and to determine that the AtPFN2 protein functions as a holddase chaperone in the HMW complex and the AtPFN1 protein does not function as a chaperone in the LMW form, thereby identifying that AtPFN2 acts as a holddase chaperone in the HMW form in a structure-dependent manner to confer resistance to external heat shock stress. Brief explanation of the drawing

[0029] Figure 1 shows the hydrophobicity of the AtPFN protein and E. coli This is a diagram showing a comparison of the tablets from. (a) Kyte-Doolittle analysis generated hydrophobicity plots for AtPFN1 (gray) and AtPFN2 (red). The y-axis represents the hydrophobicity score. Positive scores on the y-axis represent the hydrophobic region. (b) Recombinant AtPFN1 and AtPFN2 are E. coli It was isolated via expression, and purity was confirmed using 13% SDS-PAGE and Coomassie blue staining. Induction: IPTG-induced total protein, Affinity Resin: Soluble protein purified by an affinity column, SEC: Pure protein isolated from SEC. Figure 2 shows the stability of AtPFN protein under heat shock conditions. It illustrates the thermal stability analysis of AtPFN protein and maleate dehydrogenase (MDH; control). Approximately 5 µg each of AtPFN and MDH were incubated at 25°C (left; normal conditions) or 43°C (right; heat conditions) for 30 minutes, followed by centrifugation at 13,000×g for 15 minutes. The supernatant (soluble fraction) and pellet (insoluble fraction) of each protein were separated and analyzed by 13% SDS-PAGE. Figure 3 shows the structural analysis of recombinant AtPFN proteins in vitro. Concentration-dependent fractions of recombinant AtPFN proteins were analyzed using a 10% native PAGE gel and silver staining. To determine the intrinsic molecular weight of each AtPFN protein under normal conditions, two recombinant proteins were incubated at 25°C for 30 minutes and analyzed after centrifugation at 13,000×g for 15 minutes. Figure 4 shows a comparison of the holddase chaperone activities between AtPFN1 and AtPFN2. Thermal aggregation of 20 µg of maleate dehydrogenase (MDH) was irradiated at 43°C for 20 minutes in the presence of AtPFN1 or AtPFN2 proteins. (a) The molar ratios of AtPFN2 to MDH are (▲) 3:1, (■) 5:1, and (●) 10:1. (◆) indicates a negative control (MDH alone). (b) The molar ratios of AtPFN1 to MDH are (▲) 5:1, (■) 20:1, and (●) 40:1. (◆) indicates a negative control (MDH alone). Holddase chaperone activity was measured by measuring the absorbance of the solution at a wavelength of 340 nm. Figure 5 illustrates the structural analysis of AtPFN2 via size exclusion chromatography (SEC) and transmission electron microscopy (TEM). Purified recombinant AtPFN2 was isolated using MW-based SEC. The HMW (a) and LMW (b) fractions of the AtPFN2 protein isolated by SEC were collected, and the structures of each fraction were confirmed. The HMW and LMW fractions were separated using 12% SDS-PAGE electrophoresis to confirm that they were indeed AtPFN2 proteins (Inset). The oligomeric form of AtPFN2 fractionated by SEC was observed in TEM (Inset). Bars represent 50 nm. Figure 6 is a diagram showing the relationship between AtPFN2 holdingase chaperone function and protein structure. After equalizing the AtPFN2 levels in the two SEC fractions (FⅠ and FⅡ) and the total protein aliquot, the specific chaperone activity of AtPFN2 was measured at 340 nm using MDH as a substrate (A340). (a) Comparison of chaperone activities between the FI and FII fractions of AtPFN2. Heat aggregation of 20 μg MDH was performed at 43°C for 20 minutes in the presence of either the FI or FII fraction of AtPFN2 protein. The activities of the different protein fractions were compared by titration. (b) The activities of the different protein fractions were compared with the activity of the total protein. Total protein activity was measured under the inventors' analytical conditions and set to 1 (division). Representative results represent the average of at least three independent experiments. Figure 7 shows a comparison of the chaperone activity and hydrophobicity of AtPFN1 and AtPFN2 under heat shock conditions. It shows a comparison of the chaperone activity and hydrophobicity of AtPFN proteins at various temperatures. (a) Hydrophobicity analysis under normal and heat shock conditions incubated with bis-ANS for 30 minutes at 25°C, 43°C, and 60°C, respectively. Fluorescence of bis-ANS was measured using a fluorescence meter with an excitation wavelength of 390 nm and an emission wavelength of 430–630 nm. (b) Relative chaperone activity of AtPFN1 and AtPFN2 at 25°C (normal) and 43°C (heat shock). Representative results represent the average of at least three independent experiments. Figure 8 shows the antifungal activity of the HMW and LMW fractions of AtPFN2 protein against four fungal strains. After incubating fungal conidia (a: Candida crusei, b: Candida tropicalis) and proteins for 24 hours, the solution was streaked onto YPD agar and incubated for an additional 24 hours. c: control, 1: 0.5 mg / mL, 2: 0.25 mg / mL, 3: 0.125 mg / mL, 4: 0.0625 mg / mL, 5: 0.03125 mg / mL, 6: 0.0156 mg / mL, 7: 0.0078 mg / mL. After incubating fungal conidia (c: Fusarium graminarum, d: Cryptococcus genus) and proteins for 24 hours, fungal growth was observed using a microscope. Figure 9 is a diagram showing a representative model of the oligomeric state and function of the AtPFN protein. AtPFN2 functions as a chaperone and an antifungal agent in the HMW and LMW structures, respectively. However, AtPFN1, which has the LMW structure, acts as an antifungal protein. Specific details for implementing the invention

[0030] 1. Stability of Arabidopsis propylin protein in plant defense during external heat shock

[0031] Hydrophobic binding is involved in the conformational changes of proteins. Plant propylin proteins have been characterized through binding affinity, intracellular distribution, and antifungal activity. These results suggest that AtPFN proteins play an essential role in plant defense. However, the in vivo functions of Arabidopsis propylin proteins, with the exception of antifungal activity, have not yet been extensively studied.

[0033] To characterize the structure and function of AtPFN1 and AtPFN2, hydrophobicity was first determined based on amino acid information (accessed https: / / web.expasy.org / protscale / on July 22, 2022). It was confirmed that the hydrophobicity of AtPFN2 was higher than that of AtPFN1 (Fig. 1a). Additionally, the genes encoding these two proteins were amplified in Arabidopsis thaliana, and the two AtPFN proteins were overexpressed in E. coli. Finally, the recombinant AtPFN1 or AtPFN2 proteins were purified using a His-tag affinity column and SEC analysis (Fig. 1b).

[0035] According to previous reports, hazelnut propylin was reported to be heat-stable, whereas AtPFN2 was reported to exist in a high molecular weight form. To investigate the heat stability of the two Arabidopsis propylin proteins, they were incubated at 43°C for 30 minutes (Fig. 2). Maleate dehydrogenase (MDH) and AtPFN1, which are heat-sensitive proteins, aggregated, but AtPFN2 showed relatively enhanced heat stability and solubility.

[0037] Interestingly, AtPFN2 exhibited better heat shock resistance than AtPFN1 due to its larger molecular weight. Previous studies explained the structural differences between propylin isoforms using SEC, CD spectroscopy, and transmission electron microscopy (TEM). These results suggest that AtPFN2 may play an additional role in plant stability under external heat stress.

[0039] 2. Molecular chaperone function of AtPFN protein

[0041] Previous studies investigated the growth inhibitory ability of the two AtPFNs on various fungal cells and indicated that AtPFN1 has much higher antifungal activity than AtPFN2. Therefore, in the present invention, the structural form of each PFN protein was first confirmed using 10% native PAGE (Fig. 3).

[0043] The results confirmed that AtPFN2 possesses significantly more HWM forms than AtPFN1. HMW proteins are generally thought to protect denatured substrates from external stress, a characteristic of molecular chaperones. The ability of the two AtPFN isoforms to prevent thermal aggregation of MDH was explored to determine their molecular chaperone function. Heat-unstable MDH was incubated with AtPFN1 and AtPFN2 at 43°C, and heat-induced aggregation of the substrate was monitored using a spectrophotometer.

[0045] The absorbance of heat-denatured MDH increases as the degree of denaturation increases. When chaperone proteins inhibit the denaturation of MDH, the absorbance decreases. The degree of reduction in MDH absorbance indicates chaperone activity. Unstable MDH was progressively protected in a dose-dependent manner when cultured with AtPFN2 under heat shock conditions and was finally inhibited at a subunit molar ratio of 1 MDH to 10 AtPFN2 (Fig. 4a). However, AtPFN1 failed to prevent the thermal aggregation of MDH due to its LMW structure (Fig. 4b). These results suggest that AtPFN2 may possess a dual function as an antifungal agent and a molecular chaperone.

[0047] 3. Structural differences between propylene isoforms

[0049] Many plant proteins with highly oligomeric structures function as molecular chaperones that protect cells from environmental stress. The structural expansion of these proteins generally occurs due to increased hydrophobicity caused by various external factors. It is well known that AtPFN proteins function as antifungal agents.

[0051] To determine the molecular size of AtPFN2 proteins with high chaperone activity, soluble recombinant AtPFN2 was expressed and purified. First, the total AtPFN2 protein was isolated using SEC, and after collecting the HMW and LMW fractions, SEC and TEM analyses were performed again. Interestingly, the AtPFN2 protein of the FⅠ fraction obtained from SEC consisted of high molecular weight (HMW) oligomeric proteins with a molecular weight of approximately 130 to 440 kDa or more (Fig. 5a).

[0053] In contrast, the protein in the FII fraction has a lower molecular weight with a mass of approximately 100 kDa (Fig. 5b). Nevertheless, all protein fractions produced a single band with a MW of 14 kDa on the SDS-PAGE gel (Figs. 5a, b, inset). These results suggest that AtPFN2 is a homooligomeric protein with diverse structures. Next, each protein fraction was collected and concentrated to investigate the structural dependence of the molecular chaperone function of AtPFN2.

[0055] As shown in Figure 6, the FI fraction of AtPFN2 has stronger chaperone activity than the FII fraction. Specifically, compared to the 0.8 times chaperone activity of the FII fraction relative to the crude fraction, the FI fraction exhibited approximately 1.4 times higher chaperone activity compared to the unfractionated AtPFN2 protein.

[0057] Hydrophobic forces are essential for molecular chaperone activity and structural changes in proteins under external stress. The hydrophobicity of the AtPFN protein was determined using a bis-ANS probe that binds to hydrophobic regions. Spectroscopic fluorescence was used to evaluate Bis-ANS binding, which revealed the exposure of the hydrophobic regions of the AtPFN protein. When the probe bound to the AtPFN protein, the emission peak was shifted. The increase in fluorescence intensity indicates that more hydrophobic patches of AtPFN were exposed by heat treatment. The fluorescence intensity of the AtPFN2-bound bis-ANS probe was higher than that of AtPFN1, indicating that the hydrophobic regions of AtPFN2 were more exposed than those of AtPFN1 under normal conditions (Fig. 7a).

[0059] In addition, the correlation between hydrophobic effects and structural changes in proteins was verified by comparing the hydrophobicity of heat-treated AtPFN2 (Fig. 7a). It was confirmed that the degree of hydrophobicity of AtPFN2 increased compared to normal conditions. However, the hydrophobicity of AtPFN1 did not increase at various high temperatures (Fig. 7a). It can be seen that the exposure of more hydrophobic residues in the AtPFN2 protein under heat shock increased the formation of HMW complexes through hydrophobic interactions.

[0061] In addition, it was confirmed that the Holdase chaperone activity of AtPFN2 increased after heat treatment. Interestingly, fluorescence and chaperone activity continuously increased as the culture temperature increased (Fig. 7b). These findings confirmed the inventors' theory that AtPFN2 primarily functions as a molecular chaperone in the HMW complex (Fig. 6a), whereas the antifungal activity is present in the LMW structure (Fig. 8).

[0063] The present invention elucidated the unique physiological and molecular roles of AtPFN2 and demonstrated that they function as molecular chaperones and antifungal proteins to protect plants under various external conditions, such as heat shock and pathogenic attack (Fig. 9). However, the underlying mechanisms of regulation regarding structural and functional changes in AtPFN2 have not yet been verified. Therefore, further research is needed to determine how the protein structure of AtPFN2 is regulated compared to other Arabidopsis prophyllin proteins.

[0065] The present invention will be explained in more detail through the following examples.

[0067] (Example 1) Material

[0069] MDH and 1,1-bi-(4-anilinonaphthalene-5-sulfonic acid) (bis-ANS) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

[0071] (Example 2) Purification and structural analysis of AtPFN protein

[0073] Two AtPFN proteins were generated from an Arabidopsis thaliane cDNA library using PCR. The AtPFN gene was inserted into an overexpression vector (pET28(a)) and transformed into E. coli BL21(DE3). Transformed E. coli cells were grown in LB medium containing 50 µg / mL kanamycin and induced with 1 mM isopropyl-dithiogalactopyranoside (IPTG) when the absorbance at 600 nm (OD600) reached 0.6–0.8.

[0075] Subsequently, the cells were grown for 4 to 5 hours at 30°C. After harvesting the cells, the pellet was resuspended in lysis buffer (50 mM Tris-HCl, 200 mM NaCl, and protease inhibitor, pH 8.0). Meanwhile, the supernatant was centrifuged at 40,000 × g at 4°C for 30 minutes and collected by filtration through a 0.45 μm filter. AtPFN proteins bound to HisPur™ cobalt resin (Thermo F1sher ScientiF1c, Waltham, Massachusetts, USA) were eluted via imidazole treatment. The purified PFN proteins were then dialyzed against phosphate-buffered saline (PBS, pH 7.4) or 2-(N-morpholino)ethanesulfonic acid buffer (50 mM MES, 150 mM NaCl, pH 5.4).

[0077] The purity of the two isolated PFN proteins was determined using 12% or 13% SDS and 10% native-PAGE. The biochemical characteristics of the isolated proteins were analyzed as previously reported. Two SEC fractions of AtPFN2 were placed on a carbon-coated grid (Harrick Plasma, Ithaca, New York, USA), and the grid was negatively stained with 2% uranyl acetate. The structure of AtPFN2 was examined using a 200 kV FEI Tecnai 20 TEM, and images were captured with a Gatan CCD camera.

[0079] (Example 3) Enzymatic analysis of molecular chaperones and antifungal activity

[0081] Molecular chaperone activity was analyzed using the model substrate MDH. Briefly, MDH was incubated with various concentrations of AtPFN protein in 50 mM HEPES-KOH (pH 8.0) buffer at 43°C. During 20 minutes of incubation, thermal aggregation of MDH was determined by monitoring the increase in turbidity of A340 on a temperature-controlled spectrophotometer (DU800; Beckman) as previously described.

[0083] The growth inhibition assay of AtPFN2 against fungal pathogens was performed using a previously reported method. Candida crusei (accession number: CCARM-14017), Candida tropicalis (accession number: KCTC-7221), Fusarium graminarum (accession number: KCTC-16656), and Cryptococcus genus (accession number: KCTC-17072) were obtained from the Antibiotic Resistance Strain Bank (CCARM, Seoul Womans University, Seoul) and the Korea National Center for Biological Resources (KCTC, Jeongeup-si, Jeollabuk-do, South Korea).

[0085] Through this, it was measured that the AtPFN2 protein possesses antifungal activity against Candida crusei (accession number: CCARM-14017), Candida tropicalis (accession number: KCTC-7221), Fusarium graminarum (accession number: KCTC-16656), and Cryptococcus genus (accession number: KCTC-17072) in its low molecular weight structure.

[0087] (Example 4) Analysis of AtPFN hydrophobicity using bis-ANS fluorescence

[0089] The exposed hydrophobic regions of AtPFN were examined by measuring bis-ANS binding to each FPLC fraction using an SFM 25 spectrofluorometer (Kontrom, Zurich, Switzerland) as previously described. Reaction mixtures containing 10 μM of each fraction in 50 mM HEPES buffer (pH 8.0) were incubated with 10 μM bis-ANS for 30 minutes at 25°C, 43°C, and 60°C. The excitation wavelength of bis-ANS was set to 380 nm, and the emission spectrum was scanned between 400 and 650 nm.

[0091] (Example 5) Size exclusion chromatography and polyacrylamide gel electrophoresis

[0093] High-speed protein liquid chromatography (FPLC; Bio-Rad, USA) was performed using an Enrich Size Exclusion Chromatography (SEC) 650 column equilibrated with 50 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (pH 7.4) buffer at 25°C at a flow rate of 0.5 mL / min. The protein fraction peak (A280) was isolated and concentrated using a Centricon YM-10 instrument (Millipore Co., Santa Clara, USA). The protein fraction obtained from the first SEC run was concentrated and stored at 4°C until the second SEC was performed. SDS- and native-PAGE were performed using previously reported methods.

Claims

Claim 1 An AtPFN protein composition comprising an AtPFN1 protein having the amino acid sequence of SEQ ID NO: 1 and an AtPFN2 protein having the amino acid sequence of SEQ ID NO: 2, wherein the AtPFN2 protein acts as a molecular chaperone under heat shock stress and is converted into a high molecular weight complex to possess heat shock resistance. Claim 2 An AtPFN protein composition according to claim 1, characterized in that the AtPFN2 protein acts as a holdase chaperone in the high molecular weight (HMW) complex and the AtPFN1 protein does not act as a chaperone in the low molecular weight (LMW) form. Claim 3 An AtPFN protein composition according to claim 1, characterized in that the AtPFN2 protein complex is converted into a high molecular weight complex by increasing the exposure of hydrophobic residues during heat shock stress. Claim 4 delete

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